18

Plasma proteins

18

Plasma proteins

18

Plasma proteins

18

Plasma proteins

18.1 Plasma protein diagnostics

Lothar Thomas

The term plasma proteins refers to the proteins of the blood plasma circulating between the blood and the interstitial fluid compartments. Specimens are serum, plasma, urine, cerebrospinal fluid, saliva, amnion fluid, aspirated fluids (ascites, pleural exudate) and feces. Under physiological conditions, the distribution of these proteins is at a steady state between the fluid compartments /1/. The term also includes other proteins such as immunoglobulins, enzymes, and blood clotting factors.

Functionally, plasma proteins are classified into:

  • Transport proteins; they bind and transport hardly water-soluble substances; albumin, for example, is an important transport protein
  • Acute phase proteins; they are associated with inflammation; the C-reactive protein, for example, is an important acute phase protein
  • Proteins of the immune defense systems, for example immunoglobulins and complement factors
  • Proteins shed within the scope of cell membrane changes, for example the soluble transferrin receptor
  • Factors and inhibitors of plasmatic coagulation
  • Oncofetal proteins; they are produced by tumors or physiologically produced during the fetal period (e.g.,α-fetoprotein).

Diagnostically important plasma proteins are listed in Tab. 18.1-1 – Diagnostically significant plasma proteins.

18.1.1 Plasma protein synthesis and distribution

The serum concentration of plasma proteins depends on synthesis, catabolism, distribution in intracellular and extracellular fluid compartments, loss into “third space” (ascites, pleural effusion) or to the outside (proteinuria) /1/. Plasma proteins are determined in cases of:

  • Normo-, hypo- or hyperproteinemia defined by determination of total protein in serum
  • Dysproteinemia, a disturbance in the plasma protein composition. In this case, serum protein electrophoresis shows an atypical protein pattern
  • Special clinical questions as single protein (e.g., haptoglobin in presumed hemolytic anemia).

18.1.2 Plasma protein synthesis

Whereas immunoglobulins (Ig) are produced by the plasma cells, the non-Ig proteins are primarily synthesized in the hepatocytes, with the following exceptions: β2-microglobulin and the transferrin receptor, which are shed by cells as surface proteins, as well as complement factor D, a protease involved in the activation of the alternative complement pathway and produced by adipocytes.

Like lysosomal proteins and cell membrane proteins, plasma proteins are produced by the polyribosomes of the rough endoplasmic reticulum. The general mechanism for the synthesis of proteins is the same in all body cells. The synthesis of proteins includes the following steps:

Transcription 

DNA in the cell nucleus is the carrier of the genetic information, and a copy is produced in the form of RNA by RNA polymerase /2/. After the intervening sequences (or introns) are turned off, the messenger RNA (mRNA) is produced. This mRNA transfers the information regarding which protein will be synthesized from the cell nucleus to the cytoplasm. The regulation of transcription is achieved through regulatory proteins (transcription factors) that bind to specific DNA sequences of the gene to be regulated, and exert either a positive or negative effect on protein synthesis. For example, if an acute phase reaction occurs, the pro inflammatory cytokines will bind to the hepatocyte plasma membranes. This signal induces to the activation of the nuclear proteins that bind to the corresponding DNA sequences in the nucleus, thus leading to an amplified transcription of acute phase proteins such as C-reactive protein.

Translation

The mRNA base sequence is translated into a defined amino acid sequence. Translation takes place on the ribosomes of the endoplasmic reticulum, to which the mRNA becomes attached.

Protein synthesis

Plasmaprotein synthesis takes place in the ribosomes of the endoplasmic reticulum. The ribosomes are composed of ribonucleic acids and proteins that are produced by the nucleolus. Most proteins acquire two additional peptides on the N-terminal end during synthesis, a hydrophobic peptide and a pro peptide. The hydrophobic peptide is composed of 15–30 amino acids, and serves as a pilot peptide to guide the protein through the membrane of the endoplasmic reticulum in its lumen. The pilot peptide is immediately cleaved after the protein is discharged through the lumen of the endoplasmic reticulum. The folding of the peptide into its final tertiary structure takes place via the formation of disulfide bridges in the endoplasmic reticulum. Certain proteins are transported from the lumen of the endoplasmic reticulum into the lumen of the Golgi apparatus for secretion. The pro peptide remains on the protein while it matures in the vesicles of the Golgi apparatus or is stored in the secretory granules. The pro peptide is cleaved shortly before the release of the protein from these organelles /3/.

Co translational or post translational protein modification

Protein modifications start as the protein passes through the lumen and the cisterns of the endoplasmic reticulum and Golgi apparatus. The majority of the plasma proteins are glycoproteins and some are lipoproteins.

Other co translational or post translational modifications

Oxidation of C atoms 3 and 4 which results in the formation of thioester binding, phosphorylation of, for example, α2HS-glycoprotein and binding of phospholipids.

18.1.3 Glycoproteins

The glycosylation of proteins is important for the folding into a tertiary structure, and for their functioning and degradation /4/. There are two types of protein glycosylation, N-glycosylation and O-glycosylation. They are classified into:

  • N-glycosylated type: most proteins are glycosylated by forming a covalent N-bond between the amino acid asparagine and a carbohydrate residue composed mainly of mannose. This step begins in the lumen of the endoplasmic reticulum. In a subsequent process that occurs over multiple stages, a transformation of the carbohydrate chains takes place wherein the terminal position is often bound to N-acetyl neuraminic acid, while galactose is linked at the preterminal position.
  • O-glycosylated type: some plasma proteins contain sugar residues that are bound via the OH-groups of the amino acids serine and threonine. O-glycosylation takes place in the Golgi apparatus.

Microheterogeneity of glycoproteins

All glycoproteins exhibit heterogeneity, since they are derived from a population with various oligosaccharide sequences (glycoforms). The variation comes from the different numbers of carbohydrate chains, the varying chain length and the differing end substitutions that are borne by the amino acid skeleton. As a result, each glycoprotein can occur in forms having different net electric charges. This is manifested in the electrophoresis as micro heterogeneity. Thus, for example, acidic α1-glycoprotein exhibits seven bands with isoelectric focusing at acidic pH. Differences in the glycosylation of IgG have an effect on immunoregulation /5/.

18.1.4 Lipoproteins

Lipoproteins are proteins that are coupled covalently with lipids (e.g., in the cell membranes). Thus, a fatty acid can bind to an N-terminal glycine as an amide, or to cysteine as a thioester. Plasma lipoproteins are non covalent aggregates of lipids and proteins.

18.1.5 Release of proteins into the plasma

Following post translational modification, the secretory plasma proteins are stored in vesicles that are separate from the Golgi apparatus. These vesicles fuse with the plasma membrane of the cell, and the proteins are released into the extravascular compartment via exocytosis. The pro peptide plays an important role in this process.

18.1.6 Plasma protein distribution

Plasma proteins are continuously redistributed in both directions between the vascular and interstitial compartments. This takes place via diffusion through the capillary walls, pinocytotic transport through the capillary endothelium or over the intercellular connections of the tissue cells /6/. The distribution depends on molecular weight. The higher the molecular weight, the greater will be the intravascular proportion of that protein (Tab. 18.1-2 – Distribution of plasma proteins between the intravascular and extravascular compartments). Large proteins, such as fibrinogen, α2-macroglobulin and IgM remain predominantly in the extravascular compartment, and will only enter circulation to a limited extent via the intercellular connections between the endothelial cells or through pinocytosis. However, the reverse flow from the tissue into the blood vessels occurs through the lymph vessels. Glomerular filtered plasma proteins are absorbed by the cells of the proximal tubule through pinocytosis, and are degraded in these tubule cells.

The quantity of plasma proteins transferred from the intravascular to the extravascular compartment is organ-dependent; the levels are high in the liver, but quite low in the brain so that the protein concentration in the cerebrospinal fluid is about 300-fold lower than in the plasma. In the case of systemic disorders, however, the vascular permeability can change drastically and can lead to the development of exudations in the form of effusions or edema.

18.1.7 Plasma protein catabolism

Plasma protein degradation occurs in all somatic cells (see also Section 18.2.7 – Pathophysiology). The released amino acids are used by the cells for resynthesis of proteins; only essential amino acids must be taken in with food. Desialation plays an important role in the breakdown of glycoproteins. The intactness of the carbohydrate side chains protects the protein from degradation /7/. Removal of sialic acids and reduction of carbohydrates by membrane-bound or circulating enzymes favors pinocytosis and intracellular degradation by lysosomal enzymes /8/.

Organs and tissues that are significantly involved in the degradation of proteins include:

  • The hepatocytes; they are easily reached by plasma proteins since the liver sinusoids lack basement membranes and endothelial cells have marked intracellular fenestrations
  • The kidneys; following glomerular filtration, low molecular weight proteins are taken up by the brush border of tubular cells by pinocytosis and subsequently degraded by lysosomal enzymes
  • The endothelial cells of capillaries; although these cells display only minimal capacity for pinocytosis, their catabolic potential is high because of the large size of the capillary bed.

The degradation rate of plasma proteins varies and is described by their half-life (albumin 19 days, ceruloplasmin 4 days, transferrin 8 days, IgG 23 days, IgA 5 days and IgM 5 days).

Degradation of senescent proteins is non-enzymatic by way of post translational modification based on various reactions /9/:

  • Glycation; the most common reaction is the binding of glucose or other reducing substances to proteins. The carbonyl groups of the sugars and the amino groups of the protein form a Schiff base that quickly forms a stable keto amine through molecular rearrangement (Amadori product). In further reactions involving oxidation, the Amadori product is converted into an advanced glycation end product (AGE). Glycated hemoglobin is a typical Amadori product (see Fig. 3.6-1 – Glycation of N-terminal valine of hemoglobin with glucose and subsequent Amadori rearrangement).
  • Direct oxidation by reactive oxygen species forming advanced oxidation protein products (AOPPs). The main AOOPs include methionine sulfoxide from oxidation of methionine and 3-nitro tyrosine from nitration of tyrosine.
  • Carbamylation from the binding of isocyanic acid to amino acids, especially to ε-NH2 groups of lysine residues. The isocyanides form due to spontaneous dissociation of urea or reaction of thiocyanate catalyzed by myeloperoxidase in the presence of H2O2.

The organism tries to maintain the total protein concentration in the intravascular compartment at a constant level within certain limits. For instance, in infectious diseases, the elevated level of acute phase proteins and immunoglobulins is compensated for by a reduction in the concentration of negative acute phase proteins (albumin, transthyretin, transferrin, apolipoproteins). In multiple myeloma, the monoclonal increase in immunoglobulins is compensated for a long time by suppression of polyclonal immunoglobulin production.

18.1.8 Modifications in plasma protein synthesis

The daily physiological turnover of plasma proteins is about 25 g and depends on the amino acid pool available for protein synthesis. This pool, in turn, is dependent on numerous variables.

Reduced plasma protein synthesis

Genetically induced; inflammation, hepatopathies (liver cirrhosis, acute hepatitis), nutritional protein deficiency, hypothyroidism, malabsorption syndrome, alcoholism, lymphoma, metastasized carcinoma.

Increased plasma protein synthesis

Inflammation, fever, hyperthyroidism, hyper cortisolism, increased release of growth hormone, protein-losing syndrome, iron deficiency, stimulation of the immune systems and clonal increase in the number of immunoglobulin-producing plasma cells (multiple myeloma).

Genetic impacts on plasma protein synthesis

Impacts may present clinically in the form of protein deficiency, protein increase or protein dysfunction. For instance, a protein may:

  • Not be synthesized (e.g., hereditary IgA deficiency)
  • Be synthesized with a structural defect and thus not be released from the cell (e.g., hereditary α1-antitrypsin deficiency)
  • Be secreted as a structurally similar but functionally inactive variant (e.g., C1-esterase inhibitor in hereditary angioneurotic edema).

18.1.9 Diagnostic significance of plasma proteins

One-time determination

In certain clinical concerns, the determination of the concentration or activity of particular plasma proteins provides important help in the diagnosis of diseases, for example:

  • CRP if systemic inflammation is suspected
  • Haptoglobin in presumed intravascular hemolysis
  • α1-antitrypsin in pulmonary emphysema.

In addition, the finding of a normal plasma protein concentration is also of important differential diagnostic value in order to rule out certain diseases.

Plasma protein profile

The plasma protein profile allows differential diagnostic conclusions.

Monitoring

During the course of the disease (acute symptoms) or during therapy, the plasma protein concentration and its changes during the disease course may provide conclusions as to the activity, severity and possible complications of the condition. The time in point when normalization is achieved has important prognostic implications.

18.1.10 Indication

Important indications for plasma protein determination are listed in Tab. 18.1-3 – Important indications for plasma protein determination.

18.1.11 Specimen

Because of the high intravascular proportion of plasma proteins in relation to the interstitial fluid, a concentrating effect may occur before or during blood collection, thus simulating falsely elevated protein concentrations. This is the case, for example, if the patient does not sit for at least 15 min. prior to blood collection and venous occlusion during blood collection lasts for more than 3 min. The sample should be analyzed the same day. If this is not possible, it is better to store the sample at 4 °C than at –20 °C if deep-freezing at –70 °C is not feasible. Storage at 20 °C is associated with a measurable decline in protein concentration after 36 hours that affects many proteins.

Immunonephelometric determination in serum and in lithium heparin plasma yields comparable results /10/.

18.1.12 Method of determination

Principle

Immunochemical methods are used to determine plasma proteins. For the determination of an unknown plasma protein concentration a constant amount of specific antibodies is employed. Refer to Section 52.1-4 – Principles of immunochemical methods. The critical factors which determine whether the immunocomplexes which are formed can be detected in a soluble form or a precipitable form include besides the plasma protein concentration to be determined, the ratio of the plasma protein to the antibody concentration (curve according to Heidelberger and Kendall, refer to Fig. 52.1-5 – Precipitin curve. In antibody excess, soluble immune complexes are formed. The measurement of the immune complex concentration is made nephelometrically or turbidimetrically.

In immunonephelometry, light from a helium neon laser is directed through the cuvette and is scattered by the immune complexes; this scattered light is then focused on a detector by means of a lens system. The electrical signal of the detector is proportional to the light scattering intensity. The concentration of the plasma protein concentration can be determined from the light scattering signal with the help of a calibration curve. In the case of kinetic-nephelometric plasma protein determination, changes in the scattered light are measured at short intervals, while the endpoint method allows the reaction to occur for a defined period of time (e.g., 15 or 30 min.). By adding further plasma protein or antibodies, it is possible to check whether the measurement occurred on the slope of the Heidelberger and Kendall curve (see Fig. 52.1-5 – Precipitin curve). This is the case if the addition of sample (plasma protein) results in an increase of the measurement signal or if the addition of antibodies produces no change in the measurement signal.

In immunoturbidimetry soluble immune complexes are formed by the addition of the plasma protein to antibodies and a buffer containing accelerator that allows kinetic measurement according to the fixed time principle. The increase of absorption at 334 or 340 nm is measured within a defined time period.

Calibrators for the plasma protein assays are standardized using the BCR/IFCC/CAP RPPHS reference material, also know as CRM 470.

The detection limit for the immunonephelometric and immunoturbidimetric tests, which is about 10 mg/L, can be increased by a factor of 10 to 100 if the specific antibodies are bound to latex particles (latex-enhanced assays).

18.1.13 Quality assurance

Since the introduction of the reference preparation BCR/IFCC/CAP RPPHS, also known as CRM 470 (now ERM-DA470), which contains reference values for the plasma proteins listed in Tab. 18.1-4 – Plasma proteins of the ERM reference materials, the accuracy of plasma protein determinations has improved /11/. ERM-DA472/IFCC has been certified for CRP. The successor reference preparation of ERM-DA470, referred to as ERM-DA470k/IFCC, was reproduced in the same quality and spiked with CRP and β2-microglobulin /12/.

References

1. Ritchie RF, Navolotskaia O, eds. Serum proteins in clinical medicine, Vol I. Scarborough: Foundation for Blood Research, 1996.

2. Dreyfuss G, Hentze M, Lamond AI. From transcript to protein. Cell 1996; 85: 963–72.

3. Hong W. Protein transport from the endoplasmatic reticulum to the Golgi apparatus. J Cell Science 1998; 111: 2831–9.

4. Lee YC. Biochemistry of carbohydrate-protein interaction. Faseb J 1992; 6: 3193–200.

5. Hounsell EF, Davies MJ. Role of protein glycosylation in immune regulation. Ann Rheumat Dis 1993; 52: S22-S29.

6. Davis A. Plasma protein synthesis and distribution. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine, Vol II. Scarborough: Foundation for Blood Research, 1999.

7. Ciechanover A, Gonen H, Elias S, Mayer A. Degradation of proteins by the ubiquitin-mediated proteolytic pathway. The New Biologist 1990; 2: 227–34.

8. Lord JM, Davey J, Frigerio R, Roberts LM. Endoplasmatic reticulum-associated protein degradation. Cell & Developmental Biology 2000; 11: 159–64.

9. Jaisson S, Gillery P. Evaluation of nonenzymatic posttranslational modification – derived products as biomarkers of molecular aging of proteins. Clin Chem 2010; 56: 1401–12.

10. Develter M, Blanckaert N, Komarek A, Bossuyt X. Can heparin plasma be used instead of serum for nephelometric analysis of serum proteins. Clin Chem 2006; 52: 1609–10.

11. Merlini G, Blirup-Jensen S, Johnson MA, Sheldon J, Zegers I. Standardizing plasma protein measurements worldwide: a challenging enterprise. Clin Chem Lab Med 2010; 48: 1567–75.

12. Zegers I, Keller T, Schreiber W, Sheldon J, Albertini R, Blirup-Jensen S, et al. Characterization of the new serum protein reference material ERM-DA470k/IFCC: value assignment by immunoassay. Clin Chem 2010; 56: 1880–8.

13. Ritzmann SE. Serum protein abnormalities. Boston: Little, Brown, 1984.

14. Shenkin A, Cederblad G, Isaksson EM. Laboratory assessment of protein-energy status. Clin Chim Acta 1996; 253: S5–S59.

15. Ellervik C, Tybjaerk-Hansen A, Nordestgaard BG. Total mortality by transferrin saturation levels: two general population studies and a metaanalysis. Clin Chem 2011; 57: 459–66.

18.2 Total protein (TP)

Lothar Thomas

The determination of TP is based on the following premises:

  • Each individual protein reacts the same way in the method of determination as any of many other proteins in the serum, plasma or body fluids
  • All proteins are pure polypeptide chains with approximately 16% of their mass consisting of nitrogen
  • The proteins are compared to bovine serum albumin since the latter is used as a calibrator in the method of determination.

18.2.1 Indication

Presence of the following symptoms, conditions or diseases: inflammation, proteinuria, edema, polyuria, chronic renal disease, chronic liver disease, chronic diarrhea, malignant tumor, increased susceptibility to infections, bone pain, rheumatic symptoms of undeterminable localization, lymphomas, external and internal hemorrhages, pregnancy, pre- and postoperative state, monoclonal gammopathy, shock, burns, patients requiring intensive medical care, investigation of an acute decrease in hemoglobin.

18.2.2 Method of determination

The Biuret method has proven useful for quantitative determination of serum TP. Determination of TP in urine, cerebrospinal fluid (CSF) and other body fluids is performed using various methods. The most reliable method is the Biuret reaction after acid precipitation of proteins. For CSF and urine, dye-binding methods, especially the Coomassie method, and light scattering techniques are employed.

Biuret method

Principle: the Biuret method is dependent on the presence of peptide bonds in the proteins. If a protein solution is treated with Cu(II) ions in a weakly alkaline solution, a colored chelate is formed between the Cu(II) ion and the carboxyl oxygen and amide nitrogen of the peptide bond. A requirement for this reaction to take place is the presence of at least two peptide bonds (tripeptide). Amino acids and dipeptides do not react. The intensity of the resulting violet color varies in a linear fashion with the number of peptide bonds and hence with the protein concentration over a broad range. The Biuret reagent contains copper sulfate, sodium potassium tartrate, potassium iodide and sodium hydroxide. The Cu(II) ions are kept in solution as a tartrate complex at alkaline pH while potassium iodide prevents auto reduction of Cu(II).

The method is not standardized, and the molarities of the Biuret reagent components have been modified in many ways, but a candidate reference method is described /1/. Bovine serum albumin is recommended for calibrating the TP determination. If determined manually, 1 part serum is added to 50 parts Biuret reagent, and after a 30 min. incubation at room temperature the absorption of the test sample and that of the standard are measured against Biuret reagent at 546 nm.

Coomassie method

Principle: the textile dye Coomassie Brillant Blue G 250 (CBB-G250) is in its leukoform in slightly acidic solution, and has an absorption maximum at 465 nm. CBB-G250 reacts rapidly with proteins forming a protein-dye complex which causes shift of the absorption maximum to 595 nm /2/. At low levels, the absorption is approximately linear with the protein concentration. This method is acceptable for TP determinations in urine and cerebrospinal fluid.

Turbidimetric method

Principle: in specimens that are low in protein such as urine and cerebrospinal fluid are denatured by addition of trichloroacetic acid. The denatured proteins scatter short-wavelength light. The measurable light scattering signal is proportional to the TP concentration over a certain concentration range. Light scattering methods are used for TP determination in cerebrospinal fluid /3/ and urine specimens /4/.

18.2.3 Specimen

Serum, plasma (heparin), urine, CSF, aspirated fluid: 1 mL

18.2.4 Reference interval

Refer to references /56/ and Tab. 18.2-1 – Reference intervals for total protein.

18.2.5 Clinical significance

This section is limited to the clinical interpretation of TP levels in serum and plasma. For an assessment of

Deviations of serum TP from the reference interval indicate the presence of dysproteinemia or are a sign of hypoproteinemia or hyperproteinemia based on disorders in the water and electrolyte balance.

From a differential diagnostic point of view, the conditions can be distinguished by additional performance of serum protein electrophoresis and determination of hematocrit.

Electrophoretically, dysproteinemia is associated with a quantitative shift in the pattern of the protein fractions, while hematocrit is unchanged.

Both dehydration and hyper hydration lead to uniform increases or reductions in serum protein concentrations, while no shift in protein bands occurs during serum protein electrophoresis. However, the hematocrit is abnormal.

18.2.5.1 Hypoproteinemia

Hypoproteinemia is mostly due to a reduction in albumin, and is less frequently due to a decrease in antibody synthesis (Tab. 18.2-2 – Diseases and conditions that may cause hypoproteinemia). Clinical symptoms of pronounced hypoproteinemia include the development of edema and effusions in body cavities. Hypoproteinemia may be due to:

  • Disturbance in protein synthesis
  • Protein malnourishment
  • Protein malabsorption
  • Protein-losing syndrome
  • Dilution hypoproteinemia.

18.2.5.2 Hyperproteinemia

Hyperproteinemia is rarer than hypoproteinemia because in the event of an increase in globulins, a regulatory reduction in albumin ensues. Therefore, in hyperglobulinemia, the total protein concentration remains within the reference interval for a long period of time. Hyperproteinemia above 80 g/L is found in about 3.5% of clinical patient samples.

Only pronounced, monoclonal gammopathies, severe chronic-inflammatory disorders and certain auto immunological processes (e.g., autoimmune hepatitis) cause hyperproteinemia (Tab. 18.2-3 – Diseases and conditions that may cause hyperproteinemia).

18.2.6 Comments and problems

Blood collection

The blood should be collected with the patient in a supine position since blood samples obtained in an upright position lead to the measurement of up to 10% higher TP concentration. The discrepancy is even higher in patients who tend to develop edema. After more than 3 min. of venous occlusion during blood collection, the protein concentration may increase by up to 10%. Blood sampling after vigorous muscle activity may lead to a rise in TP concentration by up to 12%.

Serum or plasma can be used for TP determination. Due to fibrinogen, average TP concentration in plasma is higher than in serum (i.e., by 2.5 g/L in blood donors, by 3.6 g/L in outpatients, by 4.6 g/L in in-patients and by 6.6 g/L in in-patients with a CRP > 50 mg/L) /8/. The patient does not need to be in a fasting state for the blood collection.

Method of determination

The Biuret method is used employing many variations of the Biuret reagent. These can be roughly divided into two groups /9/:

a) Low NaOH concentration (0.1–0.2 mol/L) and high CuSO4 concentration (10–30 mmol/L).

b) High NaOH concentration (0.5–0.8 mol/L) and low CuSO4 concentration (4–6 mmol/L). The Biuret reagents of these groups have a low reagent blank value, while the determination is only linear up to a protein concentration of 1.4 g/L as a rule.

The Biuret method reacts with some amino acids, dipeptides and other substances forming a 5-membered or 6-membered ring complex with Cu. These complexes have a higher absorption maximum (blue) than peptides and proteins (pink) /10/.

Interfering factors

Infusion solutions: protein-containing infusion solutions such as oxypolygelatin as well as polypeptides that are derived from degraded gelatin and cross-linked via urea bridges are also detected in the reaction to a varying extent, depending on the Biuret reagent used. Polyglucose such as dextran and sugar solutions such as glucose, mannitol, sorbitol and fructose lead to color intensification and simulate elevated protein levels; dextran, in addition, causes turbidity. Hydroxyethyl starch and synthetic materials such as polyvinylpyrrolidone do not enter into the reaction /9/.

Other interfering substances: ammonium salts (e.g., contained in enzyme preparations) may simulate falsely low levels as a result of protein precipitation. Tris(hydroxymethyl)-aminomethane leads to falsely elevated TP concentrations since it reacts with the Biuret reagent to yield a protein-like color reaction.

Hemolysis: 0.8 g of Hb/L resemble a 2% protein increase /11/. If bovine serum albumin is used as the standard, each mg of Hb mimics approximately 2 mg of protein since the globin enters into the Biuret reaction.

Lipemia: markedly lipemic serum samples simulate elevated TP concentrations by causing turbidity of the reagent mixture and must be cleared prior to determination.

Bilirubin: serum concentrations > 5 mg/dL (85 μmol/L) cause falsely increased TP levels in the Biuret reaction unless a sample blank (with the exception of copper sulfate) is measured.

X-ray contrast media: depending on their composition, X-ray contrast media can cause falsely elevated TP concentrations.

Coomassie method and light scattering technology: while the Biuret reaction has about the same percental absorption coefficient for all proteins, this does not apply to the Coomassie method /11/. Using the light scattering technology, globulins are determined too low in comparison to albumin /12/.

Stability

In a closed container at room temperature up to 1 week, at 4 °C up to 1 month, in a deep-frozen state > 1 year.

18.2.7 Pathophysiology

Plasma TP is composed of > 100 structurally known proteins; the biological function is known in detail for about 50 proteins. Albumin, α1-, α2- and β-globulins are synthesized by the parenchymal cells of the liver, while the proteins of the γ-globulin fraction (i.e., the immunoglobulins) are synthesized by plasma cells. The half-life of proteins ranges from a few hours (e.g., acute phase proteins) to as long as 3 weeks (e.g., IgG and albumin).

The liver has a significant functional reserve capacity for protein synthesis (3-fold for the synthesis of albumin, 6-fold for the synthesis of fibrinogen), and translation and transcription are usually not disturbed with the onset of liver disease /13/.

Glucagon and malnutrition (due to a lack in nutrients and/or poor dietary habits), especially a deficiency in the amino acid tryptophan, exert an inhibitory effect on the synthesis of proteins.

Glucocorticoids, growth hormone, insulin and thyroid hormones have a stimulatory effect on the hepatic synthesis of proteins.

Hepatic protein degradation takes place after endocytic uptake of proteins by the hepatocytes. This involves, for example, binding of glycoproteins to an asialoglycoprotein receptor on the hepatocyte membrane after removal of the N-acetylneuraminic acid (NANA) that is located in a terminal position at the carbohydrate side chains. The glycoproteins are subsequently internalized by pinocytosis.

Protein degradation involves two pathways:

  • In the lysosomes mediated by the action of peptidases and proteases under acid pH conditions
  • In the cytosol, by proteolytic enzymes under neutral pH conditions. The proteins to be degraded are bound to the protein ubiquitin that is present in cytosol, and thus targeted proteins are broken down by proteases first into peptides and ultimately into amino acids /14/.

Decreases in hepatic protein degradation can result from:

  • A reduced number of asialoglycoprotein receptors on the hepatocyte. This condition has been described, for example, in liver cirrhosis. As a result of this, plasma concentration of asialylated glycoproteins is elevated.
  • Increased sialylation (covalent binding of NANA) of proteins and, thus, a delay in desialylation and a reduced uptake by the hepatocytes. Increased sialylation has been described for GGT in acute alcoholic liver injury and for ALP in primary biliary cirrhosis.

The degradation of nonsialylated albumin is still largely unknown.

The physiological importance of plasma proteins lies in the maintenance of the colloid osmotic pressure as well as in their function as a vehicle for lipids, metabolic products, hormones and minerals; several proteins display enzymatic activity.

Many abnormal processes in the organism cause an alteration in the protein composition of the plasma (dysproteinemia) but often do not lead to protein concentrations outside the reference interval.

Plasma volume-induced changes of TP concentration, as seen after infusions or severe diarrhea, may be recognized by a synchronous pattern of hematocrit and TP concentration.

Absolute changes in total protein in plasma are due to either a decrease in albumin or an increase or decrease in immunoglobulins. Proteins that physiologically migrate in α1-, α2- and β-globulin fraction during electrophoresis are rarely subject to changes that result in marked hypoproteinemia or hyperproteinemia. An absolute increase in albumin does not occur.

References

1. Doumas BT, Bayse DD, Carter RJ, Peters Jr T, Schaffer R. A candidate reference method for determination of total protein in serum. I. Development and validation. II. Test for transferability. Clin Chem 1981; 27: 1642–54.

2. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein dye-binding. Anal Biochem 1976; 72: 248–54.

3. Reiber H. Eine schnelle, einfache nephelometrische Bestimmungsmethode für Protein im Liquor cerebrospinalis. J Clin Chem Clin Biochem 1980; 18: 123–7.

4. Kirchherr H, Schiwara HW. Quantitative Proteinbestimmung im Harn mit einer empfindlichen Streulichtmethode. J Clin Chem Clin Biochem 1985; 23: 57–62.

5. Reed AH, Cannon DC, Winkelman JW, Bhasin YP, Henry RJ, Pileggi VJ. Estimation of normal ranges from a controlled sample. I. Sex-and age-related influence on the SMA 12/80 screening group of tests. Clin Chem 1972; 18: 57–61.

6. Soldin SJ, Hicks JM. Pediatric reference ranges. Washington: AACC-Press, 1995: 115.

7. Tyler RD, Cowell RL. Classification and diagnosis of anemia. Comp Haematol Int 1996; 6: 1–16.

8. Bakker AJ, Gorgels JPMC, Draaisma J,Jongendijk M, Altena L, Hamersma A, et al. Simple method for correcting total protein in plasma for actual protein content. Clin Chem 1992; 38: 2221–3.

9. Kleine TO. Interferenz von Infusionslösungen mit der Biuret-Reaktion in einem vollmechanisierten und einem manuellen System. Med Welt 1979; 30: 102–7.

10. Hortin GL, Meilinger B. Cross reactivity of amino acids and other compounds in the Biuret reaction: Interference with urinary peptide measurements. Clin Chem 2005; 51: 1411–9.

11. Sonntag O. Störung klinisch-chemischer Untersuchungen durch Hämolyse. J Clin Chem Clin Biochem 1986; 24: 127–39.

12. Nishi H, Kestner J, Elin RJ. Four methods for determining total protein compared by using purified protein fractions from human serum. Clin Chem 1985; 31: 95–8.

13. Gerok W. Protein- und Aminosäurestoffwechsel bei Leberkrankheiten. In: Seidel D, Lang H (eds). Funktion und Funktionsdiagnostik der Leber. Heidelberg: Springer, 1985: 65–97.

14. Ciecanover A, Gonen H, Elias S, Mayer A. Degradation of proteins by the ubiquitin-mediated proteolytic pathway. The New Biologist 1990; 2: 227–34.

18.3 Serum protein electrophoresis

Lothar Thomas

Serum protein electrophoresis (SPE) is performed to diagnose dysproteinemias. The separation of serum proteins takes place at alkaline pH as a function of the protein’s net charge, isoelectric point and molecular weight. For medical diagnostic purposes, proteins are separated:

  • Either on a cellulose acetate medium into the six classic factions transthyretin, albumin, α1-, α2-, β- and γ-globulins in healthy individuals
  • Or on agarose gel medium, where specimens from healthy individuals exhibit 8–11 fractions
  • Using capillary zone electrophoresis, where up to eight protein fractions can be separated in healthy individuals.

18.3.1 Indication

Diagnosis and disease monitoring in patients with

  • Monoclonal gammopathies
  • Acute and chronic inflammatory response
  • Protein-losing syndromes (kidney, gastrointestinal tract, skin, exudates and transudates).

Pathological results in basic laboratory investigations, for example:

  • Elevated erythrocyte sedimentation rate
  • Proteinuria
  • Increased or decreased serum total protein concentration.

18.3.2 Method of determination

Zone electrophoresis on cellulose acetate medium /12/

Principle: the fractionation of serum proteins on certain media, especially cellulose acetate is a function of the voltage, electroendosmosis, pH of the separation buffer and the pK value of the individual protein. The placement of the sample on the supporting medium relative to the cathode and anode will affect and determine the degree of separation and resolution of the individual proteins. A multi-sample applicator is used to apply the sample, which is most often placed near the cathode. The separation of the proteins takes place at constant voltage (200 to 250 V) with a separation time of approximately 20 min. in a separation buffer of pH 8.2–8.6. The serum proteins migrate anodically and separate into bands (zones) comprising the fractions albumin, α1-, α2-, β- and γ-globulins. The bands are stained with protein dyes (Ponceau Red S, Amido Black 10B); any non specifically adsorbed dye on the supporting medium is washed out in de colorizing baths. After staining, proteins are identifiable by a characteristic pattern of colored bands which appear on the electrophoretogram, and by the relative intensities of these bands. The quantitative evaluation of the electrophoretogram is performed by densitometric scanning after rendering the cellulose acetate support transparent.

Automatic print out of the densitometer provides following results:

  • A scan that indicates the optical density of individual bands (Fig. 18.3-1 – Zone electrophoresis on cellulose acetate sheet)
  • The percentages representing the proportions of the individual band relative to the optical density of the entire electrophoretogram
  • The protein concentration of the individual band in g/L relative to the total protein concentration in the sample.

Agarose gel electrophoresis /3/

Principle: serum protein separation using agarose as supporting medium is comparable with electrophoresis on cellulose acetate medium, except that a separation time of 30–60 min. is necessary. The higher electroendosmosis, provides a better separation of serum proteins (Fig. 18.3-2 – Separation of serum proteins on agarose gel electrophoresis).

Agarose gel electrophoresis is widely used as a basic method for the separation of proteins, which can be further supplemented by other techniques that demonstrate the proteins more sensitively (e.g., by immunochemical techniques such as immunoelectrophoresis or immunofixation electrophoresis or by enzymatic methods for the measurement of isoenzymes).

Capillary zone electrophoresis (CZE) /4/

Principle: the separation of proteins is carried out in liquid medium in a narrow-bore capillary (20–200 μm), to which a high-voltage potential is applied. In this system, the electroendosmotic separation effect on the individual proteins is greater than their electrophoretic mobility. The separation takes place in the direction of the cathode. There, the proteins are quantified through UV measurement of the peptide bonds. With the help of a data processing program, a graph comparable to that from zone electrophoresis is produced for the following 8 fractions: pre albumin, albumin, α1 acidic glycoprotein, α2-globulin, hemopexin, transferrin, complement, and γ-globulin.

18.3.3 Specimen

Serum: 1 mL

18.3.4 Reference interval

Refer to Tab. 18.3-1 – Reference intervals of serum protein electrophoresis.

18.3.5 Clinical significance

The use of serum protein electrophoresis represents an effective way to diagnose dysproteinemia.

18.3.5.1 Dysproteinemia

Dysproteinemias are quantitative or qualitative changes in the protein composition of serum and are closely related to numerous diseases. In the electrophoretogram, dysproteinemias are linked to a disease state and are mainly recognizable when albumin or protein groups are affected exhibiting an increase, or decrease of protein fractions or an extra band . Examples of such proteins or protein groups are albumin, acute phase proteins, the transthyretin-transferrin group and immunoglobulins.

Albumin

The proportion of albumin decreases in any condition associated with an absolute rise in globulins (α, β, γ); thus, total protein usually remains within the reference interval.

Acute phase proteins

Acute phase proteins migrate in the α1- and α2-globulin fraction and are elevated by 50–300% in acute inflammatory conditions but are decreased in acute hepatitis, chronic active liver disease and protein-losing syndrome.

Transthyretin-transferrin group

Pre albumin, also known as transthyretin, migrates in front of the albumin band; usually, 50–70% of the transthyretin are bound in a complex with the retinol-binding protein. Both proteins are decreased in nutritional protein deficiency or in general energy deficiency conditions due to fasting states or in patients under intensive care.

Transferrin migrates in the β-globulin fraction, is elevated in iron deficiency and decreased in a protein and energy deficiency state and in the presence of any acute and chronic inflammatory condition. This also applies to anemia in chronic disease.

Members of the transthyretin-transferrin protein group react to all cases of acute and chronic inflammation with a decrease and are termed as the negative acute phase proteins.

Immunoglobulins

These proteins have antibody function and represent the γ-globulin but in part also the β-globulin fraction. Increases in immunoglobulins are referred to as gammopathies.

Polyclonal gammopathies

In the electrophoretogram these gammopathies cause a broad-based γ-globulin band and are due to a disease that activates the humoral immune defense.

Monoclonal gammopathies

The monoclonals form a narrow-based M-spike within the globulin band. The M-spike is caused by the excessive production of an immunoglobulin or an immunoglobulin fragment by a single plasma cell clone. Clinically, this presents mainly as multiple myeloma or as Waldenström’s macroglobulinemia. The M-spike is localized in the γ– globulin band or β-globulin band, and is referred to as M-spike (M = monoclonal = myeloma = M-formation of the gradient with the albumin fraction in the electrophoretogram).

Oligoclonal gammopathies

Selective increases of one or several Ig classes or Ig subclasses but of both light chain types. The Ig class or Ig subclass has limited heterogeneity of antibodies. The γ-globulin zone displays one or mostly several bands (sawtooth pattern).

Isolated protein changes

With the exception of albumin, α1-antitrypsin and IgG, they are not detectable by cellulose acetate electrophoresis but better detectable by capillary zone electrophoresis.

Extragradient

There may be some bands that deviate from the usual electrophoretogram. These appear in SPE with a frequency of approximately 0.7%, and are either supernumerary, and thus situated atypically between the normal bands, or directly superimposed over another band. They become recognizable when their protein concentration exceeds 2 g/L. They result either from the extreme increased production of a plasma protein, or from a technical error while the SPE is being carried out.

18.3.5.2 Clinical association with the SPE

The SPE does not allow a diagnosis to be directly established; however, based on the presence of dysproteinemia and the pattern of the electrophoretogram (constellation type), the following interpretations are possible:

  • Allocation of certain diseases or groups of diseases to characteristic types of pattern
  • Evaluation of disease activity
  • Monitoring of disease course.

Because of the quantitative determination of individual plasma proteins, SPE has lost a lot of its value and is/was used:

Typical graphs of dysproteinemias are shown in Fig. 18.3-3 – Scans of serum electrophoretograms.

18.3.6 Comments and problems

Specimen

Serum should be used since fibrinogen in plasma leads to the formation of an extra gradient within the β-globulin region /8/.

Reference interval

SPE is not standardized. Cellulose acetate electrophoresis with Ponceau red staining and CZE have generally comparable reference intervals.

Capillary zone electrophoresis (CZE)

CZE has the following advantages over cellulose acetate electrophoresis and agarose gel electrophoresis:

  • Markedly lower imprecision /46/
  • CZE and immunonephelometric assays yield very similar results in the quantitative determination of albumin /4/.

α1-antitrypsin (AAT) deficiency: CZE should not be used for screening for AAT deficiency if the result is based on the lower reference interval value (Tab. 18.3-1 – Reference intervals of serum protein electrophoresis). In a study /9/, however, 86% of the ZZ phenotypes and 29% of the MZ phenotypes were detected at a cutoff ≤ 0.21 g/L (see also Section 18.5 – α1-antitrypsin (AAT)).

Hemolytic serum: causes a small peak in the anodic part of the γ-globulin band.

Monoclonal gammopathies: CZE is more sensitive in the detection of monoclonal gammopathies than cellulose acetate electrophoresis (sensitivity 74%) and agarose gel electrophoresis (sensitivity 86%). However, diagnostic sensitivity is only 95% compared to immunofixation electrophoresis /10/.

Problems with the detection of proteins by CZE /11/:

  • Low concentration of monoclonal IgA (total IgA below 3.2 g/L). The monoclonal IgA overlooked by CZE migrates in the β-globulin zone and is hidden under the C3 or transferrin band
  • Low concentration of monoclonal IgM (total IgM below 2.1 g/L)
  • Free light chains in serum
  • Monoclonal IgD
  • Monoclonal Ig with a high isoelectric point, that migrate in the cathodic part of the γ-globulin zone are readily detectable in agarose gel electrophoresis, but may be not detectable in CZE.

Specificity of SPE in monoclonal gammopathies

Monoclonal gammopathies are detected in serum protein electrophoresis by the presence of M-gradient or hypogammaglobulinemia. However, extra bands hiding monoclonal immunoprotein can infrequently occur. The laboratory must verify such electropherograms by extending the analysis order (reflex testing) and additionally perform immunofixation electrophoresis. In a study /12/, reflex testing had to be performed in 13.2% of 5992 SPEs. When doing this, numerous monoclonal gammopathies were detected that would have been missed had the assessment of the M-gradient been the sole criterion for monoclonal gammopathy (Tab. 18.3-4 – Reflex testing upon abnormalities in serum protein electrophoresis).

Interference of monoclonal antibody therapies with SPE

Therapeutically used monoclonal antibodies represent both chimeric human – mouse immunoglobulins such as rituximab (Rituxan), siltuximab, infliximab (Remicade), cetuximab (Erbitux) and humanized antibodies such as trastuzumab (Herceptin), vevacizumab (Avastin), adalimumab (Humira). Under therapy, these IgG Kappa monoclonal antibodies reach a concentration of approximately 100 mg/L and higher. They can be detected by immunofixation electrophoresis and CZE and, in SPE, migrate in the middle of the γ-globulin zone, whereas rituximab and trastuzumab migrate in the cathodic portion of the γ-globulin zone /13/.

The therapeutic monoclonal antibodies become undetectable in patients approximately 3 months after the cessation of therapy (5 half-lives).

Stability

Serum stored in a closed container at room temperature is stable for 1 day and at 4 °C for up to 1 week.

Quality assurance

In the laboratory, control sera are comparable to human sera for measurement controls of the day-to-day precision and the accuracy.

References

1. Kohne J. Zur Technik der Zelluloseazetat-Elektrophorese. Ärztl Lab 1964; 10: 269–78.

2. Thomas L (ed). Serumeiweiss-Elektrophorese. München: Urban und Schwarzenberg, 1981; 1–49.

3. Bienvenu J, Graziani MS, Arpin F, et al. Multicenter evaluation of the Paragon CZE 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing. Clin Chem 1998; 44: 599–605.

4. Jeppsson JO, Laurell CB, Franzen B. Agarose gel electrophoresis. Clin Chem 1979; 25: 629–38.

5. Grabner W, Bergner D, Wermuth G. Mikrozonenelektrophoresen auf Membranfolien. Ärztl Lab 1970; 16: 193–201.

6. Lichtinghagen R, Pietsch D, Brand K. Evaluation of an automated capillary electrophoresis system for serum protein electrophoresis with the determination of gender-specific reference values. Clin Lab 2010; 56: 119–26.

7. Wuhrmann F, Wunderly C (eds). Die Bluteiweisskörper des Menschen. Basel: Schwabe, 1957: 159–78.

8. Tate J, Caldwell G, Daly J, Gillis D, Jenkins M, Jovanovic S, et al. Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand. Ann Clin Biochem 2012; 49: 242–56.

9. Slev PR, Williams BG, Harville TO, Ashwood ER, Bornhorst JA. Efficacy of the detection of α1-antitrypsin Z deficiency variant by routine serum protein electrophoresis. Am J Clin Pathol 2008; 130: 568–72.

10. Bossuyt X, Bogaerts A, Schiettekate G, Blanckaert N. Detection and classification of paraproteins by capillary immunofixation/substraction. Clin Chem 1998; 44: 760–4.

11. Bossuyt X, Marien G. False-negative results in detection of monoclonal proteins by capillary zone electrophoresis. Clin Chem 2001; 47; 1477–9.

12. Katzmann JA, Stankowski-Drengler TJ, Kyle RA, Lockington KS, Snyder MR, Lust JA, et al. Specificity of serum and urine protein electrophoresis for the diagnosis of monoclonal gammopathies. Clin Chem 2010; 56: 1899–1900.

13. Mc Cudden CR, Voorhees PM, Hainsworth SA, Whinna HC, Chapman JF, Hammett-Stabler CA, et al. Interference of monoclonal antibody therapies with serum protein electrophoresis. Clin Chem 2010; 56: 1897–8.

18.4 Albumin

Lothar Thomas

Albumin is the most important binding and transport protein of the organism. Its physiological functions include:

  • Maintenance of colloidal osmotic pressure in the vessels
  • Binding and transport of metabolites, metal ions, bilirubin, free fatty acids, phospholipids, amino acids, hormones (steroid hormones, thyroid hormones) and drugs
  • To serve as an amino acid pool for the tissues through albumin hydrolysis
  • Major antioxidant in plasma
  • Binding and removal of substances produced during cell regeneration.

Determination of albumin is clinically relevant:

This following section is limited to a description of the significance of albumin in serum.

18.4.1 Indication

  • Protein loss (nephrotic syndrome, burns, exudative enteropathy)
  • Diagnostic investigation of edematous state
  • Prognosis in elderly, hospitalized patients as well as mortality in patients with poly trauma and patients under intensive care
  • Index of nutritional state in developing countries.

18.4.2 Method of determination

Immunonephelometric or immunoturbidimetric assays, bromocresol green or bromocresol purple method /1/.

18.4.3 Specimen

Serum, heparin anticoagulated blood: 1 mL

18.4.4 Reference interval

Refer to Ref. /2/ and Tab. 18.4-1 – Reference interval for albumin in serum.

18.4.5 Clinical significance

Only a decrease in the serum albumin concentration is of clinical significance. Hyper albuminemia based on an absolute increase in the albumin level does not occur.

18.4.5.1 Hypoalbuminemia

Hypoalbuminemia may be caused by /3/:

  • Reduced synthesis (e.g., as seen in liver dysfunction) or in protein deficient diets
  • Expansion of the extravascular compartments (e.g., as in capillary leakage, sepsis or shock)
  • Losses into “third space” (e.g., in the case of edema, ascites or pleural effusion)
  • Losses to the exterior (e.g., as observed in nephrotic syndrome, burns or exudative enteropathy)
  • Acute phase response (albumin synthesis is down regulated in favor of the acute phase proteins); albumin is a negative acute phase protein
  • Pregnancy because the plasma volume increases by 40%
  • Congenital defect of albumin synthesis.

The concentration of albumin in serum is also viewed as a global, rough indicator of the health and nutritional status of an individual. This especially applies to the elderly and to chronically ill individuals. This does not come as a surprise because albumin responds by a decrease in concentration in numerous clinical disorders (Tab. 18.4-2 – Diseases and conditions associated with hypoalbuminemia). In large epidemiological studies, albumin was associated with many health-related variables. Although the results of these studies may be partially inconsistent, they do indicate that sociodemographic, lifestyle-related and illness-related factors correlate with hypoalbuminemia /4/.

18.4.6 Comments and problems

In comparison to immunoturbidimetric and immunonephelometric assays, the bromocresol green method yields higher concentrations, but approximately 10% lower concentrations in lithium heparin plasma than in serum /15/.

If blood is not collected with the patient in a supine position or having been sitting for at least 15 minutes, a 5–10% increase in the albumin concentration should be anticipated because of hemoconcentration.

18.4.7 Pathophysiology

Albumin has a molecular weight of 66.3 kDa, is synthesized in the liver and is the only plasma protein of that size with no carbohydrate chain. Daily synthesis rate ranges from 150–250 mg/kg of body weight; this requires about 12–20% of the hepatic protein synthesis capacity /1617/.

The functional domains of albumin are shown in Fig. 18.4-1 – Albumin structure and functional domains. The N-terminal part of albumin is the binding site for divalent forms of transition metals such as iron, cobalt, nickel and copper. The N-terminus consists of an aspartate-alanine-histidine sequence and is unstable. In the presence of hypoxemia, for example in acute myocardial infarction, free radicals are produced and acidosis develops. Under these conditions, albumin is modified (ischemia-modified albumin, IMA) and the bound transition metals are released /18/. The concentration of IMA is determined based on its binding capacity for cobalt in the Albumin Cobalt Binding Test. IMA binds less cobalt than normal albumin does.

Besides binding metals, albumin transports fatty acids, acts as an antioxidant and has detoxifying capacity. These properties are due to the thiol residue of cysteine 34 (Fig. 18.4-1 – Albumin structure and functional domains). In patients with stroke, hepatic failure or spontaneous bacterial peritonitis, albumin therapy leads to improved clinical condition. It is assumed that this effect is due to the anti oxidative and detoxifying properties of albumin /7/.

Synthesis of albumin is decreased by:

  • Increase in the oncotic pressure in extracellular fluid of the liver
  • Reduced availability of amino acids
  • IL-6-induced stimulation of synthesis of acute phase proteins.

Thyroxine, glucocorticoids and anabolic steroids exert a stimulatory effect on the synthesis of albumin.

The exchangeable albumin pool ranges from 3.5–5.0 g per kg of body weight corresponding to 250–350 g in an individual weighing 70 kg. Approximately 35–40% of this quantity are located in the extravascular space with the largest portion of it in skin and muscles. The liver itself stores only approximately 0.3 g. Albumin that is synthesized by hepatocytes reaches the circulation via the hepatic vein. Approximately 10-fold the quantity of albumin synthesized daily travels from the intravascular to the interstitial space and returns again via lymph vessels.

In comparison to an upright position, plasma albumin concentration declines by about 15% after a supine position has been assumed for at least 30 min.

The half-life of albumin is 19 days. Approximately 0.1 g are lost daily via diffusion into the gastrointestinal tract and 15 mg through the kidneys. Catabolism of albumin, like that of other plasma proteins, occurs in many tissues, especially the capillary endothelial cells, and is the result of continuous pinocytosis. Catabolism is decreased in hypoalbuminemia, although fractional degradation is normal. The plasma albumin concentration mostly depends on defects in distribution and much less on defects in synthesis. If food is withheld, albumin concentration declines below the lower reference interval value after one week at the earliest. In nutritional protein deficiency, the extent of edema correlates only weakly with albumin concentration.

More pronounced albumin losses to the exterior (e.g., as seen in patients with nephrotic syndrome) lead to an increased synthetic rate. Since synthesis of albumin is linked to synthesis of cholinesterase, the activity of this enzyme is increased in serum in patients with albumin loss. Absolute albumin increases in serum do not occur. Elevated levels are almost always due to pseudo hyperalbuminemia (e.g., as in exsiccosis).

Many drugs bind to albumin. Hypoalbuminemia can, therefore, be associated with an increase in the free, pharmacologically active portion of a drug. Drugs that are strongly bound to albumin are, for example, phenytoin and valproic acid. In these patients, hypoalbuminemia can lead to an increased pharmacological effect despite constant dosage of the drug. The binding capacity of albumin for therapeutic drugs may also be altered. For instance, in the presence of renal insufficiency, albumin has a decreased binding capacity for phenytoin and salicylic acid.

Genetic structural variants of albumin are detected in serum protein electrophoresis or are incidental findings (e.g., during the determination of thyroid hormones). For instance, in familial dysalbuminemic hyperthyroidism, total T4 is elevated in the face of normal FT4. The underlying cause is an abnormal albumin with increased binding capacity for T4.

References

1. Hill PG. The measurement of albumin in serum and plasma. Ann Clin Biochem 1985; 22: 565–78.

2. Johnson AM, Guder WG. Albumin. In: Ritchie RF, Novolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996; 600-1–600-10.

3. Whicher J, Spence C. When is serum albumin worth measuring? Ann Clin Biochem 1987; 24: 572–80.

4. Reuben DB, Moore AA, Damesyn M, Keeler E, Harrison GG, Greendale GA. Correlates of hypoalbuminemia in community-dwelling older persons. Am J Clin Nutr 1997; 66: 38–45.

5. Whittacker PG, Lind T. The intravascular mass of albumin during human pregnancy: a serial study in normal and diabetic women. Br J Obstet Gynecol 1993; 100: 587–92.

6. Rothschild MA, Oratz M, Schreiber SS. Albumin synthesis. N Engl J Med 1972; 286: 816–21.

7. Jalan R, Schnurr K, Mookerjee RP, Sen S, Cheshire L, Hodges S, et al. Alterations in the functional capacity of albumin in patients with decompensated cirrhosis is associated with increased mortality. Hepatology 2009; 50: 555–64.

8. Dramaix M, Hennart P, Brasseur D, et al. Serum albumin concentration, arm circumference, and edema and subsequent risk of dying in children in central Africa. BMJ 1993; 307: 710–3.

9. Rady MY, Ryan T, Starr NY. Perioperative determinants of morbidity and mortality in elderly patients undergoing cardiac surgery. Crit Care Med 2001; 29: S163– S172.

10. Yuki RL, Bar-Or D, Harris L, Shapiro H, Winkler JV. Low albumin level in the emergency department: a potential independent predictor of delayed mortality in blunt trauma. J Emergency Med 2003; 25: 1–6.

11. Feldman JG, Gange SJ, Bacchetti P, Cohen M, Young M, Squires KE, et al. Serum albumin is a powerful predictor of survival among HIV-1-infected women. J Acquired Immune Deficiency Syndromes 2003; 33: 66–73.

12. Gamsjäger T, Brenner L, Sitzwohl C, Weinstabl C. Half-lives of albumin and cholinesterase in critically ill patients. Clin Chem Lab Med 2008; 40: 1140–2.

13. Campagnoli M, Hansson P, Dolcini L, Caridi G, Gagnino M, Candiano G, et al. Analbuminemia in a Swedish male is caused by the Kayseri mutation (c228–229delAT). Clin Chim Acta 2008; 396: 89–92.

14. Galliano M, Campagnoli M, Rossi A, Wirsing von König CH, Lyon AW, Cefle K, et al. Molecular diagnosis of analbuminemia: A novel mutation identified in two American and two Turkish Families. Clin Chem 2002; 48: 844–9.

15. Meng QH, Krahn J. Lithium heparinised blood-collection tubes give fasely low albumin results with an automated bromcresol green method in haemodialysis patients. Clin Chem Lab Med 2008; 46: 396–400.

16. Peters jr T. Serum albumin. In: Putman FW (ed). The plasma proteins. Structure, function, and genetic control, Vol I 2nd ed. New York: Academic Press, 1975: 133–81.

17. Johnson AM, Guder WG. Albumin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine, Vol I. Scarborough: Foundation for Blood Research, 1996: 600-1–600-12.

18. Collison PO, Gaze DC. Ischaemia-modified albumin: clinical utility and pitfalls in measurement. J Clin Pathol 2008; 61: 1025–8.

18.5 α1-antitrypsin (AAT)

Lothar Thomas

AAT belongs to the family of serine protease inhibitors, also referred to as serpins. These inhibitors form irreversible complexes with, and thus inactivate, serine proteases such as elastase, chymotrypsin, trypsin and thrombin. AAT is, therefore, also referred to as α1-proteinase inhibitor (α1-Pi). It is encoded by the gene SERPINA1. More than 100 genetic variants of AAT are known. AAT deficiency is usually diagnosed following the diagnosis of chronic obstructive pulmonary disease (COPD), liver disease or, in some countries, within the scope of newborn screening if familial AAT deficiency is known to exist.

Most individuals with AAT deficiency inherit two copies of the Pi*Z allele. Heterozygous individuals inheriting one of the Pi*null alleles, which encodes the absence of AAT, and a Pi*Z allele, cannot be differentiated by phenotyping from those homozygous for the Pi*Z allele. Patients of the Pi ZZ and Pi Znull genotypes are, therefore, grouped under the protein Z phenotype /1/.

Contrary to AAT deficiency, elevated AAT has no clinical relevance. However, causes of elevated AAT must be taken into consideration when assessing AAT deficiency.

18.5.1 Indication

The American Thorax Society/European Respiratory Society (ATS/ERS) /2/ recommend the examination for AAT deficiency in:

  • Patients with COPD, asthma, unexplainable liver disease and necrotizing panniculitis
  • Individuals with persistent air flow restriction
  • Siblings of individuals with known AAT deficiency.

18.5.2 Method of determination

Three strategies are followed for diagnosing AAT deficiency:

  • Determination of serum AAT concentration. If the concentration is below a certain cutoff value, the phenotype of the AAT deficiency is determined.
  • Phenotyping of AAT for determining the isoform pattern. A disadvantage of this approach is that Pi*null alleles cannot be identified because no proteins are synthesized in this variant.
  • Genotyping of SERPINA1 [Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1].

Instead of classifying the AAT type, some laboratories immediately perform AAT genotyping if the AAT concentration is lower than a specific cutoff value.

AAT concentration

Serum protein electrophoresis: among the proteins of the α1-globulin fraction, AAT is the protein that is predominantly stainable by protein dyes. In homozygous AAT deficiency, the α1-globulin fraction may be strongly diminished or absent. A normal α1-globulin band does not rule out AAT deficiency. See also Section 18.3.6 – Comments and problems.

Radial immunodiffusion, immunonephelometry, immunoturbidimetry: For principle, see Section 52.1.5 – Direct antigen or antibody assays.

Proteinase inhibitor (Pi) capacity

Principle: depending on the AAT concentration in the patient’s sample, addition of patient serum to a trypsin-catalyzed reaction inhibits the activity of a defined quantity of trypsin that was previously added /4/. In the test reaction, residual active trypsin releases p-nitroaniline from the added substrate (benzyl-arginine-p-nitroanilide or tosyl-glycyl-lysine-4-nitro anilide acetate); the absorption increase of this reaction product is spectrophotometrically measured at 405 nm. The Pi capacity is primarily determined under therapeutic aspects.

Determination of the AAT phenotype

The AAT phenotype of a patient is determined using isoelectric focusing (IEF) in polyacrylamide gel with a pH gradient of 3.5–5.0. The variants (isoforms) of AAT present in band patterns based on their migration in a pH gradient. Z0 (null), ZZ and SZ, which are the essential of 100 Pi variants with pathogenic significance, can be easily differentiated /4/. The patterns show multiple bands of different mobility, reflecting different AAT glycosylation. Interpretation of the results is influenced by artifacts such as age of the sample and storage conditions.

Classification of the AAT genotype

Genomic DNA is extracted from EDTA whole blood, and PCR is performed followed by melting point analysis. Primers are used for genes containing the Z allele and the S allele /5/. Most commercial tests for molecular identification detect the common AAT variants Pi*S and Pi*Z. However, more than 30 variants also associated with AAT deficiency are not detected.

18.5.3 Specimen

  • AAT concentration (serum): 1 mL
  • Pi capacity (citrate plasma): 2 mL
  • DNA analysis (EDTA blood): 5 mL

18.5.4 Reference interval

AAT concentration /6/

0.9–1.8 g/L* (18–35 μmol/L)

α1-Pi capacity /3/

1.4–2.4 kIU/L

* Values are 5th and 95th percentiles. Conversion: mg/L × 19.6 = μmol/L

18.5.5 Clinical significance

AAT is primarily synthesized by the hepatocytes, but also by monocytes, alveolar macrophages and granulocytes. The serum concentration depends on the genotype.

18.5.5.1 Increase in AAT

During acute phase response, AAT concentration usually increases to a maximum of 3-fold. Levels > 5 g/L are measured in patients with squamous cell cancer and adenocarcinoma. With the exception of tuberculosis, levels of such magnitude do not occur in any other pulmonary disease /7/. A 1–2-fold increase in concentration can also occur during pregnancy and when taking oral contraceptives.

18.5.5.2 AAT deficiency

AAT alleles are autosomal co dominantly inherited. More than 100 genetic variants have been described. Pi*M with six subtypes M 1–M 6 is the normal allele and is present in more than 90% of the normal population. The normal subtypes are distinguished by different amino acids due to the substitution of individual bases in the DNA. The PiMM phenotype is associated with a normal concentration and inhibitor capacity of AAT.

Clinical symptoms of AAT deficiency involve the lungs, liver and skin. The following symptoms suggest the presence of AAT deficiency:

  • Unexplainable COPD
  • Viral hepatitis-marker-negative or non-alcohol-induced liver disease
  • Necrotizing panniculitis.

Fig. 18.5-1 – Algorithm for diagnosing AAT deficiency shows an algorithm for diagnosing AAT deficiency /13/.

The majority of patients with decreased AAT concentration and COPD or AAT-deficiency-induced liver disease are either homozygous for PiZ*, compound heterozygous for S and Z alleles (PiSZ) or have a null allele.

Approximately 3.4 million people worldwide (1 in 1500 to 1 in 10,000, depending on the population group) have severe AAT deficiency, and 116 million are carriers of a Pi*Z allele or Pi*S allele, with the highest prevalence found in Europe /8/.

The following distinction is made:

  • Individuals with severe AAT deficiency due to homozygosity (PiZZ) or compound heterozygosity (PiSZ) or null alleles. These individuals have an increased risk of COPD in their 3rd to 5th decade of life and of chronic liver disease at a later age.
  • Individuals with mild to moderate AAT deficiency have a low risk of COPD. This refers to genotypes that are heterozygous for AAT deficiency-related alleles such as PiMZ (odds ratio 2.31 compared to PiMM) or the Pi*S allele /9/. The latter is more common in many European populations than the Pi*Z allele. Characteristics of selected AAT variants are listed in Tab. 18.5-1 – AAT deficiency and related diseases.

In many patients, AAT deficiency is not detected and the mean interval between initial symptoms and diagnosis is 8 years. Patients are 46 years of age on average at the time of diagnosis and have already consulted a doctor at least three times because of their symptoms /8/. In all, only about 5% of AAT deficiency-related cases are detected since many patients are asymptomatic and only 1% of the patients with COPD have AAT deficiency /1/.

18.5.5.3 Assessment of AAT concentration

The determination of the AAT concentration within the scope of screenings is an important criterion for the detection of AAT deficiency. The lower reference interval value should not be used as a cutoff for the screening because the protein concentrations of PiMM types and deficiency-related types overlap. Severe AAT deficiency is generally ruled out in concentrations above 1.0 g/L /8/, but types with mild AAT deficiency, who also have a risk of COPD, may be missed. For instance, in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) /9/, 95% of the AAT variants, including all clinically relevant ones, were detected at an AAT concentration ≤ 1.13 g/L. Classification of the patients in subgroups based on the AAT concentration led to the genotype distribution presented in Tab. 18.5-2 – AAT concentration and SERPINA1 gene variants.

The prevalence of the ZZ phenotype is 1 in 1639 newborns according to a Swedish study /10/ and 1 in 5097 in a study /11/ performed in the USA. The decrease in AAT concentration in these individuals is below 35% of the mean of PiMM types, corresponding to below 0.70 g/L. This also applies to phenotypes SZ, Szero and Zerozero.

Approximately 5–7% of the population have a deficiency-related or silent allele (MZ, M0) that is linked to a mild to moderate decrease, corresponding to an AAT concentration < 1.0 g/L (Tab. 18.5-3 – Clinical manifestations of AAT deficiency).

Liver disease should not be ruled out in the differential diagnosis of neonatal cholestasis associated with normal AAT concentrations. This is due to rare heterozygous variants. Therefore, it is recommended in neonatal cholestasis and suspected AAT deficiency to perform electrophoretic AAT phenotyping or genotyping /12/.

In carriers of a deficiency allele, the reason for an AAT concentration that still falls within the reference interval may be the acute phase function of the protein in inflammation. Such cases can be discriminated by the simultaneously elevated C-reactive protein.

In AAT concentrations < 1.0 g/L, phenotyping is performed using IFE, whereas up to 22% of the carriers of deficiency-related alleles (Mheerlen, Q0amersfoort, Mwürzburg, Q0soest) are diagnosed by genotyping /14/.

Substitution therapy in patients with AAT deficiency is performed by using highly purified AAT. Treatment consists of a once-weekly administration of 60 mg/kg of body weight. This dosage is sufficient to maintain a mean serum AAT concentration of 0.3 g/L /15/. For further information refer to Ref. /26/.

18.5.6 Comments and problems

Blood collection

Anticoagulants such as buffered citrate, potassium oxalate, sodium fluoride or EDTA lead to falsely low AAT levels when determining both the concentration and the trypsin inhibitory capacity; heparin, on the other hand, does not interference in either method /21/.

Method of determination

The genotyping of individuals with AAT deficiency using commercial assays only detects those with Pi*Z and Pi*S alleles, but not those with null alleles or other rare variants.

Reference interval

When determining the AAT concentration cutoff value, below which further genotyping and phenotyping are to be performed, care must be taken to take the reagent manufacturer into account because there still are marked differences between the individual manufacturers. In the literature, the cutoff values range between ≤ 1.1 and ≤ 1.0 g/L /89/.

18.5.7 Pathophysiology

AAT is a glycoprotein with a molecular weight of 51 kDa and occurs in approximately equal concentrations in plasma and in interstitial fluid. Its synthetic sites, besides hepatocytes, include alveolar macrophages and monocytes. The daily synthetic rate is 34 mg/kg of body weight and the half-life is 6–7 days.

AAT belongs to the serpin family, including /22/:

  • Antithrombin which causes the inactivation of released proteases of the coagulation system, such as thrombin and FXa
  • The C1 esterase inhibitor which controls the activation of the complement system
  • The plasmin activator (PA) inhibitor (PAI). The PAI inhibits the PA, which converts plasminogen into plasmin and thus activates fibrinolysis.

The AAT, also known as α1-proteinase inhibitor, inhibits the serine proteases trypsin, chymotrypsin as well as pancreatic and especially polymorphonuclear-granulocytic elastase. Elastase cleaves connective tissue structures such as collagen and elastin. This step is necessary for the formation of pus and the liquefaction of an inflammatory site. However, in tissue surrounding the inflammatory site, AAT limits the elastase effect in order to prevent tissue damage from becoming too widespread.

The serpins differ from other proteinase inhibitor families by the complex manner of protease inhibition associated with a drastic change in structure (Fig. 18.5-2 – Serine protease inactivation by α1-antitrypsin (AAT)). For instance, mutations in the gene SERPINA1 lead to a change in protein conformation so that the protein can still be synthesized but no longer secreted in the endoplasmic reticulum. This results in a conformation disorder including the precipitation of protein aggregates in the hepatocyte and hepatocellular degeneration as well as AAT deficiency /22/.

AAT exerts its effect mainly on epithelial and serous surfaces. Development of pulmonary emphysema in patients with AAT deficiency is thought to be caused by an imbalance between elastase and AAT. Imbalance is due to an activation of granulocytes by the platelet-activating factor, which is released by macrophages and granulocytes; as a mediator of inflammation, this platelet-activating factor stimulates granulocytes to release peroxidases and elastase.

A significant part of the mutants in the gene SERPINA1 has no influence on protein expression and function. However, several alleles encode an AAT that either exists in the circulation at decreased concentration or is dysfunctional.

Based on their pathomechanism, disease-associated alleles are classified in /13/:

  • Deficiency alleles such as Pi*Z. The Glu342Lys mutation changes the protein structure of the AAT protein. This results in an aggregation and polymerization of the molecule in the endoplasmic reticulum of the hepatocytes including the precipitation and formation of hepatocellular inclusion bodies.
  • PiS-protein allele. The protein of this Glu264Val mutation polymerizes but is secreted and relatively quickly eliminated from the blood; as a result, serum concentration is only 60% of the normal value.
  • Null alleles; they either produce no transcript or garbled or unstable AAT causing degradation already prior to secretion from the cell /23/.
  • Dysfunctional alleles encode an AAT that does not bind to elastase but to other proteins such as antithrombin.

Refer also to Tab. 18.5-4 – Characteristics of selected α1-antitrypsin variants.

Individuals with the PiSS phenotype always produce sufficient AAT so that severe AAT deficiency does not manifest and clinical symptoms manifest but rarely.

Carriers of a Pi*Z allele are characterized by decreased AAT concentration and decreased inhibitor capacity regarding polymorphonuclear-granulocytic elastase. This results in unrestricted elastase activity and development of COPD. The Z variant of AAT has an unstable conformation leading to the polymerization of several AAT molecules and the formation of hepatocellular inclusion bodies.

Hence, liver disease in carriers of Pi*Z alleles is not based on AAT deficiency but on abnormal AAT polymerization. The β chain of the AAT molecule in the rough endoplasmic reticulum has an altered conformation causing the entire molecule to become unstable. As a result, the peptide chain of the second AAT molecule is able to incorporate into the structure of the first molecule causing the formation of a dimer (Fig. 18.5-3 – Polymerization of two AAT molecules). The entire process can multiply, leading to the formation of polymers that can no longer be released from the endoplasmic reticulum and precipitate as a result. The resulting inclusion bodies are subject to degradation. The development of hepatocellular necrosis is thought to depend on the balance between AAT aggregation and inclusion body degradation. The fact that aggregate formation increases with increasing temperature is an explanation why newborns with the ZZ phenotype with fever more often develop liver disease than those without fever.

References

1. Silverman EK, Sandhaus RA. Alpha1-antitrypsin deficiency. N Engl J Med 2009; 360: 2749–57.

2. American Thoracic Society/European Respiratory Society Statement. Standards for the diagnosis and management of alpha1-antitrypsin deficiency. Am J Respir Crit Care Med 2003; 168: 818–900.

3. Witt J, Tritscher W. α1-antitrypsin: Referenzwerte im Serum und Plasma mit Benzoyl-D, L-arginin-p-nitroanilid, einem chromogenen Substrat. J Clin Chem Clin Biochem 1982; 20: 587–91.

4. Jeppsson JO, Franzen B. Typing of genetic variants of alpha 1-antitrypsin by electrofocusing. Clin Chem 1982; 28: 219–25.

5. Braun A, Meyer P, Cleve H, Roscher AA. Rapid and simple diagnosis of the two common α1-proteinase inhibitor deficiency alleles PiZ and PiS by DNA analysis. Eur J Clin Chem Clin Biochem 1996; 34: 761–4.

6. Luisetti M, Seersholm N. α1-Antitrypsin deficiency. 1. Epidemiology of α1-antitypsin deficiency. Thorax 2004; 59: 164–9.

7. Leonhardt L. α1-Antitrypsin: Ein Serumprotein als Marker beim Bronchialkarzinom. Diagnose und Labor 1989; 78–81.

8. Kalsheker NA. α1-antitrypsin deficiency: best clinical practice. J Clin Pathol 2009; 62: 865–9.

9. Zorzetto M, Russi E, Senn O, Imboden M, Ferrarotti I, Tinelli C, et al. SERPINA1 gene variants in individuals from the general population with reduced α1-antitrypsin concentrations. Clin Chem 2008; 54: 1331–8.

10. Sveger T. Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants. N Engl J Med 1976; 294: 1316–21.

11. US Consensus Bureau. 2000 Consensus of population and Housing; summary population and housing characteristics, PHC 1-1, Washington, DC: US Consensus Bureau, 2002.

12. Lang T, Mühlbauer M, Strobelt M, Weidinger S, Hadorn HB. Alpha-1 antitrypsin deficiency in children: liver disease is not reflected by low serum levels of alpha-1 antitrypsin – a study on 48 pediatric patients. Eur J Med Res 2005; 10: 509–14.

13. Snyder MR, Katzmann JR, Butz ML, Yang P, Dawson DB, Halling KC, et al. Diagnosis of α1-antitrypsin deficiency: an algorithm of quantification, genotyping, and phenotyping. Clin Chem 2006; 52: 2236–42.

14. Prins J, van der Meijden BB, Kraaijenhagen RJ, Wielders JPM. Inherited chronic obstructive pulmonary disease: new selective-sequencing workup for α1-antitrypsin deficiency identifies 2 previously unidentified null alleles. Clin Chem 2008; 54: 101–7.

15. McElvaney NG, Crystal RG. Therapy of α1-AT deficiency. In: Crystal RG (ed). Alpha 1-antitrypsin deficiency. New York: Marcel Dekker, 1996: 319–31.

16. Agusti A, Hogg JC. Update in the pathogenesis of chronic obstructive pulmonary disease. N Engl J Med 2019; 381: 1248–56.

17. Tanash HA, Nilsson PM, Nilsson JA, et al. Clinical course and prognosis of never smokers with severe alpha1-antitrypsin deficiency. Thorax 2008; 63: 1091–5.

18. Larsson C. Natural history and life expectancy in severe alpha1-antitrypsin deficiency PiZ. Acta Med Scand 1978; 204: 345–51.

19. McBean J, Sable A, Maude J, Robinson-Bostom L. α1-antitrypsin deficiency panniculitis. Cutis 2003; 71: 205–9.

20. Bernspang E, Carlson J, Piitulainen E. The liver in 30-year-old individuals with alpha1-antitrypsin deficiency. Scand J Gastroenterol 2009; 44: 1349–55.

21. Berninger RW, Teixeira F. α1-antitrypsin: the effect of anticoagulants on the trypsin inhibitor capacity, concentration and phenotype. J Clin Chem Clin Biochem 1985; 23: 277–81.

22. Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency – a model for conformational diseases. N Engl J Med 2002; 346: 45–53.

23. Lee JH, Brantly M. Molecular mechanisms of alpha1-antitrypsin null alleles. Respiratory Medicine 2000; 94 Suppl C: S7–S11.

24. Lomas DA. Loop-sheet polymerization: the mechanism of alpha1-antitrypsin deficiency. Respiratory Medicine 2000; 94, suppl C: S3–S6.

25. Stoller JK, Aboussouan L. Alpha1-antitrypsin deficiency. Lancet 2005; 365: 2225–36.

26. Chapman KR, Chrostowska-Wynimko J, Koczulla AR, Ferrarotti I, McElvaney NG. Alpha I antitrypsin to treat lung disease in alpha I antitrypsin deficiency: recent developments and clinical implications. Int J of COPD 2018; 13: 419–32.

18.6 Alcohol biomarkers

Lothar Thomas

Laboratory tests to detect light drinking and alcohol abuse offer the opportunity to objectively verify the information about alcohol consumption provided by a person or patient. A differentiation is made between /1/:

  • Direct biomarkers; these markers are created when ethanol is metabolized or reacts with substances in the body
  • Indirect biomarkers, which undergo typical changes in response to acute or chronic alcohol consumption e.g., increase in gamma glutamyl transferase or increase of mean cell volume (MCV) of red cells.

An overview is provided of the alcohol markers ethanol, ethyl glucuronide, carbohydrate deficient transferrin, and some indirect biomarkers.

18.6.1 Ethanol

Alcohol refers to ethanol which may carry synonyms of ethyl alcohol. The molecular weight of ethyl alcohol (chemical formula: CH3CH2OH) is 46 Da and the density is 0.79. If another alcohol is consumed, it must be specifically identified and the medical effect differently assessed /2/.

Note the following Tables and Figures:

18.6.2 Ethyl glucuronide (EtG)

Anaerobic metabolim of ethanol generates direct markers of alcohol like ethyl glucuronide (EtG). This compound is a product of the conjugation reaction catalyzed by UDP-glucuronosyltransferase (Fig. 18.6-2 – Structure of ethyl glucuronide). EtG is nonvolantile, water-soluble, stable, and detectable long after complete elimination of alcohol. EtG is concentrated by almost 200 times in urine as compared to serum; the window of detection in urine is up to 90 h. This makes EtG a preferred marker for the detection of alcohol consumption in medical and forensic diagnostics /3/.

18.6.3 Carbohydrate-deficient transferrin

The structure of transferrin (Tf), the Fe3+-transporting protein, usually consists of two branched carbohydrate chains with a sialic acid residue at the end of each of the four branches. Tetrasialo transferrin, for example, is intact Tf. Long-term continuous consumption of more than 60–80 g of pure alcohol per day causes defective Tf glycosilation. This results in an increase in Tf isoforms lacking one or two N glycans (e.g., disialo Tf, monosialo Tf and asialo Tf) (Fig. 18.6-3 – Structure of transferrin and disialotransferrin). These isoforms are correlated with chronic alcohol abuse and are collectively referred to as carbohydrate-deficient transferrin (CDT). The quantitative determination of CDT is used for diagnosing chronic alcohol abuse. CDT is currently the best indirect biomarker in this context because it yields the lowest rate of false-positive results compared to other laboratory assays /4/.

18.6.4 Indication

Based on the window between the last drink and the collection of the sample different biomarkers can show a positive result /25/:

  • Breath test: up to 10–12 h after the last drink
  • Alcohol in blood or urine: up to 10–12 h after the last drink
  • Ethyl glucuronide in blood: up to 20 h after the last drink
  • Ethyl glucuronide in urine: up to 24 h after small quantities of alcohol, up to 130 h after excessive consumption
  • Carbohydrate deficient transferrin: chronic excessive drinking.

18.6.5 Method of determination

Alcohol in blood, serum, and plasma

Principle of alcohol dehydogenase (ADH) method: in the forward direction ethanol is oxidized in the presence of NADP to aldehyde and NAPH2. The reaction is catalyzed by the enzyme ADH (EC 1.1.1.2).

Ethyl glucuronide in urine

High performance liquid chromatography coupled to tandem mass spectrometry with deuterated internal standards /6/.

Immunoassay: Refer to Ref. /7/.

CDT immunoassay in serum/plasma

Principle: the CDT immunoassay is based on the application of a monoclonal antibody that is specifically directed against CDT isoforms lacking one or several N-glycans /8/. The Tf concentration is determined simultaneously in a second immunoassay. Polystyrene particles coated with a monoclonal anti-CDT antibody are agglutinated by CDT-coated polystyrene particles. The CDT in the sample inhibits the reaction between the antibody coated and CTD-coated particles in a dose-dependent manner. The extent of agglutination depends on the CDT concentration in the sample. The increase in light scattering measured by nephelometry is recorded and %CDT is calculated based on simultaneously determined Tf.

18.6.6 Specimen

Serum: 1 mL

Urine: 5 mL

18.6.7 Reference interval

Threshold to differentiate between patients with and without alcohol dependence:

  • Ethanol in serum/plasma: 5 mg/L. Threshold for recent alcohol intake; end of drinking not longer than 1 day ago /2/.
  • Ethyl glucuronide in urine: 100–200 μg/L. Any drinking the night before should be detectable the following morning with these cutoffs /9/
  • CDT in serum/plasma: relative threshold (immunoassay): ≤ 2.5% /8/.

18.6.8 Clinical significance

Alcohol biomarkers are ordered to objectively evaluate alcohol consumption, excessive alcohol abuse, and to monitor alcohol abstinence. The selection of the biomarker is dependent on the amount of consumed alcohol and the width of the time window between the last alcohol consumption and collection of the sample to be analyzed.

The strategies for the evaluation of alcohol consumption are classified into direct and indirect methods /10/.

  • Direct methods encourage self-reporting of patients (Alcohol Use Disorder Identification Test or AUDIT).
  • Indirect methods comprise clinical tests, indirect questionnaires and laboratory investigations.

Laboratory investigations can raise the suspicion of alcohol abuse but must always be considered in combination with medical history, clinical examination and direct evaluation /11/.

18.6.8.1 Definitions

Alcohol consumption is defined according to the Dietary Guidelines for Americans 2015–2020 of the US Department of Health and Human Services and US Department of Agriculture:

  • Moderate drinking is up to 1 standard drink per day for women and up to 2 drinks per day for men
  • A heavy drinking day is defined as over 3 standard drinks for women and over 4 standard drinks for men
  • Binge drinking is a pattern of drinking that brings blood alcohol concentration levels to 0.08 g/dL (17.4 mmol/L). This typically occurs after 4 standard drinks for women and 5 drinks for men in about 2 hours.
  • Heavy alcohol use is binge drinking on 5 or more days in the past month.
  • Low risk drinking for developing alcohol use disorders is no more than 3 drinks on any single day and no more than 7 drinks per week (for women) and no more than 4 drinks on any single day and no more than 14 drinks per week (for men).
  • Alcohol use disorder is a chronic relapsing brain disease characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences.

For definition of standard drink refer to Tab. 18.6-2 – What is a standard drink?

18.6.8.2 Ethanol: marker of acute alcohol consumption

Measures of blood alcohol such as breath test, alcohol determination in blood or urine can only detect alcohol use during the preceding 12 hours, making them suitable for detection current intoxication only /12/.

18.6.8.3 Ethyl glucuronide: marker of short term alcohol consumption

Ethyl glucuronide (EtG) can be detected in blood within 45 min. after alcohol consumption and the time window in serum is by up to 8 h longer compared to ethanol. In urine EtG can be proven for up to about 24 h even after consumption of small quantities. After excessive consumption the window of detection is up to 130 hours /13/.

Results of a dose ranging alcohol challenge study have shown the following results /9/:

  • Any drinking the night before are detectable the following morning with EtG cutoffs of 100 or 200 μg/L. Twenty-four hours after drinking, sensitivity is poor for light drinking, but good for heavier consumption.
  • At 48 hours, sensitivity is low following 6 drinks or less.
  • Increasing the cutoff to 500 μg/L leads to substantially reduced sensitivity.

The results of a study using ethyl glucuronide in urine to detect light and heavy drinkers presented the following results /14/:

  • The 100 μg/L cutoff detected > 76% of light drinking for two days, and 66% at 5 days
  • The 100 μg/L cutoff detected 84% (1day) to 79% (5 days) of heavy drinking
  • The 200 μg/L cutoff detected > 55% of light drinking and > 66% of heavy drinking across 5 days
  • The 500 μg/L cutoff identified 68% of light drinking and 78% of heavy drinking for 1 day with detection of light (2–5 days < 58%) and heavy drinking (5 days < 71%) decreasing thereafter.

EtG in urine is a suitable marker both for alcohol withdrawal or quit drinking programs and for abstinence checks prior to proposed liver transplantation or inclusion in the waiting list /1/.

18.6.8.4 Carbohydrate-deficient transferrin: marker of chronic excessive alcohol intake

Carbohydrate-deficient transferrin (CDT) is the most specific biomarker of chronic alcohol abuse. Increased amounts of CDT appear with high prevalence in serum if alcohol abuse of 50–80 g of alcohol per day are consumed on at least 7 consecutive days. The %CDT decreases to within the reference interval after abstinence with a half-life of approximately 14 days /15/. The %CDT varies only slightly in unchanged alcohol consumption. During alcohol withdrawal, the decrease in %CDT in relation to the initial value is conclusive in longitudinal assessment /16/.

The diagnostic sensitivity of %CDT for detection of chronic alcohol abuse is 30–50% for women and 50–70% for men /15/.

The diagnostic specificity of %CDT is > 90% and, thus, markedly higher than that of GGT. Hence, %CDT is currently the best biomarker for excluding chronic alcohol abuse /15/.

Gender dependence

It has been found that CDT concentration (U/L, mg/L) is gender-related, while %CDT is not /16/.

Pregnant women tend to have increased CDT concentrations. However, these increases are small and, therefore, irrelevant in the clinical assessment of %CDT /17/.

Advantages of %CDT determination

No correlations have been found between the serum concentrations of CDT and Tf /15/. The proportion of CDT in Tf (%CDT) is recommended as an assessment criterion for compensating changes in Tf concentration due to electrolyte/water imbalance or an increase/decrease in Tf concentration. %CDT has the following advantages over concentration data:

  • Fewer false-positive findings (increased diagnostic specificity) in individuals with normal alcohol consumption and elevated Tf concentration (e.g., in the presence of iron deficiency) /18/.
  • Fewer false-negative findings (increased diagnostic sensitivity) in individuals with chronic alcohol abuse and low Tf concentration due to acute or chronic infections, hemochromatosis and tumor diseases /15/.

GGT and %CDT

No correlations have been found between GGT activity and %CDT. The combined determination of CDT and GGT makes sense due to the high diagnostic sensitivity of GGT and the good diagnostic specificity of CDT.

%CDT and MCV

No correlation has been found between %CDT and MCV. The combined determination of %CDT and MCV does not make sense because both have good diagnostic specificity /15/.

18.6.9 Comments and problems

Blood collection

Venous blood collection without specific preparation of the patient; CDT is not influenced by food intake or time of day. Daily intraindividual CDT fluctuation is 8%.

Method of determination

In chromatographic and electrophoretic methods, the TF phenotype D leads to falsely high %CDT and the TF phenotype B leads to falsely low %CDT values. This is not the case in the homogeneous CDT immunoassay. The latter assay is also not interfered by the serum of patients with hepatic disease.

Influence factors

CDT is largely unaffected by drugs. Disulfiram, an aldehyde dehydrogenase inhibitor used for the treatment of alcohol dependence, does not affect CDT. False-positive %CDT findings are listed in Tab. 18.6-6 – Diseases and conditions that may lead to false-positive CDT findings.

EtG can be detected in samples at low levels and can be positive after exposure to alcohol from non-beverage sources or incidental exposure, which lead to false positives. Some healthcare workers, who are exposed to alcohol containing hand washes repeatedly throughout the day, might be positive if tested shortly thereafter. Using a cutoff of 200 μg/l might reduce the risk of such false positives /9/.

Stability

CDT in serum is stable for 30 hours at room temperature, for up to 7 days at 4 °C, and for several months to years at –22 °C; CDT increases by 25% after 3 days at room temperature. CDT is not affected by repeated freezing and defrosting. When stored at 4 °C in airtight test tubes, EtG concentration remains relatively constant.

18.6.10 Pathophysiology

Alcohol

The biochemical effects of alcohol are represented in Fig. 18.6-4 – Biochemical effects of ethanol.

Alcohol content in drinks: some notes /19/

  • The amount of alcohol contained in drinks is expressed most commonly as percent by volume (% v). The (% v) multiplied with the density of ethanol gives the percentage of the drink in grams (Tab. 18.6-7, equation 1)
  • The total amount of alcohol consumed in grams is calculated according to Tab. 18.6-7, equation 2
  • The concentration of blood alcohol is calculated from the amount of alcohol consumed taking into account the reduction weight. Explanation: muscles and brain take up more alcohol in relation to bone and adipose tissue. The body weight must be corrected by the reduction factor 0.7 for calculating the relation of the amount of alcohol to the body weight (body weight × 0.7 = reduction weight). The reduction weight multiplied by the blood alcohol concentration (‰) expresses the amount of consumed alcohol (Tab. 18.6-7, equation 3).
  • Within a few hours following alcohol consumption, the blood alcohol level can be used to calculate the amount of alcohol consumed (Tab. 18.6-7, equation 4)
  • The serum alcohol concentration (mmol/L) is converted into blood alcohol concentration (‰) according to Tab. 18.6-7, equations 5.

Ethyl glucuronide (EtG)

None oxidative metabolism of ethanol generates compounds, which are called direct markers of alcohol. This group includes EtG, ethyl sulfate and phosphatidyl ethanol. EtG is formed by glucuronidation following exposure to ethanol. The molecular mass is 222 g/mol, the molecular formula is C8H14O7, and the elimination half-life is 2–3 hours. The concentration of serum EtG is a function of two opposing influences; dose of alcohol consumed and time window elapsed between consumption and sample collection. The concentration in urine is dependent on diuresis. The intake of alcohol contained in large volumes of water results in a steep decline of EtG level in urine. Therefore a minimum requirement on the urine creatinine concentration is > 20 mg/dL (1.77 mmol/L).

Carbohydrate-deficient transferrin (CDT)

Tf is a glycoprotein consisting of a single polypeptide chain containing 679 amino acid residues and carrying two N-linked carbohydrate chains at position 413 and 611 (Fig. 18.6-3 – Structure of transferrin (top) and disialotransferrin).

There are three known causes of Tf heterogeneity:

  • Variation of the amino acid structure of the polypeptide chain due to genetic polymorphism. Using starch gel electrophoresis, the wild type TfC can be differentiated from the more quickly migrating type TfB and the slower cathodic type TfD. All genetic variants have normal iron binding capacity.
  • The iron content. Among the four isoforms, diferric Tf binds iron in the N-terminal and C-terminal domains, monoferric Tf binds iron in the N-terminal and C-terminal domains and apo-Tf has no bound iron ions. The N-terminal domain has dominant iron binding capacity.
  • The carbohydrate chains. Each of the two oligosaccharide chains in the N-terminal domain structurally varies in branching, with mostly bi- or tri-antennary structure. Each antenna is terminated by sialic acid.

The following CDT isoforms are distinguished: asialo Tf (< 0.5%), monosialo Tf (< 0.9%), disialo Tf (< 2.5%), trisialo Tf (4.5–9.0%), tetrasialo Tf (64–80%), pentasialo Tf (12–18%), hexasialo Tf (1.0–3.0%) and heptasialo Tf (< 1.5%). The isoelectric point of the isoforms depends on the number of sialic acid residues.

The alcohol-induced increase in CDT concentration is due to ethanol-induced and/or acetaldehyde-induced defective synthesis of the N-carbohydrate chains of Tf. For instance, lower activities of galactosyl transferase and N-acetyl glucosaminyl transferase are measured in the serum of alcoholics.

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15. Arndt T. Carbohydrate-deficient transferrin as a marker of chronic alcohol abuse: a critical review of preanalysis, analysis, and interpretation. Clin Chem 2001; 47: 13–27.

16. Borg S, Helander A, Voltaire Carlsson A, Högström Brandt AM. Detection of relapses in alcohol-dependent patients using carbohydrate-deficient transferrin: improvement with individualized reference levels during long-term monitoring. Alcohol Clin Exp Res 1995; 19: 961–3.

17. Stauber RE, Vollman H, Pesserl L, Jauk B, Lipp R, Halwachs G, Wilders-Trusching M. Carbohydrate-deficient transferrin in healthy women: relation to estrogens and iron status. Alcohol Clin Exp Res 1996; 20: 1114–7.

18. de Feo TM, Fargion S, Duca L, Mattioli M, Cappellini MD, Sampietro M, Cesana BM, Fiorelli G. Carbohydrate-deficient transferrin, a sensitive marker of chronic alcohol abuse, is highly influenced by body iron. Hepatology 1999; 29: 658–63.

19. Ohlenschläger G. Medizinisch-biochemische Probleme zum Äthanolabbau in vivo. GIT 1977; 21: 289–92.

20. Friedmann PD. Alcohol use in adults. N Engl J Med 2013; 368: 365–73.

21. Diehl A. Mann K. Früherkennung von Alkoholabhängigkeit. Dtsch Ärztebl 2005; 102: B1894–B1900.

22. Lieber CS. Medical disorders of alcoholism. N Engl J Med 1995; 333: 1058–65.

23. Seitz HK. Wieviel Alkohol macht krank? Dtsch Ärztebl 2000; 97: A1538–A41.

24. Mallach HJ, Pedal I, Völz T. Über tödliche Alkoholvergiftungen. Med Welt 1980; 31: 1657–66.

25. Mukamal KJ, Conigrave KM, Mittleman MA, Camargo CA, Stampfer MJ, Willett WC, Rimm EB. Roles of drinking pattern and type of alcohol consumed in coronary heart disease in man. N Engl J Med 2003; 348: 109–18.

26. Sharpe PC. Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 2001; 38: 652–64.

18.7 Ceruloplasmin: Wilson’s disease and other copper disorders

Lothar Thomas

Ceruloplasmin (Cp) is the copper (Cu)-carrying protein in the circulation. A Cp molecule binds 6 Cu atoms; 90% of the Cu in plasma are present in the form of Cp-Cu complexes functioning as the source of Cu supply to peripheral organs such as the brain and kidneys.

Cp is synthesized in the liver. The hepatocytes secrete Cp as copper (Cu)-carrying protein into the circulation.Cp is an acute phase protein and, in serum protein electrophoresis, migrates in the α2-globulin band.

Moreover, the functions of Cp include:

  • Ferroxidase activity (regulation of the oxidative status of iron and other metal ions); for instance, Cp oxidizes Fe2+ to Fe3+
  • Anti oxidative effect (influence of the redox state of plasma) due to the prevention of metal-ion-catalyzed oxidation of lipids in the cell membrane
  • Oxidation of nitrogen monoxide (NO.) and influence on NO. homeostasis (see Section 19.2 – Oxidative stress).

The serum Cp concentration is diagnostically relevant because a low level suggests the presence of Wilson’s disease and Menkes disease. For diagnosing Wilson’s disease, Cp should be assessed in association with the urinary Cu excretion. See Section 10.4 – Copper.

18.7.1 Indication

  • Exclusion of Wilson’s disease in all children with suspected autoimmune hepatitis or non-alcoholic fatty liver disease (NAFLD). Wilson’s disease is to be suspected in children above 5 years of age who have sonographically detectable fatty liver or any form of acute liver failure /12/.
  • Hepatitis-marker-negative liver disease in childhood or adolescence (suspected Wilson’s disease)
  • Patients with extra-pyramidal, cerebellar or cerebral symptoms of subacute or chronic nature. The most common initial symptoms are difficulties in speaking and swallowing (suspected Wilson’s disease)
  • Neurodegenerative symptoms and signs of connective tissue disease in infants and small children (suspected Menkes disease).

18.7.2 Method of determination

Immunoassay and immunonephelometric or immunoturbidimetric assays.

18.7.3 Specimen

Serum: 1 mL

18.7.4 Reference interval

Refer to References /34/ and Tab. 18.7-1 – Reference intervals for ceruloplasmin.

18.7.5 Clinical significance

It is only the decrease in serum Cp concentration that is clinically relevant. However, when assessing the Cp concentration, diseases and conditions must be taken into consideration that may lead to an increase in Cp and mask a decrease in concentration.

18.7.5.1 Increase in Cp concentration

Elevated Cp levels in serum up to 3-fold the upper reference interval value may occur as part of the acute phase response in inflammation, especially during bacterial infections. Serum protein electrophoresis shows an increase in the α2-globulin fraction.

Depending on the dose, intake of oral contraceptives or estrogens during menopause leads to a 20–30% increase in serum Cp concentration. Cp can be elevated as high as three-fold during pregnancy.

18.7.5.2 Decrease in Cp concentration

Diseases that may be associated with a decrease in the serum Cp concentration include Wilson’s disease, nutritional copper deficiency, Menkes disease and other neurological copper disorders /5/.

18.7.5.3 Wilson’s disease (WD)

WD (primary copper excess), an autosomal recessive condition, results in a Cu deposition in the hepatocytes, brain, iris and kidney. The excretion of Cu into the bile is delayed and the incorporation of Cu in Cp is impaired. Most patients present with predominantly hepatic or predominantly neurological Cu disorders. WD is a monogenic, autosomal recessively inherited condition. The causative gene ATP7B encodes a Cu-transporting P-type ATPase /6/. The latter can be associated with symptomatic or asymptomatic involvement of the liver.

More than 500 ATP7B mutations have been described:

  • Most are missense mutations, small deletions/insertions in the coding region, or splice junction mutations /7/
  • Among Caucasians in Europe and North America, the point mutation H1069Q is the most common ATP7B mutation and 50–80% of WD patients carry at least one H1069Q allele.

Mutations resulting in completely absent or non-functional ATP7B protein activity are associated with early onset, typically hepatic, severe WD; these mutations are comparatively rare /8/ .Refer also to Tab. 18.7-2 – Common ATP7B mutations.

Firm genotype-phenotype associations for other more common ATP7B mutations do not reveal a clear correlation. In a study /9/, mutations in both chromosomes were diagnosed in 57% of the patients with WD. Approximately 15% of the patients showed no mutation; this was attributed to undetected mutations in the promoter region and exons that had not been analyzed.

There is an association between the presence of the ATP7B H1069Q mutation, the incidence of neurological symptoms and age.

For instance, patients with WD homozygous and heterozygous for the H1069Q mutation or without the H1069Q mutation had /10/:

  • Neurological symptoms in 63%, 43% and 15% of the cases, respectively
  • A mean age of 20.9, 15.9 and 12.6 years.

In another study /9/, patients with WD presenting with neurological symptoms were 20.2 ± 10.8 years of age, and those with hepatic symptoms were 15.5 ± 9.6 years of age. Diagnosing of the disease took 44.4 months in the patients with neurological symptoms and 14.4 months in the patients with hepatic symptoms.

Prevalence of Wilson’s disease

Data of a study /11/ suggested an unexpectedly high rate (approximately 1 in 40) of ATP7B heterozygote mutation carriers, predicting a 1 : 7.000 prevalence for WD in the United Kingdom population.

Diagnosis of Wilson’s disease

Evidence of WD is based on the correct assignment of clinical symptoms of hepatitis, neurological symptoms of disease, and on laboratory investigations. In a retrospective study /9/, the prevalence of diagnostic parameters in patients with WD are shown in Tab. 18.7-3 – Prevalence of findings in Wilson’s disease patients. Patients with cholestatic liver disease were found at the time of diagnosis in 173 patients with WD at a mean age of 17 years with predominantly hepatic symptoms and at a mean age of 24 years with predominantly neurological symptoms.

The spectrum of laboratory investigations according to the recommendations of the American Association for the Study of Liver Diseases (AASLD) is presented in Tab. 18.7-4 – Diagnosis of Wilson’s disease according to the AASLD practice guidelines.

A diagnostic algorithm /1/ is shown in Fig. 18.7-1 – Diagnostic procedure for Wilson’s disease in patients with unexplainable liver disease.

A laboratory test by itself does not provide evidence of, or rules out, Wilson’s disease. In genetic diagnostics of mutations, total sequencing does not cover the entire gene. Moreover, the determination of the hepatic Cu concentration leaves doubt in the case of ambiguously elevated levels /2/.

Clinical manifestation of Wilson’s disease

The spectrum of the disease comprises liver and neurological diseases. All children diagnosed in early infancy with genetically confirmed WD present with hepatic symptoms. Neurological symptoms in WD typically begin in the second or third decade.

According to the AASLD recommendations, Wilson’s disease is to be suspected if /3/:

  • The clinical picture of autoimmune hepatitis is present in childhood
  • Adults have atypical autoimmune hepatitis or poorly respond to corticosteroid therapy
  • Patients have non-alcoholic fatty liver disease or findings indicate the presence of this disease
  • Acute liver failure with Coombs-negative hemolysis and low alkaline phosphatase are present.

A scoring system for WD diagnosis developed at the international WD meeting in Leipzig 2001 is presented in Tab. 18.7-5 – Scoring system for Wilson’s disease.

Wilson’s disease and other disorders associated with a decrease in Cp level are shown in:

18.7.6 Comments and problems

Method of determination

Cp is a labile protein and is easily fragmented in serum or during determination. Immunonephelometry and immunoturbidimetry are less affected by this problem than radial immunodiffusion /3/. Serum and urinary Cu concentrations should be determined using the atomic absorption method.

Stability

Storage at 4 °C if determination is performed within 3–4 days. Longer-term storage in a deep-frozen state, only /3/.

18.7.7 Pathophysiology

The populations of industrial countries have a daily dietary intake of 5 mg Cu via the upper gastrointestinal tract. Since many components of the diet are rich in Cu, there is practically no Cu deficiency. Cu is taken up by the liver via the portal circulation within 4 hours and 6–8% Cu bound to Cp appear in the plasma within 24 hours. See also Section 10.4 – Copper.

Cp is a glycoprotein with a molecular weight of 132 kDa and has a carbohydrate content of about 9%. The Cp molecule binds 6 Cu atoms.

Cp is synthesized in the hepatocyte as apoCp. The Cu atoms are incorporated post translationally, followed by the binding of the carbohydrate side chains.

Incorporation of Cu atoms into apoCp occurs intracellularly by P1-type ATPase (ATP7B). The Cu atoms in CP are mostly in the Cu(II) state. The entrance of Cu atoms via the CTR1 receptor into the cell is not the rate-limiting step for uptake of Cu from Cp. It seems highly likely that a reductase is needed to provide Cu(I) for uptake /13/.

In comparison to Cp with a half-life of 4 days, apoCp has an intra- and extracellular half-life of only a few hours.

The body’s Cu homeostasis is critical because the liver is the only organ to eliminate enterally absorbed Cu. This is achieved via excretion into the bile. Normally all of the excess Cu intake is later excreted so that the body’s Cu pool remains constant /7/. Cu is not subject to enterohepatic circulation because it is excreted into the bile as a non-absorbable complex.

Physiological functions of Cp include:

  • Regulation of transport, availability and redox potential of iron (Fe) as a result of its ferroxidase activity. For instance, if functional iron is required for erythropoiesis, Fe3+ is released from ferritin via reduction to Fe2+. However, since the transport protein transferrin can only bind Fe3+, Fe2+ is immediately oxidized to Fe3+ by Cp. Furthermore, all Fe2+ resorbed by the intestinal mucosa is oxidized to Fe3+ by hephaestin or Cp before binding to transferrin.
  • Prevention of metal-ion-catalyzed per oxidation of membrane lipids. This per oxidation is thought to be a causative cofactor of many disorders such as atherosclerosis or neurotoxicity. Cp reacts either directly with the super oxide ion radical (O2–.), or oxidizes Fe2+ or Cu2+ and thus prevents their catalytic effect for lipid per oxidation and the damage of cellular structures /3/.

Paradoxically, Cp changes from antioxidant to oxidant in the absence of divalent cations or under acid pH conditions. For instance, it is thought to play a significant role in LDL oxidation induced by monocytes/macrophages. Thus, under monocyte/macrophage stimulation, increasingly synthesized Cp may promote atherosclerosis.

The hepatocytes play an essential role in the body’s Cu metabolism. Following binding of the Cu to the intracellular Cu transport protein ATP7B, the hepatocyte controls Cu absorption via ATP7B by mediating:

  • Binding of Cu to Cp
  • Cu excretion into the bile after the Cp is saturated with Cu.

In Wilson’s disease, the cellular Cu balance is disturbed by a mutation-related dysfunction of the Cu transporter ATP7B. This results in intracellular accumulation of Cu. If all metallothionein in the cytoplasm and Cp are saturated, the free Cu has a toxic effect. This toxic effect leads to changes in the mitochondrial structure, defective DNA synthesis, modified protein synthesis and decrease in glutathione. All processes in combination may result in hepatocellular necrosis and Cu release. As these processes usually proceed slowly in most patients, the disease remains clinically inapparent in many cases.

In Wilson’s disease, the liver can produce apo-Cp, but Cu is not incorporated into the Cp molecule and accumulates primarily in the hepatocytes and secondarily in other tissues. In general, no clinical manifestations are observed prior to 5 years of age. They usually develop around the age of 15. The following stages are distinguishable during the course of Wilson’s disease /10/:

  • Cu accumulation; Cu accumulates diffusely in the cytosol of hepatocytes. Cu content of the liver is increased, while aminotransferases are usually normal or only slightly elevated. Elevated levels are only coincidentally detected in clinically asymptomatic patients.
  • Cu redistribution; if a critical threshold of Cu accumulation has been reached in the cytosol of the hepatocytes, Cu is redistributed into the lysosomes. During this process, Cu is also released into plasma. In most patients, redistribution occurs slowly and, hence, the patients remain clinically inapparent. In some of them, redistribution takes place rapidly and a lot of Cu is released into plasma. Chronic active hepatitis occurs and may progress to liver failure. Hepatitis or liver failure is caused by intravascular hemolysis due to release of large quantities of Cu into plasma as well as toxicity-related hepatocellular necrosis. From a differential diagnostic point of view, it is important to note that in liver failure due to Wilson’s disease ALT, in relation to bilirubin, is only minimally elevated, the AST/ALT ratio is > 2, and ALP tends to be reduced /12/.
  • Development of liver fibrosis and Cu accumulation in extrahepatic organs.

In patients, in whom Cu redistribution in the hepatocyte is accompanied by a clinically inapparent course, histological findings early on include morphological nuclear changes of periportal hepatocytes and steatosis similar to that seen in alcoholic fatty liver. Subsequently, liver fibrosis develops with progression to liver cirrhosis. Among the extrahepatic changes, neurological alterations predominate. The Kayser-Fleischer corneal ring is an ocular manifestation of the disease. Because of splenomegaly, some of the patients have leukopenia and thrombocytopenia.

References

1. Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. AASLD practice guidelines. Hepatology 2008; 47: 2089–2111.

2. Dockter G. Morbus Wilson – hepatische Manifestationen. Klin Pädiatr 2009; 221: 407–8.

3. Johnson AM. Ceruloplasmin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 13.01–1–8.

4. Dati F, Schumann G, Thomas L, Aguzzi F, Baudner H, Bienvenu O, et al. Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 plasma proteins in serum based on the standardization against IFCC/BCR/ CAP reference material (CRM 470). Eur J Clin Chem Clin Biochem 1996; 34: 517–20.

5. Bandmann O, Weiss KH, Kaler SG. Wilson’s disease and other neurological disorders. Lancet Neurol 2015; 14: 103–13.

6. Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat Genet 1993; 5: 327–37.

7. www.wilsondisease.med.ualberta.ca/database.asp

8. Merle U, Weiss KH, Eisenbach C, Tuma S, Ferenci P, Stremmel W. Truncating mutations in the Wilson disease gene ATP7B are associated with very low serun ceruloplasmin oxidase activity and an early onset of Wilson disease. BMC Gastroenterol 2010; 10: 8.

9. Merle U, Schaefer M, Ferenci P, Sremmel W. Clinical presentation, diagnosis and long-term outcome of Wilsons disease: a cohort study. Gut 2007; 56: 115–20.

10. Stapelbroek JM, Bollen CW, van Amstel JK, van Erpecum KJ, van Hattum J, van den Berg LH, et al. The H1069Q-Mutation in ATP7b is associated with late and neurologic presentation of Wilson’s disease: results of a metaanalysis. J Hepatol 2004; 41: 758–63.

11. Coffey AJ, Durkie M, Hague S, McLay K, Emmerson J, Lo C, et al. A genetic study of Wilson’s disease in the United Kingdom. Brain 2013; 136: (Pt 5): 1476–87.

12. Degenhardt S, Blomhard G, Hefter H, et al. Hämolytische Krise mit Leberversagen als Erstmanifestation des M. Wilson. Dtsch Med Wschr 1994; 119: L 1421–6.

13. Ramos D, Mar D, Ishida M, Vargas R, Gaite M, Montgemery A, Linder MC. Mechanism of coppper uptake from blood plasma ceruloplasmin by mammalian cells. Plos One 2016; https://doi.org/10.1371/journal.pone.0149516.

14. Tapper EP, Rahni DO, Arnaout R, Lai M. The overuse of serum ceruloplasmin measurement. Am J Med 2013; 126: 926.e1–926.e5.

15. da Costa CM, Baldwin D, Portmann B, Lolin Y, Mowat AP, Mierli-Vergani G. Value of urinary copper excretion after penicillamine challenge in the diagnosis of Wilson’s disease. Hepatology 1992; 15: 609–15.

16. Roberts EA, Cox DW. Wilson’s disease. Baillières Clin Gastroenerol 1998; 12: 237–56.

17. Stremmel W, Meyerrose KW, Niederau C, Hefter H, Kreuzpainter G, Strohmeyer G. Wilsons disease: clinical presentation, treatment and survival. Ann Int Med 1991; 115: 720–6.

18. Huster D, Weizenegger M, Kress S, Mössner J, Caca K. Rapid detection of mutations in Wilson disease gene ATP7B by DNA strip technology. Clin Chem Lab Med 2004; 42: 507–10.

19. Twomey PJ, Viljoen A, House IM, Reynolds TM, Wierzbicki AS. Copper: caeruloplasmin ratio. J Clin Pathol 2007; 60: 441–2.

20. Das SK, Ray K. Wilson’s disease: un update. Nature Clin Practice Neurology 2006; 2: 482–93.

21. Steindl P, Ferenci P, Dienes HP, Grimm G, Pabinger I Madl C, et al. Wilson’s disease in patients presenting with liver disease: a diagnostic challenge. Gastroenterology 1997; 113: 212–8.

18.8 Haptoglobin and hemopexin: hemolytic disease

Lothar Thomas

Extracellular hemoglobin (Hb) and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. The liver synthesizes the scavenger proteins haptoglobin (Hp) and hemopexin (Hx) which bind extracellular Hb and heme respectively.

Hp binds Hb to form high-molecular weight Hp-Hb complex. In this complexed form, Hb is sequestered within the vasculature and the passage of Hb outside the vessels is prevented. Plasma clearance of Hp-Hb complexes occurs via binding to the CD163 receptor, which is expressed by hepatic and splenic macrophages /1/.

Binding of heme to Hx blocks the pre-oxidant and pro-inflammatory effects of cell-free heme and facilitates receptor-mediated uptake of heme by the liver and spleen /2/.

18.8.1 Indication

Haptoglobin

Diagnosis and monitoring of hemolytic diseases.

Hemopexin

Genetic and non-genetic diseases, e.g., sepsis, sickle cell disease, hemolytic uremic syndrome, hemolytic conditions during stages of malaria and dengue /3/.

18.8.2 Method of determination

Quantitative determination

Immunoassay and immunonephelometric or immunoturbidimetric measurement. For principle, see Section 18.1 – Plasma protein diagnostics.

Haptoglobin sub typing

Polyacrylamide gel electrophoresis, isoelectric focusing.

18.8.3 Specimen

Serum: 1 mL

18.8.4 Reference interval

Refer to References /456/ and Tab. 18.8-1 – Reference intervals for haptoglobin and hemopexin.

18.8.5 Clinical significance

Both a decrease and an increase in Hp concentrations as well as the Hp phenotype may be clinically significant. Decreased concentrations indicate in vivo hemolysis. Increased serum concentrations are mostly due to the acute phase reaction of Hp and are found to occur in conjunction with inflammation, infections and autoimmunopathy. Certain Hp phenotypes are risk factors of systemic diseases.

In serum, Hx is much less often altered than Hp and when such an alteration occurs, Hx is almost exclusively affected by a decrease, as seen in severe hemolytic anemia. As a rule, Hx is determined if Hp has declined to non measurable levels.

Elevated Hx levels are rare and are only of clinical relevance in patients with melanoma.

18.8.5.1 Clinical significance of haptoglobin

Hemolytic reaction

In any hemolytic case, the serum Hp concentration depends on the Hp type of the individual and the concentration of lysed erythrocytes. The allele frequencies (HP1 and HP2) differ significantly worldwide /7/. For instance, the HP1 frequency ranges from 7% in parts of India to 70% in parts of West Africa. The HP1 frequency is between 31% and 40% in Europe, between 20% and 40% in Asians and, in North America, is 41% in Caucasians, 52% in Afro-Americans and 31% in Asians and Orientals.

The distribution of the Hp subtypes in Southern Germany, for example, is 14% for Hp 1-1, 48% for Hp 2-1 and 38% for Hp 2-2 /8/.

Since Hp phenotypes are associated with different serum Hp concentrations, the reference interval for Hp is very broad. Without electrophoretic phenotyping and application of a phenotype-specific reference interval, it is therefore impossible to

  • Detect mild chronic hemolysis
  • Estimate the severity of a hemolytic reaction merely based on the Hp concentration.

Normal Hp concentrations are to be expected despite an underlying hemolytic disease if a concomitant inflammatory disease leads to increased synthesis of Hp as an acute phase protein. This is the case if the C-reactive protein (CRP) is elevated. Therefore, the CRP should be determined besides Hp for diagnosing a hemolytic reaction (Tab. 18.8-2 – Assessment of haptoglobin in combination with C-reactive protein).

Investigations on the diagnosis and monitoring of hemolytic disease are shown in Tab. 18.8-3 – Investigations on the diagnosis and monitoring of hemolytic anemias.

The classification of hemolytic anemias is presented in Tab. 18.8-4 – Classification of hemolytic anemias.

The behavior of Hp in hemolysis and diseases associated with various Hp haplotypes is shown in Tab. 18.8-5 – Haptoglobin in hemolysis and diseases associated with various haptoglobin haplotypes.

Further diseases and conditions associated with decreased Hp

  • Acute and chronic liver diseases
  • Malabsorption syndrome
  • Congenital Hp decrease or deficiency (e.g., as seen in 30% of black Nigerians and in 1/1,000 Caucasians).

Diseases and conditions associated with elevated Hp concentration

  • Acute phase response (e.g., acute and chronic active inflammations, acute tissue necrosis, malignant tumors)
  • Intrahepatic and extrahepatic cholestasis, Hodgkin’s disease, nephrotic syndrome, rheumatoid arthritis, iron deficiency anemia
  • New synthesis of unknown etiology, such as multiple myeloma, amyloidosis /9/.

18.8.5.2 Clinical significance of hemopexin

Decreased serum Hx concentrations are measurable if the Hp concentration has declined to non measurable levels because of pronounced hemolysis. Heme containing molecules are only bound if their concentration exceeds 6 mg/L.

Even in the presence of pronounced hemolysis, Hx is never completely undetectable in serum /10/. Hx and Hp function at different levels (i.e., initially Hp is reduced, then Hx). Causes of a divergent behavior pattern between Hp and Hx are listed in Tab. 18.8-6 – Causes of a divergent behavior pattern between Hp and Hx.

The Hx concentration in newborns is about 20% of that in adults; Hx concentration is not a criterion for detecting hemolytic reactions during the neonatal period /11/.

Hx is better suited than Hp for assessing the extent of hemolysis. The following facts are in favor of Hx:

  • Hp is too sensitive, depends on the Hp phenotype and already displays markedly reduced levels in the presence of mild to moderate hemolysis
  • As an acute phase protein, Hp increases in the presence of inflammatory processes and thus may mask a hemolytic reaction
  • Hx is still measurable even in massive hemolysis.

Reductions in Hx without an underlying hemolytic reaction

  • Chronic liver disease
  • Porphyria cutanea tarda
  • Malabsorption syndrome.

Diseases and conditions associated with elevated Hx

Rapidly growing melanomas cause an increase in Hx that correlates with tumor growth. In the event of successful treatment, levels return to within the reference interval /12/.

18.8.5.3 Heme induced oxidative modification of hemopexin

Inflammation accompanies hemolysis and injury and is associated with increased oxidative stress driven in part via heme-mediated events. The function and concentration of many proteins is impaired by oxidative modifications from reactive oxygen species (ROS). Heme-Hx complexes resist oxidative damage. Heme binding by Hx protects against heme toxicity in hemolytic diseases and conditions, sepsis and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during the heme-driven inflammation.

Apo-hemopexin is vulnerable to inactivation by reactive nitrogen species (RNS) and ROS that covalently modify amino acids. It is supposed that during inflammation apo-hemopexin is nitrated and oxidized in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing Hx protective extracellular antioxidant function. The reason is tyrosine nitration in the heme binding site of pro-hemopexin/hemopexin /3/.

18.8.6 Comments and problems

Method of determination

In radial immunodiffusion, Hp 2-1 and Hp 2-2 have slower diffusion rates than Hp 1-1 since they are more markedly polymerized.

Reference interval

Hp concentration increases continuously after birth up to the age of about 40 years. Women have higher concentrations than men.

18.8.7 Pathophysiology

Haptoglobin

The HP gene exists in two allele types, HP1 and HP2 so that there are three Hp phenotypes: Hp 1-1, Hp 2-1 and Hp 2-2. These phenotypes are differently distributed worldwide, presumably due to genetic drift and natural selection /13/.

Hp is a glycoprotein composed of four polypeptide chains, two light α-chains and two heavy β-chains. The α-chain is genetically polymorphic; the synthesis is encoded by the two above-mentioned alleles. Little is known about the polymorphism of the β-chain. The structure of the Hp molecule is shown in Fig. 18.8-1 – Structure of haptoglobin.

Hp binds oxyhemoglobin, methemoglobin, isolated hemoglobin α-chains, α/β dimers and heme-free hemoglobin H but not deoxygenated hemoglobin, heme, hemoglobin H, isolated hemoglobin β-chains or myoglobin. Its physiological function is to prevent renal losses of hemoglobin and, thus, a loss in iron; this is based on the fact that unlike Hb the Hp-Hb complex, on account of its high molecular weight, is not glomerularly filtered /14/. Hp is primarily expressed by the liver, but also produced by the lungs, kidneys, spleen, thymus and heart.

Any reduction in the erythrocyte life span leads to an increase in hemolysis. The localization of lysis depends on both the extent of the hemolytic process and the mechanism by which each erythrocyte is damaged /15/. Depending on the phenotype, 1.5 mg of Hp bind approximately 1 mg of Hb in plasma. Clearance of the Hp-Hb complex by the reticuloendothelial system is approximately 15 mg per 100 mL of plasma per hour. If more Hb occurs intravascularly in relation to the quantity of Hp produced by the liver, Hp concentration declines as a measurable sign of hemolysis. In liver parenchymal damage with reduced synthesis capacity, a decrease in Hp occurs early on. If inflammatory processes are simultaneously present, Hp reacts as an acute phase protein and, despite a mild hemolytic process, increased Hp consumption is compensated by an increase in the synthesis rate; hence, the serum concentration remains within the reference interval.

Elimination of free hemoglobin by Hp

Intravascular hemolysis changes Hb from being an intracellular O2/CO2 transporter to a highly toxic substance in plasma. The pathologic effect of free Hb arises /13/:

  • From the hem iron, which can react with endogenous H2O2 to produce free radicals, which in turn may cause oxidative tissue injury, especially in the kidney
  • From the potency of hemoglobin to be a scavenger of NO, a signaling molecule that functions as a regulator of smooth muscle relaxation, endothelial adhesion molecule expression, and platelet activation and aggregation. The reaction of hemoglobin with NO is irreversible, leading to the production of nitrate and methemoglobin. NO scavenging limits the bioavailability of NO and thereby impairs NO homeostasis.

The effects of free hemoglobin are neutralized by binding to Hp. The hemoglobin-Hp complex is directed to the CD163 receptor expressing macrophages, which internalize the complex (Fig. 18.8-2 – Utilization of iron from the haptoglobin/hemoglobin complex). In the macrophage, the globin portion of the hemoglobin is degraded in the lysosomes, while heme is degraded by hem oxygenase-1 to Fe2+, CO and biliverdin. Fe2+ induces the synthesis of apoferritin, which binds Fe2+. Thus, Fe2+ is prevented from forming reactive oxygen species via the Fenton reaction.

In excessive hemolysis, the Hp-CD163-mediated scavenging mechanism is exhausted and fHb and heme are present in plasma. Both are important contributors to disease states associated with hemolysis which is characterized by free hemoglobin, heme induced inflammation and toxic cell damage. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) impaire antioxidant functions. For instance, 50% of patients with sickle cell disease have endothelial dysfunction due to the constant presence of fHb and heme.

In inflammatory conditions (extra corporeal circulation, sickle cell anemia) the synthesis of Hp and CD163 is up regulated by interleukin-6 to increase the fHb removal capacity. Hp does not bind to CD163. FHb binds with low affinity to CD163, provided that fHb was not oxidized by H2O2 /16/. Hp-bound Hb cannot be oxidized because it is protected. In contrast to the high-affinity Ca2+-dependent binding of the Hp-Hb complex to CD163, the low-affinity binding of non-oxidized fHb is a possibility for Hb removal if fHb concentrations are high.

The Hp phenotypes differ in their free hemoglobin removal functionality, also regarding CD163 interaction. The protective capacity of the phenotype Hp 1-1 against free hemoglobin-mediated oxidation is thought to be better than that of Hp 2-2. This might explain the different diseases associated with the Hp haplotypes.

Elimination of free hemoglobin by Hx and albumin

If Hp binding capacity has been exhausted, free hemoglobin occurs in plasma. Free hemoglobin is either oxidized to methemoglobin, followed by its dissociation into heme and globin, or oxidation of heme follows after dissociation. In both cases, hematin derivatives with Fe3+ are present. Hx binds these hematin derivatives with high affinity and albumin does with low affinity (Fig. 18.8-3 – Intravascular hemoglobin and hematin transport in Hp excess and Hp deficiency). Albumin is not involved until large quantities of hemoglobin are released; in this case, as methemalbumin, it gives the serum a coffee-brown color. Like Hx, albumin transports hematin derivatives to the reticuloendothelial system. If, during degradation of erythrocytes, heme is directly cleaved from hemoglobin due to enzymatic action Hx is mainly consumed while Hp may remains unaffected (e.g., as observed in hemorrhagic pancreatitis).

Oxidation of hemoglobin to methemoglobin and further degradation is achieved by the reticuloendothelial system. Detection of methemoglobin in plasma is always a sign of massive hemolysis.

Free Hb in plasma is glomerularly filtered at a clearance rate of 5 mL/min. In the proximal tubule, Hb is reabsorbed and heme iron released from hemoglobin is stored in the form of ferritin and hemosiderin, followed by its re utilization. If tubular epithelia are overloaded due to pronounced hemolysis, these cells degenerate, are sequestered and appear in urine as hemosiderin-carrying epithelial cells that are stainable with the Prussian blue reaction. Hemosiderinuria is, therefore, considered to be a valuable indicator of severe acute and chronic intravascular hemolysis.

Hemoglobinuria occurs when tubular reabsorption capacity is exhausted because of a massive hemolysis.

Hemopexin

Hx has a molecular weight of 80 kDa and, like Hp, is synthesized in the liver. Whereas Hb is exclusively bound by Hp, heme and heme derivatives are only bound by Hx and albumin. Hx has a high affinity toward heme and removes these substances from the plasma after cleavage of fHb into a heme and a globin portion. It only responds in the presence of pronounced hemolysis (i.e., if free Hp is no longer available). Hx is not an acute phase protein. Hx deficiency develops in patients with sepsis that is life-threatening.

Several diseases in which Hx plasma levels increase are associated with inflammation, increased activity of nitric oxide synthase, and oxidative stress. Thus Hx is expected to be exposed to high levels of oxidative species. Because proteins can be inactivated by oxidative modifications from exposure to ROS and RNS it is supposed that the increased levels of modified Hx might be a physiological response to damage, in which case Hx is functional, i.e., capable of binding to heme or to Hx receptors. The protective extracellular antioxidant function of Hx is impaired during heme-driven inflammation because apo-Hx is is vulnerable to inactivation by RNS that cause tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of Hx /3/.

References

1. Kristiansen M,Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law SK, Moestrup SK. Identification of the haemoglobin scavender receptor. Nature 2001; 409: 198–201.

2. Tolosano E, Fagoonee S, Morello N, Vinci F, Fiorito V. Heme scavening and the other facets of hemopexin. Antioxid Redox Signal 2010; 12: 305–20.

3. Hahl P, Hunt R, Bjes ES, Skaff A, Keightley A, Smith A. Identification of oxidative modifications of hemopexin and their predicted physiological relevance. J Biol Chem 2017; 292: 13658–71.

4. Jeppsson JO. Haptoglobin. In: Ritchie RF, Navolotskaia O (eds). Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research 1996: 7.04-1–6.

5. Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996; 42: 1589–1600.

6. Meiers HG, Lissner R, Mawlawi H, Brüster H. Hämopexinspiegel bei Männern und Frauen in verschiedenen Lebensaltern. Klin Wschr 1974; 52: 453–7.

7. Carter K, Worwood M. Haptoglobin: a review of the major allele frequencies worldwide and their associations with diseases. Int Jnl Lab Hem 2007; 29: 92–110.

8. Braun HJ. Eigenschaften und Funktion menschlicher Serumproteine bei intravasaler Hämolyse. Dtsch Med Wschr 1971; 96: 595–601.

9. Aeschlimann A, Mall TH, Rieder HP. Hyperhaptoglobinämie bei Plasmozytom und Amyloidose. Dtsch Med Wschr 1985; 110: 1128–9.

10. Braun HJ. Eigenschaften, Funktion und Serumkonzentration des menschlichen Hämopexins. Klin Wschr 1971; 49: 445–51.

11. Lundh B, Oski FA, Gardner FH. Plasma hemopexin and haptoglobin in hemolytic diseases of the newborn. Acta Paediat Scand 1970; 59: 121–6.

12. Manuel Y, Defontaine MC, Bourgoin JJ, Dargent M, Sonneck JM. Serum hemopexin levels in patients with malignant melanoma. Clin Chim Acta 1971; 31: 485–6.

13. Nielsen MJ, Moestrup SK. Receptor targeting of hemoglobin mediated by haptoglobins: roles beyond heme scavenging. Blood 2009; 114: 764–71.

14. Marchand A, Galen RS, van Lente F. The predictive value of serum haptoglobin in hemolytic disease. JAMA 1980; 243: 1909–11.

15. Zvi B, Levy AP. Haptoglobin phenotypes, which one is better and when? Clin Lab 2006; 52: 29–35.

16. Buehler PW, Abraham B, Vallelian F, Linnemayr C, Pereira CB, Cipollo JF, et al. Haptoglobin preserves the CD 163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification. Blood 2009; 113: 2578–86.

17. Kretschmer V, Mueller-Echkardt C. Relevanz verschiedener Untersuchungsmethoden bei Immunhämolysen. Lab Med 1982; 6: 133–8.

18. Tabbara IA. Hemolytic anemias. Med Clin North Am 1992; 76: 649–68.

19. Garby L, Noyes WD. Studies on hemoglobin metabolism. 1. Kinetic properties of the plasma hemoglobin pool in normal man. J Clin Invest 1959; 38: 1479–83.

20. Shih AWY, McFarlane A, Verhovsek M. Haptoglobin testing in hemolysis: Measurement and interpretation. Am J Hematol 2014; 89: 443–7.

21. Nakagawa T, Muramoto Y, Hori M, Mihara S, Marubayashi T, Nakagawa K. A preliminary investigation of the association between haptoglobin polymorphism, serum ferritin concentration and fatty liver disease. Clin Chim Acta 2008; 398: 34–8.

22. Levy AP, Hochberg I, Jablonski K, Resnick HE, Lee ET, Best L, et al. Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: The Strong Heart Study. J Am Coll Cardiol 2002; 40: 1984–90.

23. Graw JA, Mayeur C, Rosales I, Liu Y, Sabbisetti VS, Riley FE, et al. Haptoglobin or hemopexin therapy prevents acute adverse effects of resuscitation after prolonged storage of red cells. Circulation 2016; 134; 945–60.

24. Hulikova A, Kramer H, Khan H, Swietach P. Detection of intravascular hemolysis in newborn infants using carbonic anhydrase I immunoreactivity. JALM 2020; September: 921–34.

18.9 Immunoglobulins (Ig)

Lothar Thomas

Immunocompetent individuals have an acquired immune system that is divided into the following two functional mutually cooperative but developmentally independent units:

  • Thymus (T) lymphocyte system; it represents a functionally heterogeneous group of cells concerned with immune regulation and antigen elimination. The T lymphocyte system is primarily responsible for cellular immunity.
  • Bursa or bone marrow (B) lymphocyte system; B lymphocytes differentiate into plasma cells which synthesize and secrete immunoglobulins (Ig) after an antigenic stimulus.

Ig represent a heterogeneous group of proteins with antibody functions (i.e., they are capable of binding antigens). Their synthesis is the adaptive response to the antigen structure that will be bound. The structure of the antibody binding site is synthesized to correspond with the configuration of the antigen with which the antibody will react.

Ig have the following effector functions:

  • Formation of immune complexes with antigens that are amenable to phagocytosis
  • Binding to the membrane receptors of immune cells in order to activate them
  • Reaction with plasma proteins (e.g., with complement components, and activation of these proteins in order to eliminate the antigen).

18.9.1 Structure and function of Ig

Ig have a common basic structure, consisting of two identical heavy (H) chains and two identical light (L) chains (Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding). The chains are joined together by disulfide bonds. The amino terminal ends of the light and heavy chains of the Ig molecule form the variable region. The variable regions of the Ig differ in their amino acid sequences, since these are responsible for the specificity towards the antigen determinants. Within the variable regions, there are areas of lesser and greater variability in the amino acid composition; these latter are also referred to as the hyper variable regions /123/.

The heavy chains of the Ig molecule consist of a variable and a constant region. Each of the two polypeptide chains is comprised of a series of globular regions also referred to as domains, with substantial amino acid homology.

N-terminal domains of H and L chains contain variable amino acid sequences (V region) determining the antigen specificity. The N-terminal amino acids of the V region form a functional pocket into which the epitope of the antigen fits. Surfaces of the antigen binding site and epitope are complementary to each other, so that the concavities of the one are filled by the convexities of the other. The complementarity is not only physical but also chemical in nature. The contacts of binding site and epitope include van der Waals forces, hydrogen bridge bonds between polar groups and ion pair bonds between differently charged side chains /45/.

The constant regions of the heavy chains include domains (CH regions) with certain structural and antigenic differences that allow their classification into Ig subclasses.

In the IgG molecules, for example, the domains have the following functions /45/:

  • The first (CH1) and the second domain (CH2) form the hinge region. Because of this region, the Ig molecule is capable of transforming from the T form, in which it is in solution, into the V form, which it assumes during antigen binding.
  • The CH2 domain binds the complement protein C1q, which activates the classic pathway of the complement system
  • The third domain CH3 mediates binding of Fc fragment to Fc receptor of immune cells such as granulocytes, monocytes/macrophages and antigen dependent cytotoxic cells (ADCC)
  • The combination of CH2 and CH3 acts as an additional domain for binding, for example, to granulocytes and natural killer (NK) cells.
  • Papain cleaves the Ig molecule into three fragments: Two identical Fab fragments consisting of the complete L-chain, the variable region and a portion of the constant region of the H-chain
  • A fragment consisting of the constant regions of the two heavy chains, linked via disulfide bridges. Since this fragment crystallizes, it is also referred to as Fc fragment (fragment crystallizable).

Refer to Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding:

18.9.1.1 L-chain

Kappa- and lambda-L-chain types are differentiated. Each Ig molecule has either two kappa- or two lambda-L-chains since a B cell can only synthesize one type of L-chain. L-chains have a molecular weight of about 22 kDa and consist of an amino-terminal, variable region and a carboxy-terminal, constant region. The L-chain is connected to the heavy chain via a disulfide bond between a cysteine molecule in the constant regions of both chains. B cells produce approximately twice as many kappa chains as lambda chains. The term monoclonal free light chain or, if detectable in urine, Bence Jones protein refers to L-chains of one type that are not bound to heavy chains and are synthesized and secreted by malignant B-cell proliferation. Because of the cysteine molecules, they are prone to dimerization.

Polyclonally synthesized free light chains are of the kappa and the lambda types and are detected in about equal amounts in a sample; traces are present in the urine (e.g., associated with infectious diseases).

18.9.1.2 H-chain

The structure of the H-chain determines the Ig class of an Ig molecule. Differences between individual Ig classes concern their amino acid sequence, molecular weight, carbohydrates, antigenicity, allotypic heterogeneity and electrophoretic mobility. Five major Ig classes are distinguished, with each of them displaying two identical H-chains: IgG has γ-heavy chains, IgA has α-heavy chains, IgM has μ-heavy chains, IgD has δ-heavy chains and IgE has ε-heavy chains.

18.9.1.3 J-chain

The J-chain is a peptide connecting two Ig molecules of the same class (connection piece). Ig molecules of M and A classes exhibit polymorphism. IgM occurs mostly as a pentamer and IgA as a dimer. IgM monomers as well as IgA monomers are each only connected via a single J-chain. J-chains are glycoproteins with a molecular weight of 15 kDa and bind via disulfide bonds near the carboxy-terminal end of the heavy chains (Fig. 18.9-2 – Structure of immunoglobulins).

18.9.1.4 Secretory component

As a dimer, secretory IgA, which is synthesized by plasma cells of the respiratory, genitourinary and gastrointestinal tracts and released into secretions, contains a secretory component that protects the Ig molecule from peptidase-mediated degradation (Fig. 18.9-2 – Structure of immunoglobulins). The secretory component is a glycoprotein with a molecular weight of 60 kDa. Dimeric IgA is not bound to the secretory component until it penetrates the epithelial layer, where the secretory component is synthesized by epithelial cells. This secretory component is detectable in secretory IgA deficiency.

18.9.1.5 Antibody heterogeneity

The heterogeneity of antibodies can be subdivided into isotypic, allotypic and idiotypic variations.

Isotypic variation

Isotypes have the same constant domains. Isotypic variations consist of differences in H-classes, L-types and domains, which are present in all healthy members of a given species. The synthesis of isotypes is encoded by genes, which are present in all human beings.

Allotypic variation

Allotypic variation is defined as an allele-induced variation of the Ig within a species. Allotypes usually occur as variants of the constant region of the H-chains. For instance, Gm factor is responsible for the regulation of the constant H-chain region of IgG. More than 25 different Gm (genetic marker) allotypes are known. Allotypic variants are often the result of an amino acid substitution within the H-chains.

Idiotypic variation

Idiotypes have the same variable domains. The idiotypic variation relates to the diversity at the antigen binding site. The Ig synthesized by a plasma cell clone is normally completely identical (i.e., it consists of a uniform idiotype). An alteration (e.g., an amino acid exchange in the variable region of the antigen binding site) may lead to idiotypic variation. Such antibodies differ even if they belong to the same isotype and allotype. Many monoclonal components (para proteins) are probably idiotypic variations.

18.9.2 Ig classes

Ig classes IgG, IgA, IgM, IgD and IgE are present in descending order of concentration in the serum of healthy individuals /12/. Within the IgG class, the subclasses IgG1, IgG2, IgG3 and IgG4 are differentiated, and the class IgA includes the subclasses IgA1 and IgA2, while the IgM class contains the subclasses IgM1 and IgM2. The physicochemical and biological properties of the individual Ig classes are listed in Tab. 18.9-1 – Physicochemical and biological properties of the Ig classes.

18.9.2.1 IgG antibodies

IgG consists of two identical H-chains with a molecular weight of about 50 kDa and two identical L-chains with a molecular weight of about 22 kDa. In primary infections (primary antibody response), IgG are usually secondary antibodies and, in repeat infection with the same organism, primary antibodies (secondary antibody response). Approximately one half of total body IgG is present in plasma, while the other half is distributed in the body fluids. When performing serum protein electrophoresis, IgG is located in the γ-globulin fraction. Under standardized conditions, the subclasses IgG2 and IgG4 migrate toward the anode, whereas IgG1 and IgG3 migrate toward the cathode. Fetal IgG originates from maternal blood and during the first 20 weeks of gestational age has a concentration corresponding to approximately 10% of that found in the mother.

Fetal infections during this time period do not cause an increase in IgG. Between the 22nd and the 28th week of gestational age, placental permeability markedly increases, thus accounting for the fact that at this point of time the fetal IgG concentration is equal to that in maternal blood. Concentrations of IgG1, IgG3 and IgG4 are the same, whereas fetal IgG2 concentration is lower than the maternal one. After delivery, maternal IgG decreases in the circulation of the newborn with a half-life of approximately 30 days, thus resulting in a residual concentration of only 3.5–4.0 g/L by the 3rd to 4th month. As infant’s own IgG synthesis gets under way, serum IgG concentration slowly increases, reaching a concentration of 7–8 g/L by the end of the first year and adult levels prior to the age of 16 years (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood/2/.

The synthesis of IgG subclasses during the course of an immune response depends on the nature of antigens, their site of entry and the duration of antigen exposure. The antibody response is directed against:

  • Protein antigens, such as bacteria and viruses, mediated by IgG1 and IgG3 and induced by CD4+T-cells
  • Polysaccharide antigens (e.g., encapsulated bacteria such as Pneumococcus, Streptococcus group A but also H. influenzae) mostly mediated by IgG2 and partly IgG1 and not induced by CD4+T-cells
  • Polyvalent antigens such as snake venoms, parasites, insects and food components in the case of chronic antigen stimulation and mediated by IgG4. The IgG4 antibodies, like IgE, bind to surface receptors of mast cells.
  • Native DNA, mediated by IgG1 and IgG3 autoantibodies.

The IgG subclass response is regulated and modulated by interleukins; the number of existing B-cell sub populations in a tissue region also plays an important role. For instance, in the blood of healthy individuals, B cells with cytoplasmic IgG1 and IgG2 dominate, whereas in the tonsils B cells with IgG1 and IgG3 are predominant.

Because of its binding to the Fc receptor of the immune cell, the Fc fragment of the IgG molecule is of significant importance in the immune response. The Fc fragment mediates:

  • The uptake of IgG coated bacteria by macrophages. The Fc fragment of the IgG molecule is bound by the Fc receptor of the macrophage; the bacterium is engulfed and incorporated into the macrophage in a zipper-like manner
  • Clearance of IgG-containing immune complexes according to the aforementioned mechanism
  • The antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as monocytes/macrophages, granulocytes and lymphocytes. These cells have Fc receptors to which the Fc fragment of the IgG-loaded target cell binds. These cell is subsequently destroyed.

The IgG catabolism is proportional to the plasma concentration (i.e., enhanced in the case of a high IgG level and low in the case of a low concentration). Accordingly, the half-life is 70 days in case of a low IgG synthetic rate, but may be shortened to 11 days if the IgG synthetic rate is high.

18.9.2.2 IgM antibodies

IgM circulates in the serum as a pentamer, has a molecular weight of 971 kDa and consists of five monomers that are covalently linked via disulfide bonds and connected via five connection pieces (J-chains). Small amounts of monomers and hexamers are also present in the circulation. Each μ-chain contains one V region and four C regions.

Serum IgM comes in two flavors, pre-immune without exposure to exogenous antigen also known as natural IgM that is spontaneously produced, and the second type is exogenous antigen induced or immune IgM antibodies /3/.

The majority of IgM in serum is comprised of natural IgM antibodies. The two prominent features of natural IgM are poly reactivity and auto reactivity. The natural IgM antibodies are reactive with many conserved epitopes that are shared by microbes and self antigens. The production of natural IgM appears to be triggered by interaction with self antigens. In addition to providing early defense against microbes, natural IgM plays an important role in immune homeostasis, and provides protection from consequences of autoimmunity and inflammation.

Immune IgM is the first antibody secreted during an initial immune response to an exogenous antigen. Mature naive B cells in response to antigens undergo clonal expansion and differentiation into Ig secreting cells.

75–80% of IgM are located intravascularly. In serum protein electrophoresis, IgM migrates between the γ-globulin and the β-globulin fraction. Because of slight differences in the μ-chain, two IgM subclasses are distinguishable. The pentameric form of the IgM molecule has ten antigen binding sites, but only five of these are usable for antigen binding due to steric hindrance. The pentameric IgM can be split into monomers by SH group cleaving reagents.

Besides pentameric and monomeric IgM, a secretory IgM with a secretory component is present in body secretions much like IgA. The essential functions of IgM in immune response are the agglutination of pathogens and the activation of the classic complement pathway.

Maternal IgM does not cross the placental barrier. The healthy fetus as well as the newborn have an IgM concentration that is about 10% of the adult level and is produced by the fetus itself.

After birth, IgM synthesis rises markedly: 50% of the adult IgM concentration are reached after the first 4 months of life and the adult level is reached at the age of 8–15 years (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood). The fetus by itself can synthesize IgM in larger quantities from the 20th gestational week. Intrauterine infection can, therefore, cause a steep increase in the IgM concentration, and IgM levels > 0.2 g/L in cord blood are a criterion pointing to such an infection.

The IgM class includes the natural antibodies such as AB0 blood group isohemagglutinins, cold agglutinins (anti-i, anti-I), heterophilic antibodies, saline erythrocytic antibodies as well as antibodies to IgG (e.g., rheumatoid factors).

The catabolism of IgM is independent of the serum concentration.

18.9.2.3 IgA antibodies

IgA antibodies occur as serum IgA and as secretory IgA.

Serum IgA

Approximately 90% of IgA are present in a monomeric form with a molecular weight of 160 kDa and 10% in a polymeric form. Serum IgA tends to form complexes, especially with albumin, but also with enzymes, thus forming macroenzymes. Half of the serum IgA is located intravascularly and occurs in two subclasses. The ratio of the two IgA isotypes IgA1/IgA2 is 9/1. In serum protein electrophoresis, IgA migrates in the cathodic part of the β-globulin and the anodic part of the γ-globulin fraction.

Since IgA does not cross the placental barrier, it is not present in fetal blood. After birth, the synthesis of IgA begins slowly; by the end of the first year, the infant has about 25%, by 3.5 years of age approximately 50% and by 16 years of age 100% of the adult serum level (Fig. 18.9-3 – Course of immunoglobulin serum levels during fetal period and in childhood). The function of serum IgA is not known in further detail. It activates complement via the alternative pathway and has specific antibody functions. The catabolism of IgA is independent of the serum concentration.

Secretory IgA

This IgA molecule is composed of a unit of two IgA molecules, which are connected via a J-chain and have a secretory component (Fig. 18.9-2 – Structure of immunoglobulins). Secretory IgA is produced by plasma cells that are located in the lamina propria of mucous membranes. It is synthesized independently from serum IgA. Therefore, deficiency in serum IgA does not necessarily imply deficiency in secretory IgA. Secretory IgA is the predominant Ig of body secretions such as saliva, tears, colostrum, nasal secretions, tracheobronchial mucus, gastrointestinal secretions and breast milk. Newborns and infants are supplied with IgA via breast milk and, hence, are passively immunized against gastrointestinal infections. Essential functions of secretory IgA are: the binding of microorganisms on mucous membranes, activation of the alternative complement pathway and activation of inflammatory reactions. Inside epithelial cells, IgA is thought to neutralize intracellular microorganisms. In the lamina propria of mucous membranes, IgA binds antigens in the form of immune complexes and secretes them on the mucomembranous surfaces. This prevents the circulation from being overloaded with immune complexes. Individuals with secretory IgA deficiency are found to suffer more commonly from mucosal infection, atopy and autoimmune disease.

18.9.2.4 IgD antibodies

IgD has a high carbohydrate content with numerous oligosaccharide chains. Due to the fact that the two δ-chains are only held together via disulfide bonds and due to the abundance of lysine and glutamic acid in the hinge region, the molecule is prone to proteolysis.

Together with IgM antibodies, IgD antibodies are located on the cell membrane of B cells as antigen receptors. In cord blood, IgD is not detectable or only detectable in minimal concentrations; adult levels are reached at 2–5 years of age.

The distribution of IgD in the body is identical to that of IgM. The exact function of IgD is unknown, but this Ig class is thought to include antinuclear antibodies (ANA) as well as antibodies to insulin and penicillin. The higher the serum level of IgD, the slower its catabolism (i.e., the plasma concentration of IgD is inversely related to IgD catabolism).

18.9.2.5 IgE antibodies

The ε-heavy chain, like the μ-chain of the IgM, possesses five domains, which explains the molecular weight of 72.5 kDa. IgE binds via the domains Cε3 and Cε4 to the high-affinity receptor FcεRI located on mast cells and basophil granulocytes. Moreover, it binds to the low-affinity receptors FcεRII or CD23 present on monocytes, lymphocytes and eosinophil granulocytes.

IgE antibodies are also referred to as reagins. Their distribution in the body is identical to that of IgA and their catabolism, at a half-life of 2.5 days, is high. The serum IgE level does not represent the effective IgE load of the organism since the predominant IgE synthesis sites are located in the respiratory tract, gastrointestinal tract and lymph nodes. Because of its affinity to mast cells, some IgE is bound to the IgE receptors of these cells.

IgE antibodies mediate type I hypersensitivity reaction of the immediate type. Harmless, polyvalent antigens, such as pollen or house dust mites, stimulate mucosal B cells at the site of entry to synthesize specific IgE. This process is mediated by CD4+T cells. Specific IgE binds via Fc receptors to mast cells that are now sensitized. During the next contact of the polyvalent antigen with the sensitized mast cell, bound IgE antibodies are cross-linked, the cell is de granulated and mediators are released, which cause, for example, symptoms of hay fever, asthma and atopic eczema.

18.9.3 Quantitative determination of immunoglobulins

IgG, IgA, IgM, IgD

Immunoassay and immunonephelometric or immunoturbidimetric measurement /6/.

IgE

Immunoassays such as enzyme, fluorescence or luminescence immunoassays.

IgG subclasses

Immunoassay and immunonephelometric or immunoturbidimetric measurement mostly using monoclonal antibodies (Section 18.11 – Cryoglobulins and cryofibrinogen).

IgM in cord blood

Latex agglutination test for rapid determination of IgM; latex particles are coated with anti-IgM.

Secretory IgA

Radial immunodiffusion, rocket immunoelectrophoresis, radioimmunoassay and enzyme immunoassay /7/.

If secretory IgA levels are measured in saliva by employing radial immunodiffusion and specific plates (LC-partigen plates), which contain antiserum to serum IgA, the measured levels are multiplied by 3.25 in order to compensate for the molecular weight. Reducing pretreatment of saliva using dithiotreitol is recommended /8/.

18.9.3.1 Specimen

IgG, IgA, IgM, IgD, IgE

Serum, body fluids: 1 mL

IgG subclasses: same as for IgG.

Secretory IgA: saliva, tear fluid, intestinal juices and/or feces.

If immunoglobulin determination has to be performed in a neonate, as much cord blood should be collected as possible.

18.9.3.2 Reference interval

Refer to Ref. /1/ and Tab. 18.9-2 – IgG, IgA, IgM reference intervals in serum.

  • IgD in serum: 0.03–0.14 g/L /9/
  • IgE in serum: Below 0.24 mg/L (100 KU/L)

1 U IgE = 2.4 ng

Secretory IgA in saliva /7/: 0.08–0.20 g/L.

18.9.3.3 Clinical significance

Changes in serum Ig concentrations are usually classified into /2/:

  • Hypogammaglobulinemias that may be associated with numerous diseases. The decrease in Ig can be due to reduced synthesis, increased loss or hyper catabolism of Ig or a combination of causes.
  • Polyclonal gammopathies that may be due to an increase in antibodies of one or several Ig classes. The spectrum of diseases causing polyclonal gammopathy is broad and includes, for example, infections, chronic liver diseases, autoimmune disorders.
  • Monoclonal gammopathies that are characterized by a narrow band (M-gradient) in the γ-globulin fraction in serum protein electrophoresis. M-gradients are caused by excessive proliferation of a B-cell clone, which synthesizes Ig of one class and one type (Chapter 22 – Monoclonal plasma cell proliferative diseases).
18.9.3.3.1 Hypogammaglobulinemia

If hypogammaglobulinemia is present, one should

  • Determine the extent of the antibody deficiency by measurement of the Ig of each class and subclass
  • Perform more detailed examinations concerning differentiation of the antibody deficiency, see Chapter 21 – Immune system.

Extent of antibody deficiency

Ig deficiencies can be of varying extent:

  • One or all Ig classes or Ig subclasses are absent or strongly decreased
  • The Ig of one Ig class or Ig subclass are moderately reduced as compared to an age-matched group of healthy individuals
  • The Ig of the Ig classes and of some of the Ig subclasses are normal but, within a certain subclass, an antibody response cannot be established. For instance, a selective antibody synthesis defect can be present in the IgG2 subclass involving only antibodies to pneumococcal polysaccharide.

The behavior of Ig in primary immunodeficiency diseases is presented in Chapter 21 – Immune system.

The behavior of Ig in secondary immunodeficiency diseases is shown in Tab. 18.9-3 – Laboratory findings in secondary immunoglobulin deficiency.

A decrease in serum Ig level due to hyper catabolism occurs in hyper metabolic states. For example, such a state is present in patients with hyperthyroidism; it can be associated with a reduction in Ig of all Ig classes. In patients with myotonic dystrophy, serum Ig are also reduced because of a shortened half-life. Antibodies to Ig are also rarely a cause for the rapid elimination of Ig from the circulation.

18.9.3.3.2 Polyclonal and oligoclonal gammopathies

With an influx of antigens into the organism, clonal selection operates to activate those B cells with antigen receptors for which the antigen has a high affinity. Usually, after primary contact of the immune system with such an antigen, several B cells are activated to a varying extent. They proliferate and mature into antibody-producing plasma cell clones /2/.

During an infection with complex agents (e.g., microorganisms); many antigens are produced because of antigen processing and presentation to the B cells. The selection of several T-cell stimulated B cells leads to their clonal proliferation and specific antibody production. This results in polyclonal immune response.

Polyclonal immune response

Polyclonal immune response leads to an increase in serum Ig, involving either one or several Ig classes and both Ig types. A broad-based increase in the γ-globulin fraction is detectable in serum protein electrophoresis.

Oligoclonal immune response

The oligoclonal immune response results from limited activation of B cells, also referred to as limited heterogeneity. Possible causes include: nature of the antigen, lack in reactivity of the immune system or contact of the antigen with tissues that are poor in immunocompetent cells (e.g., the central nervous system).

Diagnosis of polyclonal and oligoclonal gammopathies

The diagnosis of polyclonal or oligoclonal Ig increases is based on screening by serum protein electrophoresis; a more differentiated diagnosis is possible by quantitative determination of Ig classes and Ig subclasses or, in cerebrospinal fluid, by isoelectric focusing and subsequent immunofixation.

In diseases causing hypergammaglobulinemia, quantitative Ig determination in conjunction with the clinical picture as well as serological and clinical chemistry findings can provide results that contribute to a diagnosis, differential diagnosis, disease monitoring and formulating a prognosis.

This may be the case for:

  • Acute and chronic infections
  • Liver diseases
  • Diseases of the central nervous system
  • Intrauterine and perinatal infections.
  • Ig determination in hypergammaglobulinemia

The indications are limited, in which the differentiation of hypergammaglobulinemia by means of quantitative Ig determination is clinically useful for the diagnosis of a disease. Usually, especially in infectious diseases, an increase in one or several Ig classes (with a similar Ig pattern) occurs /2/. No specific Ig pattern exists that occurs exclusively in a certain disease and by itself is of diagnostic value. However, the Ig pattern may be an important supplemental finding and may, in combination with the overall clinical picture, contribute to differential diagnosis, assessment of the course of the disease and formulating a prognosis.

18.9.3.3.3 Differential diagnostic value of Ig determination in hypergammaglobulinemia

An isolated polyclonal Ig increase in one Ig class or a more pronounced increase in one Ig class within the Ig pattern are valuable in differential diagnosis.

Isolated IgM increase

In combination with the clinical picture and especially if present for several days, an isolated IgM increase is a sign for an initial infection of the organism by a pathogen (primary reaction).

In newborns, an IgM increase in cord blood is considered to be a nonspecific sign of an infection acquired in utero.

Isolated IgG increase

In an acute infectious disease with normal or only mildly elevated IgM, an isolated increase in IgG is a sign of secondary response of the immune system to an already known pathogen. Chronic infections elicit primarily to an isolated IgG increase, while chronic active infections cause an increase in IgG, occasionally accompanied by elevated IgM and/or in IgA.

Isolated IgA increase

In liver diseases, a relatively higher IgA increase or an isolated IgA elevation suggests toxic damage (e.g., due to alcohol, hormonal contraceptives, antidepressants).

Ig pattern

The Ig pattern is not very conclusive if two or more Ig classes are increased. While increases in all three Ig classes are often found in liver cirrhosis, conclusions concerning the etiology of chronic liver disease can only be reached to a limited extent based on the Ig pattern (Tab. 18.9-4 – Pattern of IgG, IgA, IgM in liver diseases).

18.9.3.3.4 Monitoring of hypergammaglobulinemia

The assessment of the course of an inflammatory event that employs quantitative Ig determination should be based on the Ig pattern. Persistently elevated Ig concentrations argue for a sustained assault on the organism by the antigen, and the Ig will decline in the course of overcoming the infection process and the normalization for eliminating the extracellular antigen. The magnitude of the IgG concentration is a measure of the activity of the inflammatory process, especially in virally induced chronic liver disease, chronic bacterial infection, connective tissue disease and other autoimmune diseases. In toxic liver damage, the intensity of inflammation correlates with IgA concentration /10/.

During the course of infectious diseases affecting certain organs, the Ig concentration, and especially that of IgM, is of prognostic value. Persistence of elevated IgM levels at a time when a decline is expected suggests the transition to a chronic process, while continuously rising IgG levels suggest the transition to a chronic active process. In some infections such as borreliosis, patients can have persistent IgM antibodies for years without any clinical symptoms.

18.9.3.3.5 Screening for prenatally acquired infections

Because of the transplacental transfer of IgG, the newborn has the same IgG antibody pattern as the mother. An infection of the newborn cannot be recognized by methods that only detect IgG antibodies because maternal IgG antibodies are not distinguished from those produced by the fetus as result of an infection /11/.

From the 20th week of gestational age, the fetus is capable of producing IgM antibodies and from the 30th week it can also synthesize IgA. From this time point, intrauterine infections, due to various viruses (rubella, cytomegaly, herpes simplex, varicella, mumps, measles, influenza, hepatitis B, parvovirus B19), treponema pallidum or toxoplasma gondii are detectable by elevated IgM and/or IgA concentrations in the newborn blood (cord blood). The incidence of infections in newborns is reported to be 2–4% /11/.

Because of the simple and rapid measurement, the IgM concentration in cord blood serum is considered to be a good screening test. A concentration > 0.20 g/L is regarded to be an indicator of an infection.

A significant percentage of cases with intrauterine infection remains unrecognized because of the lack of clinical manifestations in the newborn or exhibits only a slight IgM increase.

In the presence of elevated IgM, clinical symptoms are only found in one third of newborns. The reason for the low diagnostic specificity is either placental leakage or contamination of cord blood with maternal blood. An infection is unlikely if the IgA concentration is also elevated and if, during a second test after 5 days, the levels of IgA and IgM in neonatal blood have markedly decreased (half-life: 5 days).

A persistence or increase in IgM indicates the presence of an infection acquired in utero or during the perinatal period. In such a case, infection-specific IgM or IgA antibodies should be determined by enzyme-linked immunosorbent assay (ELISA).

18.9.3.3.6 IgE increase

The increase in IgE is a characteristic sign of atopic diseases (see Chapter 23 – Atopy and allergy). Diseases associated with an IgE increase that is not induced by atopic diseases are presented in Tab. 18.9-5 – IgE increases in non-atopic diseases.

18.9.3.3.7 Oligoclonal Ig increase

The Ig increase is not infrequently limited to increases in certain antibody populations. These antibodies may belong to one or several Ig classes or to only one Ig subclass although they are still of polyclonal origin (i.e., they are present as kappa- and lambda-light chain types). In serum protein electrophoresis on cellulose acetate but more commonly on agarose, one or several distinct bands are visible against the diffuse background of the γ-globulin band (sawtooth pattern).

Diseases and conditions leading to oligoclonal increases in Ig in serum are listed in Tab. 18.9-6 – Causes of oligoclonal IgG increases.

Oligoclonal Ig in cerebrospinal fluid is described in Chapter 46 – Laboratory diagnosis of neurological diseases.

18.9.3.3.8 Monoclonal gammopathy

Refer to Section 22.3 – Monoclonal gammopathy.

18.9.3.4 Comments and problems

Standardization and quality assurance

Refer to Section 18.1.13 – Quality assurance.

Method of determination

Radial immunodiffusion: this method is relatively resistant to interferences. An antigen excess phenomenon must be kept in mind, recognizable by the presence of fuzzy precipitation along the margin. Aggregated Ig simulate falsely low Ig concentrations, while Ig fragments simulate falsely high ones.

Immunonephelometry: interferences in this methodology include light-scattering contaminants such as micro clots, cells from inadequately centrifuged samples, particles derived from proteins of the cerebrospinal fluid and microbial contaminations. In principle, problems should be anticipated in samples after deep-freezing or in hyperlipidemic samples. Nephelometric assays properly detect antigen excess and are approximately 10 times more sensitive than turbidimetric assays that are not latex particle-enhanced.

Immunoturbidimetry: turbidimetric Ig determinations, usually measured with clinical chemistry analyzers, are prone to interference by samples with high absorbance, as seen in hyperbilirubinemic, hemolytic or hyperlipidemic sera. Antigen excess is easily overlooked and inappropriately low Ig levels are determined.

References

1. Bienvenu J, Whicher J, Chir B, Aguzzi F. Immunoglobulins. In: Ritchie RF, Navolotskaia O. Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 11.01-1–16.

2. Ritzmann SE, Daniels JC, eds. Serum protein abnormalities. Diagnostic and clinical aspects. New York: AR Liss, 1982.

3. Gupta S, Gupta A. Selective IgM deficiency: an underestimated primary immunodeficiency. Frontiers in Immunology 2017: 8: article 1056.

4. Sheehan C, ed. Clinical immunology. Principles and clinical diagnosis. Philadelphia: Lippincott, 1990.

5. Kalden JR, Burmester GR. Das Immunsystem des Menschen. Göttingen: Edition SK + F, 1984.

6. Thomas L. Quantitative immunchemische Plasmaproteinbestimmung mittels Nephelometrie und Turbidimetrie. Lab Med 1990; 14: 313–20.

7. Johnson jr RB, Liu J. The application of enzyme immunoassay to the study of salivary IgA. J Immunoassay 1982; 3: 73–89.

8. Alaluusua S, Grönblad EA, Tölö H. Quantitation of IgA in human whole saliva: a comparison of three immunoassays. Acta Odontol Scand 1981; 39: 155–61.

9. Warsy AS, Bahakim AM. Immunoglobulin D – What do we know about it? Saudi Med J 1986; 7: 106–15.

10. Fateh-Moghadam A. Die Bedeutung der Immunglobuline G, A, M und E in der Diagnostik von Lebererkrankungen. Bayr Internist 1982; IV: 1–5.

11. Rosanelli K. Present diagnostic value of IgM-determination in newborns. In: Bethke K, Riegel K, Belohradsky BH, eds. Diagnostics in perinatal infections. Marburg: Med Verlagsges 1984: 174.

12. IgE, allergies and helminth parasites: a new perspective on an old conundrum. Immunology and Cell Biology 1996; 74: 337–45.

13. Renner ED, Belohradsky BH, Grimbacher B. Hyper-IgE-Syndrom. Monatsschr Kinderheilkd 2002; 150: 1186–79.

14. Einsele H, Saal JG, Dopfner R, et al. Hochmalignes Non-Hodgkin-Lymphom bei Hyper-IgE-Syndrom. Dtsch Med Wschr 1990; 115: 1141–4.

15. Heremans JF, Masson PL. Specific analysis of immunoglobulins. Techniques and clinical value. Clin Chem 1973; 19: 294–8.

18.10 Immunoglobulin G subclasses

Lothar Thomas

Immunoglobulin G (IgG) accounts for about 10–20% of plasma proteins. IgG can be further divided in 4 subclasses, named, in order of decreasing abundance IgG1, IgG2, IgG3, and IgG4. Although they are more than 90% identical on the amino acid level, each subclass has a unique profile with respect to antigen binding, immune complex formation, complement activation triggering of effector cells, half-life, and placental transport /12/. Differences in the properties are presented in Tab. 18.10-1 – Properties of the IgG subclasses :

18.10.1 Indication

  • Suspicion of a defective immune response in patients with frequent infections
  • Monitoring of immunotherapy with inhalative antigens
  • Suspicion and monitoring of IgG4-related disease

18.10.2 Specimen

Serum, plasma (heparin, EDTA), body fluids (cerebrospinal fluid, bronchoalveolar lavage): 1 mL

18.10.3 Method of determination

The most common method measuring IgG subclasses is immunonephelometry There are two major vendors. Some laboratories use the IgG subclasses LC-MS/MS method. The methods are calibrated to the international reference material ERM-DA470K.

18.10.4 Reference interval

Refer to Ref. /34/ and Tab. 18.10-2 – IgG subclass reference intervals.

18.10.5 Clinical significance

Quantitation of the amount of each IgG subclass in a given serum sample allows identification of selective IgG subclass deficiencies and IgG subclass increase i.e., IgG4 related disease.

18.10.5.1 IgG subclass deficiency

IgG subclass deficiency is present if the concentration of one or several IgG subclasses decreases below the age-related reference interval values. In childhood, IgG subclass deficiencies are three times more common in boys than in girls. This changes during puberty so that in adults the female/male ratio is 2 : 4. In children, IgG2 deficiency occurs most commonly, while in adults IgG1 and IgG3 deficiencies are the most common /5/.

Most patients with IgG subclass deficiency suffer from frequent respiratory tract infections /6/. Therefore, IgG subclass analysis is part of the routine diagnostic examination in patients who are susceptible to respiratory tract infections. The most important diseases associated with IgG subclass deficiency are described in Tab. 18.10-3– Diseases commonly associated with IgG subclass deficiency. IgG subclass deficiencies may occur in an isolated form or associated with other immune defects (IgA, IgM, IgG, complement deficiencies, and T-cell defects, ataxia teleangiectatica).

More commonly, a certain Gm phenotype is associated with low IgG subclass concentrations. Individuals who are homozygous for G3m(21) have very low IgG3 concentrations /7/. G2m(23)-negative individuals not only have low IgG2 levels but, after vaccination with polysaccharides (pneumococci), also show poorer vaccination response as compared to heterozygote carriers of this defect /8/. Occasionally, familial clusters are encountered.

In many cases, decreased IgG subclass concentrations may be clinically manifest as infectious diseases and may also occur secondarily after therapy with steroids, sulfasalazine and carbamazepine /910/.

As in IgA deficiency, many patients with IgG subclass deficiency are healthy. Especially in children, a temporary decrease (delayed maturation) of IgG2 is often present /11/. For diseases associated with IgG subclass deficiency, see Tab. 18.10-4 – IgG subclass deficiency associated diseases.

In many cases, the cause for IgG subclass deficiency is a regulatory defect in the immune response. In some patients with IgG2 deficiency, an impaired synthesis of interferon has been described /12/. Deletions on chromosome 14 have been found but in very rare cases in the area of the gene cluster encoding the constant region of the H chain /13/.

Further testing to characterize the immune deficiency

IgG subclass deficiency is primarily considered to be an indicator of impaired immune response /67/. Further, more detailed examinations are required in order to characterize the underlying immune defect and to estimate its clinical relevance /14/.

For instance in patients with IgG subclass deficiency:

  • The synthesis of specific antibodies to proteins (tetanus, diphtheria) is usually not affected
  • The production of polysaccharide-specific antibodies (e.g., pneumococcal antigen) is reduced in some patients. Detection of naturally acquired polysaccharide antibodies is not very conclusive for the identification of patients with an actual specific immune defect. Vaccination against pneumococci is recommended.

Assessment of functional activity of the immune system following pneumococcal vaccination

An adequate pneumococcal vaccination response is present if the pneumococcal antibody titer in the global test (ELISA, 23-valent vaccine as antigen) rises to > 1,000 U/mL 4–6 weeks after the vaccination or if a significant vaccination response (> 1 μg/mL, calibrated according to the WHO standard 89 SF) is detectable involving five examined pneumococcal serotypes /15/. An adequate vaccination response is detectable in the majority of the patients with IgG2 subclass deficiency /1617/. If a vaccination response < 500 U/mL is measured in the global test, no significant vaccination response to any of the individual serotypes is usually detectable. If a titer < 500 U/mL is confirmed by repeat vaccination, relevant IgG subclass deficiency is diagnosed including a defect of polysaccharide-specific immunity. Depending on the clinical presentation, such a disorder necessitates prolonged therapy (antibiotic prophylaxis or immunoglobulin replacement therapy).

18.10.5.2 Increases in IgG subclasses

Abnormally increased IgG subclasses are common in chronic antigen stimulation, for instance, patients with infections. For further information, see Tab. 18.10-5 – Increases in IgG subclasses.

18.10.5.3 IgG4-related disease

IgG4-related disease (IgG4-RD) was described originally in the pancreas as sclerosing pancreatitis, now referred to as type 1 IgG4-related autoimmune pancreatitis (AIP). Shortly thereafter, however, the identification of a variety of extra-pancreatic organ involvement linked by unique histopathological features led to the recognition that AIP was part of a systemic condition /1819/.

The concept of IgG4-RD encompasses a wide range of organs sharing two characteristics /20/:

  • A set of unique histopathological features. The hallmarks are lymphoplasmacytic infiltrate, storiform fibrosis, obliterative phlebitis, and mild to moderate tissue eosinophilia.
  • Elevations in serum IgG4 concentrations.

IgG4-RD can involve almost any organ e.g., the pancreas biliary tree, salivary glands, periorbital tissues, kidneys, lungs, lymph nodes, meninges, aorta, breast, prostate thyroid, pericardium, and skin /20/.

There is no evidence that the IgG4-autoantibodies described so far in IgG4-RD contribute directly to pathogenesis. The role of IgG4 itself in the disease process remains unclear. IgG4-RD tends to form tumefactive lesions. As a result, patients are often suspected of having malignancy.

IgG4-RD presents in a subacute fashion in most patients, without rapid onset of constitutional symptoms such as fever. The clinical presentation is usually indolent, with signs and symptoms becoming evident over months or even years. High spiking fevers and other manifestations of systemic inflammation that mimic infections are classically absent. IgG4-RD typically comes to medical attention because of single-organ involvement, but more widespread disease is often observed following a detailed workup. Involvement by IgG4-RD of different organs can occur either simultaneously or metachronously, with the emergence of one newly affected organ following another. IgG4-RD has a predilection for middle-aged to elderly men /1820/.

Diagnosis of IgG4-RD

Diagnosis of IgG4-RD requires both histopathological confirmation and clinicopathological correlation. Serological findings are largely non-specific. Acute phase reactants such as erythrocyte sedimentation rate and C-reactive protein are usually elevated to a moderate degree. Peripheral blood eosinophilia and increased serum IgE occur in almost 30% of patients. Some patients have positive low-titre anti-nuclear antibodies. Although serial measurement of serum IgG4 is often useful in the assessment of disease activity, they should never be used as the sole determinant of treatment decisions /18/.

High serum IgG4 concentrations occur in 60–70% of patients, typically in those with multi-organ involvement. Unfortunately, elevation of IgG4 can be associated with conditions other than IgG4-RD e.g., systemic vasculitides, connective tissue disease, infections and malignancies /21/. Some IgG4-RD patients have normal serum IgG4 concentrations despite histopathologic and immunohistochemical findings in tissue /20/.

According to Japanese study groups a definitive diagnosis of IgG4-RD is obtained if the patient fulfills the following criteria /2223/:

  • Elevated serum IgG4 > 135 mg/dL
  • Histopathology: IgG4+/IgG+ cell ratio > 0.4
  • Swelling or damage of affected organ
  • Histopathology: more than 10 IgG4-positive cells per high power field
  • Even in the absence of elevated serum IgG4, as long as there is organ involvement plus more than 10 IgG4-positive cells per high power field, and the IgG4+/IgG+ cell ratio > 0.4, a diagnosis of IgG4-RD may still be made.

18.10.6 Comments and problems

The sum of the individual IgG subclasses should not deviate by more than 10% from the measured total IgG (plausibility check). If this is not the case, in an abnormal distribution of the IgG subclasses, a control of both measured parameters (IgG subclasses and total IgG) should be performed.

Method of determination

LC-MS/MS should be the preferred method for measurement of IgG subclasses. In a study /24/, using immunonephelometric IgG subclass reagents analytic errors were noted in patients with increases in IgG4. The sum of the individual IgG subclasses was substantially greater than the measured total IgG concentrations, and the IgG4 concentration was always less than the IgG2 concentration. Using a tryptic digest LC-MS/MS method as reference to quantify the IgG subclasses biases of the immunonephelometric measurements compared with the correspondent LC-MS/MS measurements was observed. The biases potentially reflect two analytical phenomena caused by immunonephelometry:

  • Cross reactivity of sample IgG4 with IgG2 reagents
  • Measurement of IgG1 and IgG2 that represent an aggregate of the target Ig and nonspecifically IgG4 (IgG4 bound to either the target Ig or the reagent Ig).

In each case, these proposed phenomena would explain the observation of IgG4-dependent positive biases with immunonephelometric IgG1 and IgG2 concentration measurements as compared with the corresponding LC-MS/MS measurements /24/.

Using immunonephelometric methods contaminations inducing light-scattering interfere with this method (e.g., micro clots, cells from inadequately centrifuged samples, particles derived from proteins of the cerebrospinal fluid and microbial contaminations). In principle, problems should be anticipated in samples after deep-freezing or in hyperlipidemic samples.

Reference intervals of IgG subclasses

The reference intervals of the IgG subclasses for children are age-dependent. By 6 months of age, IgG1 and IgG3 concentrations are about 50% of the adult level which is reached by 3 years of age. IgG2 and IgG4 are produced in a delayed fashion; during the 1st year of life, their concentrations are 25% and during the 3rd year of life 50% of the adult level.

Subclass assays yield different results. Whereas the reference intervals for IgG1 and IgG2 from Siemens and The Binding Site are similar /25/, there is a marked difference in those for IgG3 and IgG4. This is due to the fact that the standards established by The Binding Site are based on the material ERM-DA470k, while those established by Siemens are based on WHO 67/97, later replaced by Sanquin M1590. Hence, the reference intervals of a given manufacturer should be used consistently in a given case.

18.10.7 Pathophysiology

The function of the 4 IgG subclasses is to eliminate invading pathogens and their products. The Ig structure is adapted to these functions. Ig have the following functional units:

  • A Fab portion with a variable region for antigen detection
  • An Fc portion mediating the effects of the molecule. The Fc portion binds complement and reacts with Fcγ -receptors on the surface of the defense cells such as polymorphonuclear granulocytes and monocytes/macrophages. This results in the inactivation and/or elimination of the Ig-bound antigen. The fact that 4 IgG subclasses do not have a uniform Fc portion causes their functional differences /12/.

The four IgG subclasses have the following functional differences (Tab. 18.10-1 – Physicochemical and biological properties of the IgG subclasses).

IgG1

Antibody response to membrane proteins and soluble proteins (T cell-dependent antigens such as, for example, viral and bacterial antigens) primarily induce IgG1 and with lower levels IgG3 and IgG4. IgG antibodies against proteins (tetanus toxin) and viral antigens are predominantly IgG1 and IgG3. IgG1 deficiency is associated with recurrent infections. IgG1 reaches an adult serum level at the age of 1–4 years; the other Ig subclasses reach approximately 50% of the adult level at this age. Adult levels are finally reached in the adolescent period.

IgG2

T cell independent antigens like the polysaccharide capsule of H. influenzae and S. pneumonia lead mostly to an IgG2-restricted antibody response. Antigens are pneumococcal antigen, teichoic acids, dextran HIB-PrP. Low concentrations of IgG2 often occur in association with a deficiency in IgG4 and/or IgA1 and IgA2.

IgG3

Being a potent pro-inflammatory antibody IgG3 is particularly effective in the induction of effector functions. Decreased IgG3 levels are frequently associated with other subclass deficiencies.

IgG4

Allergens are often good inducers of IgG4 and IgG1 in addition to IgE. In a non-infectious setting IgG4 antibodies are often formed following repeated or long term exposure to antigens. Allergen-specific antigens (bees’ venom) under conditions of hyposensibilization stimulate antibody production primarily in the IgG4 subclass. Helminth or filarial parasite infections may result in the formation of IgG4 antibodies.

IgG4-RD is a chronic inflammatory condition that affects almost any organ in the same patient. Molecular mimicry has been proposed to play a role. The target antigens for these IgG4 antibodies seem evolutionarily conserved, because purified patient IgG4, but not IgG4 from healthy donors are deposited at the base of acini or pancreatic duct cells /23/.

References

1. Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Frontiers in immunology 2014; 5: article 520.

2. Maguire GA, Kumararatne DS, Joyce HJ. Are there any clinical indications for measuring IgG subclasses? Ann Clin Biochem 2002; 39: 374–7.

3. Schauer U, Stemberg F, Rieger CHL, Borte M, Schubert S, Riedel F, et al. IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the Binding Site reagents. Clin Chem 2003; 49: 1924–9.

4. Lepage N, Huang SHS, Nieuwenhuys E, Filler G. Pediatric reference intervals for immunoglobulin G and its subclasses with Siemens immunonephelometric assays. Clin Biochem 2010; 43: 694–6.

5. Herrod HG. Clinical significance of IgG subclasses. Curr Opin Pediatr 1993; 5: 696–9.

6. Oxelius VA, Carlsson AM, Hammarstrom L, Bjorkander J, Hanson LA. Linkage of IgA deficiency to Gm allotypes; the influence of Gm allotypes on IgA-IgG subclass deficiency. Clin Exp Immunol 1995; 99: 211–5.

7. Preud’homme JL, Hanson LA. IgG subclass deficiency. In: Rosen FS, Seligman M, eds. Immunodeficiencies. Chur; Harwood 1993: 61–76.

8. Sarvas H, Rautonen N, Käyhty H, Kallio M, Mäkelä O. Effect of Gm allotypes on IgG2 antibody responses and IgG2 concentrations in children and adults. Int Immunol 1990; 4: 317–22.

9. Ito J, Nakagawa T, Morita Y, Okudaira H. Immunologic analysis of steroid-dependent asthma. Annals of Allergy 1989; 62: 15–20.

10. Leickly FE, Buckley RH. Development of IgA and IgG2 subclass deficiency after sulfasalazine therapy. J Pediatr 1986; 108: 481–2.

11. Zielen S, Ahrens P, Kotitschke R, et al. IgG-Subklassenspiegel bei gesunden Kindern. Monatsschr Kinderheilkd 1990; 138: 377–80.

12. Inoue R, Kondo N, Kobayashi Y, Fukutomi O, Orii T. IgG2 deficiency associated with defects in production of interferon-gamma; comparison with common variable immunodeficiency. Scand J Immunol 1995; 41: 130–4.

13. Plebani A, Ugazio AG, Meini A, et al. Extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is IgG subclass deficiency clinically relevant? Clin Immunol Immunopathol 1993; 68: 46–50.

14. Warshaw BL, Check IJ. IgG-subclasses in children with nephrotic syndrome. Am J Clin Pathol 1989; 92: 68–72.

15. Rijkers GT, Sanders LAM, Zegers BJM. Anticapsular polysaccharide antibody deficiency states. Immunodeficiency 1993; 5: 1–21.

16. Gross S, Blaiss MS, Herrod HG. Role of immunoglobulin subclasses and specific antibody determinations in the evaluation of recurrent infection in children. J Pediatr 1992; 121: 516–22.

17. Macdermott RP, Nash GS, Auer IO, Shlien R, Lewis BS, Madassery J, Nahm MH. Alterations in serum immunoglobulin G subclasses in patients with ulcerative colitis and Crohn’s disease. Gastroenterol 1989; 96: 764–8.

18. Della-Torre E, Lanzilotta M, Doglioni C. Immunology of IgG4-related disease. Clinical and Experimental Immunology 2015; 181: 191–206.

19. Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med 2012; 366: 539–50.

20. Mahajan VS, Mattoo H, Deshpande V, Pillai SS, Stone JH. IgG4-related disease. Annu Rev Pathol Mech Dis 2014; 9: 315–47.

21. Carruthers MN, Khosroshahi A, Augustin T, Despande V, Stone JH. The diagnostic utility of serum IgG4 concentrations in IgG4-related disease. Ann Rheum Dis 2014; 74: 14–8.

22. Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, Saeki T, et al. Comprehensive diagnostic criteria for IgG4 related disease (IgG4-RD), 2011. Mod Rheumatol 2012; 22: 21–30.

23. Kawa S, Sköld M, Ramsden DB, Parker A, Harding SJ. Serum IgG4 concentration in IgG4-related disease. Clin Lab 2017; 63. 1323–37.

24. Van den Gugten G, Demarco ML, Chen LYC, Chin A, Carruthers M, Holmes DT, Mattman A. Resolution of spurious immunonephelometric IgG subclass measurement discrepancies by LC-MS/MS. Clin Chem 2018; 64: 735–42.

25. Wilson C, Ebling R, Henig C, Adler T, Nicolaevski R, Barak M, et al. Significant, quantifiable differences exist between IgG subclass standards WHO 67/97 and ERMDA470k and can result in different interpretation results. Clin Biochem 2013; 46: 1751–5.

26. Bartmann P. IgG-Subklassenmangel. Allergologie 1990; 13: 398–403.

27. French MA, Denis KA, Dawkins R, Peter JB. Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG2 and/or IgG4. Clin Exp Immunol 1995; 100: 47–53.

28. Buckley RH. Immunoglobulin G subclass deficiency: fact or fancy? Current Allergy and Asthma Reports 2002; 2: 356–60

29. Oehling AK, Sanz ML, Resano A. Importance of IgG4 determination in in vitro immunotherapy follow-up of inhalant allergens. Invest Allergol Clin Immunol 1998; 8: 333–9.

30. Hamano H, Shigeyuki K, Horiuchi A, Unno H, Naoyuki F, Akamatsu T, et al. High serum IgG concentrations in patients with sclerosing pancreatitis. N Engl J Med 2001; 344: 732–8.

31. Detlefsen S, Drewes AM. Autoimmune pancreatitis. Scand J Gastroenterol 2009; 44: 1391–1407.

32. Nishimori I, Otsuku M. Autoimmune pancreatitis and IgG4-associated sclerosing cholangitis. Best Practice & Research Clin Gastroenterol 2009; 23: 11–23.

18.11 Cryoglobulins, cryofibrinogen

Lothar Thomas

Cold-precipitable proteins

This group of proteins includes cryoglobulins, cryofibrinogen and fibronectin containing complexes /1/.Cold-precipitable proteins must not be confused with cold agglutinins. Cold agglutinins belong to the IgM class and are capable of agglutination and hemolysis of erythrocytes via complement activation (cryohemolysin).

Cryoglobulins

Cryoglobilins are immunoglobulins or immunoglobulin complexes that precipitate at temperatures below 37 °C and redissolve on rewarming (Tab. 18.11-1 – Types of cryoglobulins).

Cryoglobulins have long been classified as /23/

  • Type I (monoclonal). This type represents individual monoclonal immunoglobulins, generally associated with lymphoproliferative diseases.
  • Type II (mixed type). Type II consists of mixed immunoglobulins with a monoclonal component and is strongly associated with hepatitis C infection.
  • Type III (polyclonal). Type III is constituted by a mixture of polyclonal immunoglobulins and is associated mainly with a wide range of infectious, autoimmune, and liver disease.

Cryofibrinogen

Cryofibrinogen is a complex of fibrin, fibrinogen and fibrin cleavage products with albumin, immunoglobulins and other proteins that become insoluble at cold temperatures. Cryofibrinogen forms a clot with thrombin at 4 °C that dissolves again at 37 °C.

Fibronectin complexes

Fibrinonectin and C-reactive protein-albumin complexes. These complexes are of no pathophysiological significance.

Cold agglutinins

Contrary to cryoglobulins cold agglutinins are capable of agglutination and hemolysis of erythrocytes via complement activation (cryohemolysin). Cold agglutinins belong to the IgM class.

18.11.1 Indication

Suspected cryoglobulinemia

In the presence of the following symptoms and/or conditions/diseases: purpura, symptom complex consisting of purpura, weakness, arthralgia, neurological disorders, glomerulonephritis, Raynaud’s phenomenon, arthritis, sicca syndrome, chronic hepatitis C /4/.

Suspected cryofibrinogenemia

In the presence of the following symptoms and/or conditions/diseases: cutaneous ischemia such as purpura, livido reticularis, ecchymosis, ulceration, ischemic necrosis and gangrene (rare).

18.11.2 Method of determination

Detection and quantification of cryoglobulins and cryofibrinogen by the precipitation method

Cryoprecipitation of cryoglobulins is detected in serum, cryoprecipitation of cryofibrinogen and cryoglobulins is detected in plasma.

Principle: traditionally cryoprecipitation is detected and quantified in vitro by incubating serum samples at 4 °C for 2–15 days and the result of the assay is reported as cryocrit %, which is the percent of the precipitate in respect to the total serum value /5/.

Cryoglobulin and cryofibrinogen is detected and quantified in vitro by incubation of plasma samples at 4 °C for 2–15 days and the result of the assay is reported as cryocrit. The proportion of precipitate in relation to the serum/plasma volume is assessed (Tab. 18.11-2 – Measurement and differentiation of cryoglobulins).

Procedure:

  • For the determination of cryoglobulins, a prewarmed tube is filled with serum and incubated at temperatures of 4 °C for 2 to 7 days
  • For the determination of cryoglobulin and cryofibrinogen, plasma is incubated into a second tube at temperatures of 4 °C for 2 to 7 days
  • The same amount of precipitate in the serum and plasma tubes is based on cryoglobulins
  • Precipitate only in the plasma tube points to cryofibrinogen and usually has a concentration > 1 g/L; lower concentrations become visible after centrifugation /6/. Determination of cryofibrinogen is confirmed by warming the content of the tube to 37 °C.

If precipitation is detected in the plasma and/or serum tubes, the tubes are centrifuged. The quantitative determination of cryoprecipitates is performed either as cryocrit in % or by measuring the protein concentration of the washed precipitate using the Biuret method. Monoclonal cryoglobulins (type I) and mixed type II cryoglobulins precipitate at 4 °C usually within 24 hours. Mixed cryoglobulins (types III) often require more than 72 hours and are often barely visible upon precipitation. Cryofibrinogen forms a clot within 72 hours. The precipitate of cryoglobulins can be floccular, gelatinous or crystalline. A gelatinous form of precipitate in the serum after blood collection often points to cryoglobulinemia type I.

Differentiation of precipitate cryoglobulins

The goal of cryoglobulin differentiation is their classification into types I–III. The cryoglobulin type allows conclusions regarding etiopathogenesis and, thus, facilitates the interpretation of clinical symptoms.

Procedure: after removal of the supernatant the precipitate is washed three times with cold physiological NaCl by centrifugation and resuspension. Finally, the precipitate is dissolved in warm 0.9% NaCl for further processing. The precipitated components are identified by immunofixation electrophoresis using specific antisera (e.g., antihuman serum, anti-Ig/kappa, anti-Ig/lambda, anti-Ig/α-chain, anti-Ig/μ-chain, anti-Ig/γ-chain). A lane without antiserum should be run as a sample blank in order to ensure that the proteins do not precipitate at the application site of the cold gel.

Spectrophotometric detection of cryoglobulins

Two methods are published:

  • An assay was described /7/ based on the spectrophotometric detection of a difference in optical density between two aliquots of serum sample incubated at 4 °C and 37 °C respectively.
  • In a second study /2/ the increase of turbidity during the cryoglobulin aggregation was determined. Measurements were performed using a dual-beam spectrophotometer with a cell whose temperature was maintained at 10 °C.

18.11.3 Specimen

  • Whole blood without additive (cryoglobulins): 10 mL
  • EDTA, citrate, oxalate whole blood (cryofibrinogen and cryoglobulins): 10 mL

Blood is drawn by venipuncture in two pre warmed tubes at 37 °C and left for 2 h (clotting of the serum). Serum and plasma are separated at 37 °C by centrifugation (2,500 × g for 10 min at 37 °C). An aliquot of each sample is transferred in a graduated tube for precipitation.

18.11.4 Reference interval

Cryocrit /9/

< 0.4%

Protein concentration of the cryoglobulins /8/

< 80 mg/L

Protein concentration of cryofibrinogen /8/

< 60 mg/L

18.11.5 Clinical significance

The incidence of cryoglobulinemia is much higher than that of cryofibrinogenemia. For instance, in the Sheffield Protein Reference Unit, 887 samples were analyzed for cryoglobulinemia in 2004–2008 /9/. As a result of the analysis, 188 cryoglobulinemias and 5 cryofibrinogenemias were detected. The prevalence of types is listed in Tab. 18.11-3 – Cryoglobulinemias at the Sheffield Protein Reference Unit.

Negative evidence of cryoglobulin does not exclude cryoglobulinemia because, in many cases, improper handling of the samples yields false-negative results. The concentration of C4 is a surrogate marker. Low concentrations can point to cryoglobulin-induced immune complex activity; normal concentrations do not allow any conclusions. About 16–70% of patients with chronic hepatitis C and cryoglobulinemia have positive rheumatoid factor /10/.

18.11.5.1 Cryoglobulinemia

Cryoglobulin-induced diseases are associated with immunoglobulin precipitation during cold exposure. Physiologically, small amounts of cryoglobulins are produced and eliminated by the hepatocytes via a specific receptor. Cryoglobulin deposits in the glomeruli are removed by mono­cytes/macro­phages /11/. Precipitation of cryoglobulins in the vessels causes the development of vasculitis, especially in the skin, kidneys and peripheral nervous system. Clinical manifestation of cryoglobulinemia depends on the organs affected. Histologically, a leucoclastic vasculitis secondary to vascular deposition of immune complexes is seen /3/. This leads to ischemia, necrosis and purpura. About 50–70% of symptomatic patients with cryoglobulinemia have liver involvement, arthralgia and asthenia, and about 25% have renal involvement. The incidence of nervous system involvement is 36%.

There is a lack of correlation between serum cryoglobulin concentration, and severity of disease. Many patients have cryoglobulinemia without symptoms even when serum cryoglobulin levels are high /12/. Cryoglobulins in most of the patients exist in low concentration (100–300 mg/L).

Cryoglobulins undergo reversible condensation upon changing temperature and concentration. Various morphologies of IgG cryoglobulin condensates (e.g., including crystals, amorphous aggregates and gels) exist in different patients /13/.

Mixed cryoglobulinemia, an autoimmune disease characterized by the formation of cold-precipitable cryoglobulin complexes composed of immunoglobulins, is recognized as the most common extrahepatic disease induced by hepatitis C infection. Based on the immunoglobulin composition mixed cryoglobulinemia has two types of immunoglobulins: type II cryoglobulins consisting of polyclonal IgG and monoclonal IgM with rheumatoid factor (RF) activity and type III cryoglobulins characterized by polyclonal IgG with polyclonal IgM /15/. Mixed type II and polyclonal type III are generally associated with diseases. Approximately 95% of cryoglobulins are immune complexes that contain rheumatoid factor. Usually the RF is monoclonal or polyclonal IgM, although other immunoglobulins may be found. Type II and type III cryoglobulins account for about 50–60% and 25–30%, respectively. Mixed type II is generally associated with diseases where there is a chronic infection such as HCV, chronic hepatitis B or human immunodeficiency virus infection, and in autoimmune diseases such as Sjögren syndrome. More than 90% of patients with mixed type II cryoglobulinemia are infected with hepatitis C virus /14/.

In HCV-related mixed cryoglobulinemia, the majority of patients have asymptomatic cryoglobulinemia, and only 10–15% of these patients will develop cryoglobulinemic symptoms characterized by small vessel vasculitis, glomerulonephritis, and neuropathy due to immune complex deposition and activation of the complement cascade in small blood vessels. The prevalence of cryoglobulin is 2–4 times higher in HCV than in HBV /14/.

A differentiation is made between monoclonal and mixed forms of cryoglobulinemia. The mixed forms (type II and type III) account for up to 90% of cryoglobulinemias, the monoclonal form (type I) accounts for approximately 10%. About 95% of the mixed forms are based on chronic hepatitis C, the remaining forms are of different nature.

18.11.5.1.1 Monoclonal cryoglobulinemia (type I)

Monoclonal cryoglubilinemia is usually associated with plasma cell dyscrasia such as multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstroems disease and accounts for 10% of cryoglobulins.

Type I cryoglobulins precipitate at higher temperatures (≤ 32 °C) than mixed cryoglobulins. Concentrations can range between 60 mg/L and 60 g/L.

Clinical manifestations are hyper viscosity syndrome with symptoms of peripheral vascular obstruction, purpura or dermal manifestation (Raynaud’s phenomenon). Type I is rarely associated with vasculitis. Joint manifestations are seen if the cryoglobulin (usually IgG3) precipitates in crystallized form.

Laboratory findings

The cryocrit is usually high and protein concentrations are > 1 g/L in many cases. Examination for evidence of monoclonal IgG or IgM. The rheumatoid factor is negative, complement decreases are not continuously detectable.

18.11.5.1.2 Mixed cryoglobulinemias (types II and III)

Mixed cryoglobulinemia, an autoimmune disease characterized by the formation of cold-precipitable cryoglobulin complexes composed of immunoglobulins, is recognized as the most common extrahepatic disease induced by hepatitis C infection.

Based on the Ig composition mixed cryoglobulinemia has two types of immunoglobulins /15/:

  • Type II cryoglobulins consisting of polyclonal IgG and monoclonal IgM with rheumatoid factor (RF) activity
  • Type III cryoglobulins characterized by polyclonal IgG with polyclonal IgM.

Mixed type II and polyclonal type III are associated with diseases. Approximately 95% of cryoglobulins contain rheumatoid factor (RF). Type II and type III cryoglobulins account for about 50–60% and 25–30%, respectively. Mixed type II is generally associated with diseases where there is a chronic infection such as hepatitis C, chronic hepatitis B or human immunodeficiency virus (HIV) infection, and in autoimmune diseases such as Sjögren syndrome. A shared feature of these disorders is chronic inflammation, high antigen load and antigen-driven dysregulation of the B-cell system /16/. More than 90% of patients with mixed type II cryoglobulinemia are infected with hepatitis C virus /14/. According to a study /15/ higher frequencies of TH1 cells and activated memory B cells were associated with type II asymptomatic mixed cryoglobulinemia in HCV infection.

In HCV-related mixed cryoglobulinemia, the majority of patients have asymptomatic cryoglobulinemia, and only 10–15% of these patients will develop cryoglobulinemic symptoms characterized by small vessel vasculitis, glomerulonephritis, and neuropathy due to immune complex deposition and activation of the complement cascade in small blood vessels.

If the RF is monoclonal the mixed cryoglobulinemia is referred to as type II. If the RF is polyclonal, the cryoglobulinemia is referred to as type III. Infections can cause either type II or type III cryoglobulinemia. Associated diseases with type III cryoglobulinemia are post streptococcal glomerulonephritis, chronic infections and essential cryoglobulinemia /17/.

Diseases and symptoms associated with mixed cryoglobulins are listed in Tab. 18.11-4 – Diseases and conditions associated with mixed cryoglobulinemia.

Laboratory findings

As a rule, mixed cryoglobulins precipitate at temperatures ≤ 23 °C. Positive evidence of rheumatoid factor in type II, complement decrease is continuously detectable.

18.11.5.1.3 Heat-insoluble cryoglobulinemia

In the presence of this cryoglobulinemia, the precipitated protein dissolves at temperatures higher than 37 °C, for example at 56 °C. These forms of cryoglobulinemia are rare but have been described in association with multiple myeloma, Sjögren’s syndrome and cryoglobulin-occlusive membranoproliferative glomerulonephritis /11/.

18.11.5.2 Cryofibrinogenemia

Cryofibrinogenemia is diagnosed when EDTA plasma that is clear at 37 °C develops a precipitate when it is cooled to 4 °C. Cryofibrinogenemia is subdivided into an essential (primary) and a secondary form /18/. The primary form is rare. The prevalence of secondary cryofibrinogenemia without clinical symptoms is said to be 13% in hospitalized patients. Primary cryofibrinogenemia often develops spontaneously in healthy individuals, while secondary cryofibrinogenemia is associated with an underlying chronic inflammatory disorder in the context of a malignant tumor, diabetes mellitus, inflammation, collagen vascular disease or active infection. The female/male ratio is 1.4 : 1.

The main clinical symptoms in patients with cryofibrinogenemia are cold intolerance, cutaneous ischemia such as purpura, livido reticularis and acral skin ulcerations. These patients live in colder climate zones and report a temporal association between cold exposure and the onset of symptoms.

According to one of the few cases described to date, the laboratory-diagnostic findings in essential cryofibrinogenemia included a cryocrit of 5% and a protein concentration of the cryoprecipitate of 850 mg/L. The cryofibrinogen precipitate must be distinguished from the heparin-precipitable fraction, which also forms in normal individuals during cold exposure if heparinized plasma is used instead of EDTA plasma /19/.

18.11.6 Comments and problems

Temperature control

Temperature control is particularly important in the preanalytical phase of type I cryoglobulins because these cryoglobulins precipitate at higher temperatures and can also be present at concentrations > 5 g/L. High concentrations of monoclonal cryoglobulins tend to precipitate earlier at higher temperatures. For type III cryoglobulins, temperature control is not crucial because they slowly precipitate over a period of days /9/.

Cryoglobulin detection using the precipitate method is due to false negative results because the standard pre analytical procedures to collect blood (tube preheating, transport in container, sedimentation, and/or centrifugation at 37 °C ) are not exist in many laboratories /4/.

Cryoprecipitate

Several components can be found in cryoglobulin precipitates in addition to immunoglobulins (e.g., rheumatoid factor, albumin, fibrinogen, fibronectin viruses and bacteria) /18/.

Washing of the cryoprecipitate

The cryoprecipitate must be washed at cold temperature prior to protein determination to remove contamination by other serum proteins.

Cryoproteins

Cryoproteins are differentiated as follows:

  • Cryoglobulins
  • Cryofibrinogen is a complex of fibrin, fibrinogen and fibrin cleavage products with albumin, immunoglobulins and other proteins that becomes insoluble at cold temperatures. Cryofibrinogen forms a clot with thrombin at 4 °C that dissolves again at 37 °C.
  • Fibrinonectin and C-reactive protein-albumin complexes. These complexes are of no medical significance.

References

1. Merlini G, Aguzzi F, Whicher JT, Chir B, Navolotskaia O. Cryoglobulins. In: Ritchie RF, Navolotskaia O, eds. Serum proteins in clinical medicine. Scarborough: Foundation for Blood Research, 1996: 11.05-1–05.

2. Romitelli F, Pucillo LP, Basile U, Di Stasio E. Comparison between the traditional and a rapid screning test for cryoimmunoglobulins detection. Biomed Res Int 2015; Article ID 783063

3. Voma CB, Levinson SS. Analysis, detection and quantitation of mixed cryoglobulins in HCV infection. Brief review and case examples. Clin Chem Lab Med 2016; 54: 1853–9.

4. Brouet JC, Clauvel JP, Danon F, Klein M, Seligman F. Biologic and clinical significance of cryoglobulins. Am J Med 1974; 57: 775–88.

5. Vermeersch P, Gijbels K, Marien G, Lunn R, Egner W, White P, Bossuyt X. A critical appraisal of current practice in the detection, analysis and reporting of cryoglobulins. Clin Chem 2008; 54: 39–43.

6. Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004; 116: 332–7

7. Kalovidouris AE, Johnson RL. Rapid cryoglobulin screening and aid to the clinician. Ann Rheumat Dis 1978; 37: 444–8.

8. Blain H, Cacoub P, Musset L, et al. Cryofibrinogenemia: a study of 49 patients. Cin Exp Immunol 2000; 120: 253–60.

9. Sargur R, White P, Egner W. Cryoglobulin evaluation: best practice? Ann Clin Biochem 2010; 47: 8–16.

10. Cicardi M, Cesana B, Del Nino E, et al. Prevalence and risk factors for the presence of serum cryoglobulins in patients with chronic hepatitis C. J Viral Hepat 2000; 7: 138–43.

11. Meng QH, Chibbar R, Pearson D, Kappel J, Krahn J. Heat insoluble cryoglobulin in a patient with essential type II cryoglobulinemia and cryoglobulin-occlusive membranoproliferative glomerulonephritis: case report and literature review. Clin Chim Acta 2009, Aug 406; 170–3.

12. Cesur S, Akin K, Kurt H. The significance of cryoglobulinemia in patients with hepatitis B and C virus infection. Hepatogastroenterology 2003, 50: 1487–9.

13 Wang Y, Lomakin A, Hideshima T, Laubach JP, Ogun O,Richarson PG, et al. Pathological crystallization of human immunoglobulins. PNAS 2012; 109: 13359–61.

14. Minopetrou M, Hadziyannis E, Deutsch M, Tampaki M, Georgiadou A, Dimopoulou E, et al. Hepatitis C virus (HCV) related cryoglobulinemia: cryoglobulin type and anti-HCV profile. Clin Vaccine Immunol 2013, 20. 698–703.

15. Kong F, Zhang W, Feng B, Zhang H, Rao H, Wang J, et al. Abnormal CD4+ T helper (Th) 1 cells and activated memory B cells are associated withtype III asymptomatic mixed cryoglobulinemia in HCV infection. Virology J 2015; 12: 100. https://doi.org/10.1186/s12985-015-0324-2.

16. Charles ED, Dustin LB. Hepatitis C virus-induced cryoglobulinemia. Kidney International 2009; 76: 818–24.

17. Agarwal A, Clements J, Sedmak DD, Imler D, Nahman NS Jr, Orsinelli DA, et al. Subacute bacterial endocarditis masquerading as type III essential mixed cryoglobulinemia. J Am Soc Nephrol 1997; 8: 1971–6.

18. Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004; 116: 332–7.

19. Smith RT. A heparin-precipitable fraction of human plasma. II. Occurence and significance of the fraction in normal individuals. J Clin Invest 1957; 36: 605–16.

20. Dammacco F, Sansonno D. Therapy for hepatitis C virus-related cryoglobulinemic vasculitis. N Engl J Med 2013; 369. 1035–45.

18.12 β2-microglobulin (β2-M)

Lothar Thomas

β2-M forms the non-variable light chain of the class I major histocompatibility complex (MHC) and is found on the surfaces of nearly all nucleated cells (Fig. 18.12-1 – HLA antigens on the cell membrane of nucleated cells). When MHC is degraded, the MHC associated β2-M is released into circulation. This results in a constant production rate of free β2-M of 2.4 mg/kg/day. β2-M is a non-glycosylated polypeptide with a molecular mass of 11.8 kDa. Due to its small size, β2-M is present in the glomerular filtrate of the kidney and other body fluids. Elevated β2-M serum levels are measured in chronic renal failure, lymphoproliferative disorders, conditions with high cell turnover, inflammations and infections.

18.12.1 Indication

  • Monitoring and assessment of therapy in lymphoid neoplasia, especially non Hodgkin lymphoma, Hodgkin’s lymphoma and multiple myeloma
  • Assessment of the glomerular filtration rate, especially in children
  • Diagnosis and monitoring in tubulointerstitial kidney damage
  • Monitoring of β2-M in dialysis patients
  • Assessment of renal function after kidney transplantation and early detection of Cytomegalovirus infection
  • Detection of a rejection episode after allogenic bone marrow transplantation
  • Monitoring of disease progression in patients with HIV infection
  • Diagnosis of fetal infections.

18.12.2 Method of determination

Immunoassay and immunonephelometric or immunoturbidimetric assays /1/.

18.12.3 Specimen

Serum, plasma: 1 mL

Random urine sample to which 0.5 mL of 2n NaOH is added in order to obtain a pH > 6. 10 mL of this urine sample need to be delivered to the clinical laboratory. It is used, for example, as part of occupational-medical examinations.

Urine collection, 6–8 h collection period; collection to be performed only during the daytime. This allows monitoring of the urine pH and, if necessary, alkalinization of the urine by adding 2n NaOH. The urine collection over the specified period of time is indicated for detection of acute, toxic injury to tubules.

18.12.4 Reference interval

Refer to References /2, 3, 4, 5, 6/ and Tab. 18.12-1 – Reference intervals for β2-microglobulin.

18.12.5 Clinical significance

The concentration of β2-M in serum and its excretion in urine provide valuable information only:

  • In the presence of specific clinical problems
  • If other diseases that may also have an impact on synthesis or excretion of β2-M, have been ruled out first. For example, if the glomerular filtration rate is to be estimated, no lymphoid neoplasia should be present or the presence of a tubulointerstitial renal disease should be ruled out when the therapy of a lymphoid neoplasia is to be assessed.

Refer to Tab. 18.12-2 – β2-microglobulin in various diseases

In the presence of constant serum levels, an acute rise in concentration or fractional urinary excretion of β2-M indicates tubular damage. In serum concentrations > 6 mg/L and normal GFR, the tubular reabsorption capacity is exceeded and the concentration in urine cannot be used as an indicator of tubular damage /7/. In such cases, it is better to determine the excretion of α1-microglobulin. Refer to Section 12.9.6.8.3 – α1-microglobulinuria. This is recommendable in general because α1-microglobulin in urine is more stable than β2-M.

18.12.6 Comments and problems

Stability

In the presence of a pH < 6.0, β2-M is denatured within a period of 2 hours /6/, even in the bladder. As a result of such degradation, it can no longer be immuno-chemically determined. Consequently, examination of the urine should not involve the first morning urine (usually with a pH < 6.0) but instead should employ a random daytime urine sample or a urine sample collected during the daytime. The pH must be checked after voiding and, if necessary, alkalinization should be performed by adding a few drops of 2n NaOH.

Reference interval

Children have slightly higher serum concentrations than adolescents and adults as well as individuals over the age of 60.

18.12.7 Pathophysiology

β2-M is a peptide of 100 amino acids. The peptide chain is linked via a disulfide bond between amino acids 25 and 81 (cysteine in each case) /25/.

β2-M is the light chain protein of the MHC class I antigens and thus present on the membrane of all nucleated cells. It is located on the outside of the cell membrane and is not covalently bound to the heavy chain of the MHC class I antigen. Thus, it is free to exchange with β2-M in the body fluids, where more than 98% are present in the form of a free monomer.

The expression of MHC class I antigens and, thus, of β2-M on the cell membranes of immune defense cells (especially of lymphocytes) is stimulated by cytokines of the hemolymphatic system. The β2-M production is increased in all diseases associated with activation of the immune system such as bacterial infections, certain viral infections and autoimmune diseases (e.g., rheumatoid arthritis). The main synthesis site for β2-M is lymphocytes. Healthy individuals with a body weight of 70 kg synthesize 9 mg of β2-M/hour. The half-life is 40 min. The kidney is the main elimination site and glomerular filtration is approximately 210 μg/min. and 99.9% of β2-M are reabsorbed in the proximal tubules.

Changes in serum β2-M level or β2-M excretion are caused by increased production, reduction in GFR or tubular reabsorption.

Since the lymphatic system is the main synthesis site of β2-M, all conditions with an increased proliferation rate of lymphocytes are associated with elevated serum concentrations; this applies, in particular, to multiple myeloma, Hodgkin’s lymphoma, chronic lymphocytic leukemia and other malignant non Hodgkin lymphomas. In these diseases, the determination of β2-M is a good indicator for monitoring the disease and assessing the treatment outcome. Monoclonal Ig level and serum β2-M concentration show a parallel course in about 70% of the patients. The β2-M concentration is not elevated in some patients despite marked monoclonal gammopathy. This is explained by changes in renal function in myeloma patients /26/. Other diseases associated with pronounced activation of the cellular immune response and elevated serum β2-M are certain autoimmune diseases, infectious mononucleosis, and transplant rejection episodes /8/.

A reduction in GFR prolongs the half-life of β2-M, and serum level increases exponentially i.e., is inversely related to GFR (this relationship applies to the entire filtration range). An increase in serum concentration may occur with a GFR decline to below 80 [mL × min–1 × (1.73 m2)–1]. Reports concerning a correlation between abnormal serum creatinine and serum β2-M concentrations differ. The usefulness of β2-M for assessment of GFR is limited by the fact that other diseases may also cause an elevation /12/.

β2-M is reabsorbed in the brush border of the proximal tubule, followed by its catabolism into amino acids. Excretion of β2-M is increased in tubular damage (e.g., due to bacterially induced interstitial nephritis, cadmium nephropathy and amino glycoside-induced tubular necrosis). Patients with amino glycoside-related nephrotoxicity have a urinary excretion > 10 mg in an 8-h urine collection.

In cadmium-exposed individuals, a relationship exists between exposure duration, the cadmium concentration in blood and β2-M excretion. In individuals with prolonged occupational exposure to cadmium, increased β2-M excretion should be expected after an exposure period of about 10 years /3/.

In Cytomegalovirus infection, an increase in CD8+ T lymphocytes occurs, which is responsible for an increase in serum β2-M concentration.

Increased serum β2-M serum levels in malignant lymphomas are presumably due to increased cell turnover. Another theory /26/ is based on the fact that in malignant lymphomas the cell membrane of lymphocytes carries less β2-M than in healthy individuals. This is thought to be caused by a defect in the α-chain of MHC class I antigens, which subsequently binds less β2-M. Because an intact MHC class I antigen structure is a prerequisite for recognition of altered cells by cytotoxic lymphocytes, these altered cells are not recognized and, hence, they are able to proliferate.

References

1. Terrier N, Bonardet A, Descomps B, Cristol JP, Dupuy AM. Dermination of β2-microglobulin in biological samples using an immunoenzymometric assay (chemiluminescence detection) or an immunoturbidimetric assay: comparison with radioimmunoassay. Clin Lab 2004; 50: 675–83.

2. Evrin PE, Wibell L. Serum levels and urinary excretion in apparently healthy subjects. Scand J Clin Lab Invest 1972; 26: 69–74.

3. Lammers M, Gentzer W, Reifferscheidt G, Schmidt B. Dermination of β2-microglobulin with a particle enhanced immunonephelometric assay. Clin Chem 2002; 48: A-119 (abstract).

4. Schaller KH, Gonzales J, Thürauf J, Schiele R. Früherkennung von Nierenschäden durch Blei, Quecksilber und Cadmium bei beruflich exponierten Personen. Zbl Bakt Hyg, I Abt Orig 1980; B 171: 320–35.

5. Jonasson LE, Evrin PE, Wibell L. Content of β2-microglobulin and albumin in human amniotic fluid. Acta Obstet Gynec Scand 1974; 53: 49–58.

6. Schardijn J, v Eps LWS, Swaak AJG, et al. Urinary β2-microglobulin in upper and lower urinary tract infections. Lancet 1979 I; 805–7.

7. Grützner FJ. Diagnostik mit β2-Mikroglobulin. Inn Med 1982; 9: 45–56.

8. Späti B, Child JA, Kerruish SM, Cooper EH. Behaviour of β2-microglobulin and acute phase reactant proteins in chronic lymphocytic leukaemia. Acta Haemat 1980; 64: 79–86.

9. Litam P, Swan F, Cabanillas F, et al. Prognostic value of serum β2-microglobulin in low-grade lymphoma. Ann Int Med 1990; 114: 855–60.

10. Child JA, Späti B, Illingworth S, et al. Serum beta 2 microglobulin and C-reactive protein in the monitoring of lymphomas. Cancer 1980; 45: 318–26.

11. Bien E, Balcerska A. Serum soluble interleukin-2 receptor, β2-microglobulin, lactate dehydrogenase and erythrocyte sedimentation rate in children with Hodgkin’s lymphoma. Clinical Immunology 2009; 70: 490–500.

12. Diem H, Fateh-Moghadam A, Lamerz R. Prognostic factors in multiple myeloma: role of β2-microglobulin and thymidine kinase. Clin Investig 1993; 71: 918–23.

13. Kult J, Lämmlein Ch, Röckel A, Heidland A. β2-Mikroglobulin im Serum, ein Parameter des Glomerulumfiltrates. Dtsch Med Wschr 1974; 99: 1686–8.

14. Portman RJ, Kissane JM, Robson AM. Use of β2 microglobulin to diagnose tubulo-interstitial renal lesions in children. Kidney International 1986; 30: 91–8.

15. Drüeke TB, Massy ZA. Beta2-microglobulin. Semin Dial 2009; 22: 378–80.

16. Steinhoff J, Feddersen A, Wood WG, et al. β2-Mikroglobulinurie bei Cytomegalievirus-Infektionen nach Nierentransplantation. Dtsch Med Wschr 1991; 116: 1008–12.

17. Uthmann U, Geisen HP. Beta-2-Mikroglobulin. Dtsch Med Wschr 1981; 106: 782–6.

18. Hümpfner A, Heidegger H. β2-Mikroglobulinurie und Lysozymurie: Bedeutung als diagnostischer und therapeutischer Kontrollparameter bei Dilatation der oberen Harnwege in der Schwangerschaft. Nieren- und Hochdruckkrankheiten 1989; 18: 545–51.

19. Moss AR, Bacchetti P, Osmond D, et al. Seropositivity for HIV and the development of AIDS or AIDS-related condition: three year follow up of the San Francisco General Hospital cohort. Brit Med J 1988; 296: 745–50.

20. Norfolk DR, Forbes MA, Cooper EH, Child JA. Changes in plasma β2 microglobulin concentrations after allogeneic bone marrow transplantation. J Clin Pathol 1987; 40: 657–62.

21. Nishimaki S, Shima Y, Sato M, An H, Hashimoto M, Nishiyama Y, et al. Urinary β2-microglobulin in premature infants with chorioamnionitis and chronic lung disease. J Pediatr 2003; 143: 120–2.

22. Dreux S, Rousseau T, Gerber S, Col JY, Dommergues M, Muller F. Fetal β2-microglobulin as a marker for fetal infectious diseases. Prenat Diagn 2006; 26: 471–4.

23. Vree TB, Guelen P, Jongman-Nix B, et al. The relationship between the renal clearance of creatinine and the apparent renal clearance of β2-microglobulin in patients with normal and impaired kidney function. Clin Chim Acta 1981; 114: 93–6.

24. Wibell L, Evrin PE, Berggard I. Serum β2-microglobulin in renal disease. Nephron 1973; 10: 320–31.

25. Ljunggren HG. Role of β2-microglobulin in cancer. Cancer J 1992; 5: 308–15.

26. Swan F, Ordonez N, Manning J, et al. Beta 2 microglobulin cell surface expression as an indicator of resistance in lymphoma and its relation to the serum level. Blood 1988; 72: 258a.

Table 18.1-1 Diagnostically significant plasma proteins /13/

Comment

Albumin

Albumin is the binding and transport protein for numerous substances in blood (e.g., amino acids, hormones, drugs and trace elements). Albumin determination is of clinical significance for estimating protein synthesis in the liver, in patients with protein malnutrition, and in cases with presumed oncotic pressure.

C-reactive protein (CRP), serum amyloid A (SAA), α1-acid glycoprotein, haptoglobin (Hp)

These proteins belong to the group of acute phase proteins. The plasma concentration of acute phase proteins increases in inflammation (e.g., tissue damage, microbial infection). The extent of elevation depends on the severity of tissue damage as well as the synthesis rate and half-life of the individual acute phase proteins. Accordingly, the pattern of any change following a stimulus is different for each protein. Hp has a protective function by forming a complex with hemoglobin (Hb) and, thus, preventing renal loss of iron. The Hp/Hb complex is the source of iron and amino acids for protein and heme neosynthesis and is catalyzed by the reticuloendothelial system. Low Hp concentrations are measured in acute episodes of intravascular hemolysis.

α1-antitrypsin (α1-AT), α2-macroglobulin 2-M)

These proteins are proteinase inhibitors. α1-AT is a ember of the group of serine protease inhibitors (serpins). See also Section 18.5 – α1-antitrypsin.

α1-AT: Homozygous deficiency may be associated with pulmonary emphysema in adults and liver cirrhosis in early childhood.

α2-M: This protein inhibits the activity of many proteases and, together with C1-esterase inhibitor, regulates the formation of kinins. Decreased concentrations of α2-M are found in conditions associated with a release of proteases (e.g., in acute pancreatitis).

C3, C4, C1-esterase inhibitor (C1-INH)

These proteins are factors of the complement system.

C3: this protein represents the central component of the complement system. Cleavage of C3 results in the formation of anaphylatoxins, chemotaxins, opsonins and the membrane attack complex, all of which serve an important function in the inflammatory process that is part, for example, of an infection.

C4: important component for activation of the classical pathway of the complement cascade. C4 deficiency is the most common hereditary deficiency within the complement system and is often decreased in systemic lupus erythematosus.

C1-INH: this protein belongs to the group of serpins and is an inhibitor of C1r and C1s. C1-INH deficiency is the second most common genetic defect in the complement system and results in uncontrolled activation of the classical pathway. Clinically, the picture of hereditary angioneurotic edema may occur.

Ceruloplasmin (Cp)

Cp, also referred to as plasma ferroxidase, is important for the transport and availability of iron in tissues. In the presence of Cp, Fe2+ is oxidized to Fe3+. Besides the determination of copper in serum and urine, Cp is also a parameter employed for diagnosing Wilson’s disease.

Immunoglobulins (Ig)

Ig are effectors of humoral immune response. They bind to specific antigens to form immune complexes. These complexes trigger defense mechanisms (e.g., activation of the complement system, activation of phagocytes) and thus lead to elimination of the antigen. Determination of individual Ig classes is clinically relevant for the diagnosis of immunodeficient states, diagnostic evaluation of hypergammaglobulinemias and diagnosis of allergies. See Chapter 21 – Immune system and Chapter 22 – Monoclonal plasma cell proliferative diseases.

Lipoproteins (Lp)

Water-insoluble lipids are bound to proteins, the so-called apolipoproteins (apo Lp); this results in the formation of a water-soluble lipoprotein particle. The following apolipoproteins can be bound in the form of such particles: apo AI, AII, AIV, B-100, B-48, CI, CII, CIII, D, E and J. Changes in serum apolipoprotein concentrations may be indicators of certain defects in fat metabolism.

Transthyretin (TTR), retinol-binding protein (RBP)

Determination of TTR and RBP is indicated for assessing the energy supply in suspected malnutrition.

TTR: this protein has an MW of 55 kDa, binds to RBP and has a reference interval of 220–450 mg/L. TTR is a negative acute phase protein, whose concentration decreases in the presence of inflammation and protein-deficient diet. TTR is helpful in early diagnosis of malnutrition because it quickly responds to restricted energy supply with lower plasma concentrations. TTR is not suited for diagnosis and monitoring of chronic adaptive malnutrition such as anorexia nervosa. Due to its half-life of only 2 days, TTR is a good parameter for assessing response to treatment, such as enteral or parenteral energy supply. The assessment of malnutrition should only be based on TTR concentration if CRP concentrations are normal because TTR is a negative acute phase protein /14/.

RBP: has an MW of 21 kDa and binds to TTR in plasma. Binding of retinol causes RBP to lose its binding capacity for TTR. The half-life of RBP is 12 h and plasma concentration is 30–80 mg/L. RBP decreases in hyperthyreosis, chronic liver disease, vitamin A deficiency and zinc deficiency. It is elevated in alcoholism and reduced glomerular filtration rate. In the assessment of malnutrition, lower RBP concentration rather indicates insufficient energy supply than protein restriction. The response of RBP and TTR to energy supply is almost identical /14/.

Transferrin (Tf)

Tf is the iron-transport protein in the circulation; based on its total iron binding capacity (TIBC), Tf is normally 30% saturated with iron (transferrin saturation, TfS 30%). In the presence of iron deficiency, the Tf concentration is elevated and the TfS is below 16%. In conditions of iron overload, such as hemochromatosis, the TfS is ≥ 50%. In the population, individuals with TfS ≥ 50% have an increased risk of early mortality (odds ratio 1.3 in men and 1.5 in women) compared to individuals with lower values /15/. Tf is a negative acute phase protein, whose concentration decreases in the presence of systemic inflammation. See also Section 7.5 – Transferrin saturation (TfS).

Table 18.1-2 Distribution of plasma proteins between the intravascular and extravascular compartments /1/

Protein

MW
(kDa)

Intra-
vascular (%)

Albumin

66

42

Haptoglobin 1-1

85

50

Haptoglobin 2-2

160

75

IgA

160

41

IgG

144

44

IgM

971

77

α2-macroglobulin

720

92

Transferrin

80

32

Table 18.1-3 Important indications for plasma protein determination

Indication

Plasma protein

Acute and chronic liver disease, edema

Albumin

Early childhood liver cirrhosis, pulmonary emphysema in adults

α1-antitrypsin

Chronic alcoholism

Carbohydrate-deficient transferrin

Hereditary angioedema, Capillary leak syndrome

C1-esterase inhibitor

Immune complex disease

Complement C3, C4

Acute phase response (APR)

C-reactive protein

Virally induced APR

Serum amyloid A protein

Acute hepatitis of non-viral genesis, Wilson’s disease

Ceruloplasmin

Hemolytic disease

Haptoglobin, hemopexin

Risk of atherosclerosis

Lipoprotein (a), apolipoprotein A-I, apolipoprotein B

Chylomicronemia syndrome

Apolipoprotein C-II

Hyperlipoproteinemia type IIi

Apolipoprotein E

GFR of 80–30 [mL × min–1 × (1.73 m2)–1

Cystatin C

Protein and energy nutrition status in seriously ill patients or in famine regions

Retinol-binding protein, transthyretin, albumin

Allergy diagnostics

IgE

Humoral immunodeficiency

IgM, IgG, IgA

Chronic, active, cholestatic or alcoholic liver disease

IgM, IgG, IgA

Extent of immune activation in inflammatory and autoimmune disease

Neopterin

Iron turnover in cases of reduced iron storage (low ferritin) or inflammation, hemochromatosis

Transferrin and/or transferrin saturation

Table 18.1-4 Plasma proteins of the ERM reference materials /12/

ERM-DA470/IFCC

  • Albumin

Haptoglobin

  • α2-macroglobulin

IgA

  • α1-acid glycoprotein

IgG

  • α1-antitrypsin

IgM

  • C3c

Transferrin

  • C4

Transthyretin

ERM-DA472/IFCC

  • C-reactive protein (CRP)

Table 18.2-1 Reference intervals for total protein

Serum/
plasma

Adults /5/

66–83

Children /6/

  • 1–30 days

42–62

41–63

  • 31–182 days

44–66

47–67

  • 183–365 days

56–79

55–70

  • 1–18 yrs

57–80

57–80

Urine

Up to 0.15

CSF

0.2–0.4

Extra-
vascular
fluids

See Chapter 47 – Extravascular fluids

Data expressed in g/L. Values are 2.5th and 97.5th percentiles.

Table 18.2-2 Diseases and conditions that may cause hypoproteinemia

Clinical and laboratory findings

Defective synthesis – Antibody deficiency syndrome (ADS)

Both the congenital ADS and the transitory form of ADS become clinically evident when the synthesis of antibodies fails. However, ADS can also first appear in adulthood (late manifestation type) or secondarily as the result of another, underlying primary disease.

– Analbuminemia

Rare disorder that shows familial occurrence or is observed in conjunction with nephrotic syndrome. Familial analbuminemia is of minor clinical relevance.

– Severe liver disease

Hypoproteinemia of 40–50 g/L is measured in patients with massive damage to hepatic parenchymal cells (e.g., as seen in viral hepatitis with a fulminant course or in toxic liver injury). Albumin is reduced to concentrations as low as 15 g/L. In liver cirrhosis as well as in acute and chronic hepatitis, TP is nearly normal.

Nutritional protein deficiency – Fasting states, psychogenic anorexia, gastrointestinal tumors, failure to grow in children due to malnutrition

Hypoproteinemia due to malnutrition (dietary deficiencies or poor dietary habits) occurs mainly if insufficient or no animal protein is consumed. Under such circumstances, total protein (TP) does not decline within a few days, but instead decreases several weeks later because albumin is initially replenished from the extravascular pool. Hypoproteinemia does not occur until the albumin pool is reduced by two thirds. Protein and caloric deficiencies are present in marasmus whereas kwashiorkor and flour-related malnutrition are only associated with protein deficiency. TP decrease is not an indicator for early detection of nutritional protein deficiency.

Malabsorption syndrome

In intestinal diseases with chronic diarrhea (celiac disease, food allergy, disaccharidase deficiency, mucoviscidosis, selective IgA deficiency), hypoproteinemia develops because of protein malabsorption and intestinal protein losses. During the acute stage of sprue, concentrations of 30–40 g/L are measured. Clinical symptoms may already be preceded by a reduction in serum protein; occasionally, the diagnosis of agammaglobulinemia is made.

Protein loss syndrome – Glomerulonephritis with proteinuria, nephrotic syndrome

Hypoproteinemia is due to albuminuria. However, exogenous protein loss is not directly related to hypoalbuminemia. The cause can vary among individuals, where only in some patients does the liver exhibit an elevated albumin synthesis rate, while in others the increased rate of albumin degradation, as seen in patients with nephrotic syndrome, varies from patient to patient.

– Exudative enteropathy

In exudative enteropathy (ulcerative colitis, Crohn’s disease, Ménétrier syndrome, colonic polyposis and diverticulosis, lymph drainage abnormalities), there is a protein effusion into the small intestine; serum TP concentration is 30–50 g/L. Contrary to patients with nephrotic syndrome, protein loss is nonselective. Therefore, the erythrocyte sedimentation rate is not or only insignificantly elevated. Diagnosis is confirmed by measuring the radioactivity excreted into feces after intravenous administration of 51Cr-labeled albumin. In the feces of healthy individuals, less than 1% of the albumin that was administered i.v. is found.

– Ascites, pleural exudate

TP can be reduced in the case of burns, weeping eczemas and bullous dermatoses. Dysproteinemia resembles that seen in cases of enteral protein losses. In bullous dermatoses, protein composition of the bullae is almost identical to that of serum.

– Chronic hemodialysis

Hypoproteinemia occurs in these conditions, especially if effusions are repeatedly tapped.

– Chronic hemodialysis

TP reduction as a result of ongoing albumin losses.

Pseudohypoproteinemia

Infusion therapy, polydipsia and pregnancy are associated with a physiologically or therapeutically induced expansion of intravascular volume (e.g., during pregnancy by about 50%).

Hemorrhagic anemia

In acute external hemorrhages, TP is usually decreased because protein is lost together with red blood cells. After 12–24 hours, the red blood cell count falls below the lower reference interval value, reaches the nadir after 48–72 hours and returns to its pre hemorrhagic level after 2–4 weeks. After 4–6 hours, TP falls below the lower reference interval value, reaches the nadir after 12–24 hours and returns to its pre hemorrhagic level after 1–3 weeks. In hemolytic anemia, TP does not decrease and in acute internal hemorrhage (e.g., bleeding into the gastrointestinal tract) declines but slightly /7/.

Table 18.2-3 Diseases and conditions that may cause hyperproteinemia

Clinical and laboratory findings

Multiple myeloma, Waldenström’s macroglobulinemia

Pronounced hyperproteinemia may occur in these disorders. However, elevated serum protein levels occur relatively late. In targeted laboratory testing, only about 10% of the patients with multiple myeloma have protein values > 80 g/L at the time of diagnosis. Light-chain myeloma may be associated with subnormal TP concentrations.

Chronic inflammatory disease

Hyperproteinemias in chronic inflammatory disorders are due to an increase in γ-globulins and are rarely associated with total protein (TP) concentrations > 90 g/L. Such diseases include, for example, autoimmune hepatitis, active sarcoidosis, certain types of pulmonary tuberculosis, some cases of sepsis, syphilis, leprosy, malaria, lymphogranuloma inguinale, schistosomiasis, kala-azar.

Liver cirrhosis

In some patients with liver cirrhosis, the compensated stage is associated with elevated TP concentrations. In these cases, the increase in γ-globulins is not compensated for by an equal reduction in albumin. As the duration of liver cirrhosis extends, TP levels gradually decline; during the decompensated stage, the transition to hypoproteinemia is marked by the occurrence of ascites and edema.

Pseudohyperproteinemia due to dehydration

In the presence of an unchanged amount of protein, plasma volume is reduced as a result of water losses. Hematocrit is usually increased as well. Causes for dehydration include diarrhea, vomiting, inadequate oral fluid intake, sweating, diabetes insipidus, polyuric phase of acute renal failure.

Table 18.3-1 Reference intervals of serum protein electrophoresis

Distribution of albumin and globulin fractions in % of the total protein for adults

Stain

Albumin

α1-globulin

α2-globulin

β-globulin

γ-globulin

Cellulose acetate
(Amido black) /2/

60.6–68.6

1.4–3.4

4.2–7.6

7.0–10.4

12.1–17.7

Cellulose acetate
(Ponceau red) /5/

55.3–68.9

1.6–5.8

5.9–11.1

7.9–13.9

11.4–18.2

Capillary zone
electrophoresis /6/

53.1–66.4

3.2–5.7

7.5–12.4

9.0–13.7

10.3–19.6

* Values are the 2.5th and 97.5th percentiles.

Distribution of albumin and globulin fractions in g/L /2/*

Band

Newborns

Infants
up to 1 year

Children
up to 6 years

School-age
children

Adults

Albumin

32.7–45.3

35.7–51.3

33.1–52.2

40.0–52.5

35.2–50.4

α1-globulin

1.1–2.5

1.3–2.5

0.9–2.9

1.2–2.5

1.3–3.9

α2-globulin

2.6–5.7

3.8–10.8

4.3–9.5

4.3–8.6

5.4–9.3

β-globulin

2.5–5.6

3.5–7.1

3.5–7.6

4.1–7.9

5.9–11.4

γ-globulin

3.9–11.0

2.9–11.0

4.5–12.1

5.9–13.7

5.8–15.2

* The values apply to protein bands stained with Ponceau red S on cellulose acetate sheet.

Table 18.3-2 Reaction patterns in serum protein electrophoresis /7/

 

Reaction constellation

Type

Albu-
min

α1-G

α2-G

β-G

γ-G

Acute
inflammation

()

n

Chronic reactive
inflammatory
and proliferative
processes

n

n

Acute viral
hepatitis

n

n

n

Liver cirrhosis

↓↓

n

n

n

↑↑

Obstructive
jaundice

Nephrotic
syndrome

↓↓

n

↑↑

Malignant tumor

γ-globulin
multiple myeloma

n

n

n

↑↑

G, globulin; description of symbols: increased, decreased, ↑↑ strongly increased, ↓↓ strongly decreased

Table 18.3-3 Dysproteinemias with M-spike and extragradient /2/

Position
of the band

Disease and/or cause

Prealbumin

Two pre albumin bands may occur if the serum has been stored for several weeks.

Acid substances (e.g., drug metabolites such as salicylic acid) that are firmly bound to albumin, may cause the separation of a rapidly migrating albumin band whose final position is within the pre albumin zone.

Rapidly migrating albumin bands in certain types of albumin allomorphism (bisalbuminemia).

Albumin

Slowly migrating albumin bands associated with the heterozygous type of albumin allomorphism.

Slowly migrating albumin band after serum contamination with heavy metals (e.g., copper).

More rapidly migrating albumin band after parenteral therapy with excessive doses of penicillin.

Pronounced hyperbilirubinemia, usually not as an extra band but as an increased anodic front.

Monoclonal Ig (extremely rare; not confirmed in the literature).

α1-zone

M-spike (very rare or not observed).

α2-zone

In pronounced intravascular hemolysis, an extra band may appear in the cathodic portion as a result of haptoglobin-hemoglobin complexes. M-spike (very rare).

α2-β-
intermediary
zone

M-spike (rare).

Free hemoglobin due to intravascular or in vitro hemolysis (e.g., as a result of blood collection) the extra band has a reddish tint.

Denatured protein on the application site if the serum application was not placed at the cathodic end of the supporting medium (cellulose acetate sheet); the result resembles a double α2-band.

M-spike (monoclonal Ig or free light chains).

β-zone

Abnormally elevated concentrations of β-lipoprotein (hypercholesterolemia) may cause an extra band that is located in the cathodic portion of the β-zone.

The second most common position for M-gradient is within the β-zone. Often, this band represents monoclonal IgA.

β–γ-
intermediary
zone

Fibrinogen results in extra bands if plasma (obtained with heparin, EDTA, citrate) is separated (e.g. in dialysis patients and intensive care patients).

M-spike in Waldenström’s macroglobulinemia. Often also observed in lymphoreticular diseases that may be associated with monoclonal gammopathy. The third most common position for M-spike is within the β–γ-intermediary zone.

γ-zone

The most common position of M-spike, especially in IgG multiple myeloma. In Waldenström’s macroglobulinemia, the M-spike is not infrequently located in the cathodic portion of the γ-globulin zone or even at the application site if serum was applied at the cathodic end of the supporting medium. Extra gradients of mild to moderate intensity can also occur in the presence of high concentrations of immune complexes (e.g., as seen in patients with immune complex disorders, and also in those with liver cirrhosis, cardiovascular diseases and cancer). Immunoglobulin degradation products in old serum samples as well as high concentrations of plasma expanders may also cause extra gradients. Moreover, therapy with monoclonal antibodies may cause minor M-spikes.

Table 18.3-4 Reflex testing upon abnormalities in serum protein electrophoresis (n = 5,992) /13/

Serum protein
abnormalities

Reflex
tests

Immunofixation
positive cases

Number of cases

790
(13.2%)

341 (43%), including
9 free light chains

M-spike

169

169 (100%)

Extragradients

206

112 (54%), including
4 free light chains

Unclear
extragradients

263

47 (18%)

Hypogamma
globulinemia
(< 5.5 g/L)

85

10 (12%), including
5 free light chains

Increased
β-fraction
(16–19 g/L)

10

1 (10%)

Increased
α2-fraction
(≥ 14 g/L)

48

2 (4%)

Broad or extra
α2-fraction

9

0

Table 18.4-1 Reference interval for albumin in serum /2/

Adults

≤ 60 yrs

35–53

> 60 yrs

34–48

> 70 yrs

33–47

> 80 yrs

31–45

> 90 yrs

30–45

Children

Newborns

35–49

1st yr of life

36–50

2–20 yrs

37–51

Data expressed in g/L; values are 2.5th and 97.5th percentiles

Table 18.4-2 Diseases and conditions associated with hypoalbuminemia /23/

Clinical and laboratory findings

Pregnancy

Albumin serum concentration decreases continuously during pregnancy due to an average increase in plasma volume by 40%. Albumin concentration is about 20% lower in the last third of pregnancy than that prior to pregnancy despite an absolute increase in albumin mass by 20% /5/. Albumin excretion is elevated in pregnancy and increases with the duration of pregnancy.

Acute phase response

Albumin is a negative acute phase protein; its synthesis is compensatorily reduced during acute phase response. Besides decreased synthesis, the following other mechanisms are important: increased migration into the interstitial space, hemodilution, increased catabolism and increased oncotic pressure due to elevated plasma concentration of acute phase proteins. The presence of hypoalbuminemia due to inflammation can be determined by means of serum protein electrophoresis or C-reactive protein measurement. Tumor-induced inflammatory reactions also cause hypoalbuminemia.

Polyclonal and monoclonal gammopathies

Chronic inflammation with polyclonal gammopathy causes hypoalbuminemia. This also applies to multiple myeloma with a high M-gradient. A decline in albumin in connection with this disease indicates a poor prognosis.

Liver cirrhosis

In patients with liver cirrhosis, hypoalbuminemia is usually present. The albumin reduction is caused, on the one hand, by a rise in oncotic pressure due to an increase in plasma immunoglobulin concentration and, on the other hand, by albumin losses into the third space. Liver function is not correlated with albumin concentration. In these patients, alcohol causes an acute decrease in the synthesis of albumin. In liver cirrhosis, albumin concentration is not correlated with the severity of the disease; however, an albumin level below 30 g/L indicates a poor prognosis. In acute liver injury, albumin concentration may remain normal for a long time and does not decline until the parenchymal cells are severely damaged /6/.

In liver cirrhosis, the functional activity of albumin is markedly reduced, resulting in impaired liver function. In addition, the decrease in albumin concentration leads to further functional impairment. The two causes combined are associated with reduced survival rate /7/.

Nutritional protein deficiency

In elderly patients and in epidemiological studies involving populations in developing countries, albumin and/or retinol-binding protein and/or transthyretin are employed as indicators of nutritional protein deficiency. According to a study /8/ involving starving children in Zaire, an albumin concentration < 16 g/L was the best indicator of impending death.

Postoperative mortality

Besides various cardiovascular parameters, an albumin concentration below 40 g/L is a preoperative risk factor for increased mortality in patients over 75 years of age undergoing cardiac surgery /9/.

Acute trauma

Emergency patients with an albumin concentration below 34 g/L upon arrival at the hospital are 2.5 times more likely to die compared to patients with higher values /10/.

HIV infection

Serum albumin is a predictor of survival among HIV-infected women. Female patients with an albumin concentration below 35 g/L have 5-fold more medical problems than those with an albumin concentration above 42 g/L, and the proportion of patients with a 3-year survival is markedly lower /11/.

Critically ill patients

Hypoalbuminemia is a common finding in intensive care unit patients. Patients, who do not survive, have lower albumin concentrations than those with a positive outcome; this is why albumin is a criteria in the Acute Physiology and Chronic Health Evaluation score (APACHE III). Hypoalbuminemia is thought to be caused by increased catabolism, reduced synthesis and capillary leakage. Another cause is the reduction in half-life which rather presupposes an increased rate of albumin synthesis. For instance, in a study /12/, the plasma half-life for albumin in critically ill patients was reduced from the normal value of about 19 to 11.8 (10.8–12.9) days.

Analbuminemia

Analbuminemia (MIM 103600) or idiopathic hypoalbuminemia should be suspected if the serum albumin concentration lies within an interval of 0.001–10 g/L. Incidence in the population is lower than 1 in 1 million. Analbuminemia is a hereditary disorder. Afflicted individuals must have inherited an abnormal allele from each parent. Overall, 13 different defects in the encoding region of the Albumin gene and its intron-exon junctions have been detected to date /13/. Analbuminemia is a heterogeneous allelic disease with a homozygous or compound-heterozygous mode of inheritance. The most common cause is said to be Kaysei mutation due to a homozygous AT deletion at nucleotides c.228–229 and the 91st and 92nd bases of exon 3 /14/.

Symptoms are mild; patients have mild edema, suffer from fatigue and have hyperlipidemia with elevated cholesterol and phospholipids due to transport of these lipids being dysfunctional. Compensatory increase in γ-globulins maintains the colloid osmotic pressure. Atherosclerosis may develop due to the elevated concentration of LDL cholesterol.

Table 18.5-1 AAT deficiency and related diseases

Genotype

AAT
(g/L)

Risk
of COPD

Risk of
liver disease

ZZ

≤ 0.30

Very high

High

ZZero

≤ 0.30

Very high

Unknown

MZ

≤ 1.0

Possibly
elevated

Possibly
elevated

MZero

≤ 1.0

Unknown

None

SZ

≤ 0.70

Elevated

Possibly
elevated

Zerozero

Not
quantifiable

Very high

None

Table 18.5-2 AAT concentration and SERPINA1 gene variants /9/

AAT (g/L)

Variants

> 1.03 to ≤ 1.13

86.5% with PIMM

> 0.93 to ≤ 1.03

53.5% with PIMM

> 0.83–0.93

54.9% with PIMS

> 0.73 to ≤ 0.83

76.4% with PIMZ

Table 18.5-3 Clinical manifestations of AAT deficiency

Clinical and laboratory findings

Chronic obstructive pulmonary disease (COPD) /16/

The classic clinical presentation of congenital AAT-deficiency is severe panacinar emphysema in adults that occurs with distal predominance at an early age. It is also seen diffusely distributed in the upper lobes. Bronchiectasis with or without emphysema is less common /1/. COPD induced by AAT deficiency rarely occurs before the age of 30. Risk ratios for COPD range from 1.5–12-fold, depending on whether the Z allele is present in heterozygous or homozygous combinations. Among normal individuals, only 1 in 100 have a Z allele. However, among individuals with COPD, the prevalence of the Z allele is 10%.

Clinical manifestation: dyspnea is the predominant symptom, but chronic coughing and whooping are also common. COPD in smokers usually begins during the 6th to 8th decade of life and mainly involves changes in the upper regions of the lungs. By contrast, nonsmokers or light smokers with AAT deficiency already present with COPD during the 4th to 5th decade of life if they carry the genetic variants ZZ, Zzero and Zerozero. The risk of COPD is high in individuals with the PiSZ genotype who smoke, but is generally lower than in those carrying the PiZZ genotype. COPD is caused by AAT deficiency in 1–3% of the cases. Most patients with AAT deficiency who develop COPD are smokers, and about 70% die around the age of 50. Some of these patients had presented with hepatic disease already in their childhood. Individuals with severe AAT deficiency who neither smoke nor are exposed to harmful inhalative substances may have a normal life expectancy /17/. In children up to the age of 7 years, no pulmonary changes are present that are detectable by pulmonary function tests. According to a study /18/, 85% of 246 hospitalized PiZZ smokers and 58% of the nonsmokers had COPD. Median age at the onset of clinical symptoms was 40 years in smokers and 53 years in nonsmokers. Mean age at death was 53 years in smokers and 63.5 years in nonsmokers.

Laboratory findings: approximately 10–15 AAT variants are associated with concentrations below 0.45 g/L. The Pi*Z allele is involved in 95% of the cases. An AAT concentration ≤ 0.30 g/L practically confirms the presence of a ZZ phenotype. The first test is the serum level of AAT. A low level indicates a genetically determined COPD endotype that responds to long-term replacement of the missing protein. The second test is blood eosinophil count. In patients with frequent exacerbations despite appropriate bronchodilator treatment, the blood eosinophil count helps predict the response to inhaled glucocorticoids. Eosinophil count higher than 300 × 106/L indicate a good response, values between (100–300) × 106/l suggest a moderate response, and a low eosinophil count (< 100 × 106/L) is associated with minimal benefit in the risk of pneumonia) /16/.

Liver disease

AAT deficiency is the most common congenital liver disease. AAT deficiency-associated liver injury is based on hepatocellular inclusion bodies. Homozygous carriers of the ZZ phenotype can synthesize AAT, but are not able to clear it from the rough endoplasmic reticulum. As a result, AAT aggregates in the hepatocytes in patients with the alleles Z, Mmalton and Siijama. Failure to degrade the aggregated protein leads to necrosis of the hepatocytes and possibly to liver cirrhosis.

Neonatal period: about 35% of all hepatic diseases occurring during this period are caused by AAT deficiency. 10% of homozygous carriers of the ZZ phenotype develop neonatal cholestasis which progresses to liver cirrhosis in up to 13% of the cases. Evidence of liver disease was found in 18% of the newborns with the ZZ phenotype within the first 6 months of life; after 2 years, liver function tests still gave pathologic results (ALT, AST) in 25% of these infants /10/. The risk of cirrhosis in those with abnormal liver function was 50%; 25% of the children died within the first decade of life, and 2% developed liver cirrhosis at a later age. The high incidence of liver disease in newborn ZZ phenotypes compared to the sporadic occurrence in childhood and adolescence is explained by the impaired ability of the newborn liver to degrade the protein aggregates /19/. Although liver function tests in these infants may give normal results, the liver disease may progress continuously due to fibrotization, and might only become symptomatic later in life.

Adults: in patients with severe AAT deficiency, about 20% develop liver cirrhosis and 7% develop hepatocellular carcinoma. In many adults with the ZZ phenotype, globular precipitation and degradation of the aggregated AAT are balanced. Liver cirrhosis may not develop until late in life. For example, liver cirrhosis was diagnosed in only 2% of the PiZZ individuals aged 20–50-years, but in 19% of those over the age of 50 /18/. In a study /20/, the liver function in a cohort with individuals aged 30 years, who had been identified as carriers of the genotypes PiZZ and PiSZ in a neonatal screening program, was investigated by determining the aminotransferases and GGT. The enzyme activities in all of these individuals were within the normal range, but markedly higher than in the cohort with healthy control subjects.

Panniculitis

Necrotizing AAT deficiency panniculitis, a rare form of panniculitis, is associated with phenotypes ZZ, SZ and SS. Prevalence is 1 in 1,000 individuals with AAT deficiency. The disease preferentially involves the trunk and the upper extremities /19/. It is characterized by painful skin nodules and areas with fat tissue necrosis.

Table 18.5-4 Characteristics of selected α1-antitrypsin variants, according to Ref. /25/

Alleles

Type of
mutation

Cellular
disorder

Disease
association

Normal
alleles

Substitution (1bp)

None

Normal

Deficiency alleles

  • S

Glu264Val

Intracellular
degradation

Lung

  • Z*

Glu342Lys

Intracellular
accumulation

Lung

liver

  • Mmalton

Phe52del or Phe51del

Intracellular
accumulation

Lung

liver

  • Siijama

Ser53Phe

Intracellular
accumulation

Lung

  • Mheerlen

Pro63Leu

Intracellular
degradation

Lung

  • Mprocida

Leu41Pro

Intracellular
degradation

Lung

  • Mmineral springs

Gly67Glu

Intracellular
degradation

Lung

Zero alleles

  • Q0granin falls

Tyr160X

No mRNA

Lung

  • Q0ludwigshafen

Ile92Asn

No protein

Lung

liver

  • Q0hong kong

Leu318LeufsX17

Intracellular
accumulation

Lung

  • Q0isola di procida

17 kb deletion
in exon 2–5

No mRNA

Lung

Dysfunctional alleles

  • F

Arg223Cys

Defective
elastase
inhibition

Lung

  • Pittsburg

Met358Arg

Antithrombin
activity

Bleeding
diathesis

  • Mmineral springs

Gly67Glu

Defective
elastase
inhibition

Lung

  • Z*

Glu342Lys

Defective
elastase
inhibition

Lung, liver

* Dysfunction regarding the inhibition of the elastase activity of polymorphonuclear granulocytes.

Table 18.6-1 Effects of alcohol (ethanol) consumption

Clinical and laboratory findings

Ethanol metabolism

Ethanol is converted to acetaldehyde by dehydrogenation via alcohol dehydrogenase in the cytoplasm of the hepatocyte and oxidized to acetaldehyde by an H2O2-dependent peroxidase in the peroxisomes of the smooth endoplasmic reticulum. Acetaldehyde can then be used for energy production in the mitochondria (Fig. 18.6-1 – Metabolisation of ethanol). Ethanol oxidation leads to increased NADH production, which may cause the following metabolic disorders /20/:

  • Hyperlactatemia and metabolic acidosis
  • Hyperuricemia due to acidosis-induced reduced uric acid excretion
  • Inhibition of gluconeogenesis with increased production of α-glycerophosphate, inhibition of the citrate cycle and fatty acid oxidation (Fig. 18.6-1). Inhibition of fatty acid oxidation promotes steatosis of the liver and hyperlipidemia associated with an increase in all lipoprotein classes including HDL.

The amount of alcohol per drink is shown in Tab. 18.6-2 – Pure alcohol per drink .

Acute changes in laboratory findings are shown in Tab. 18.6-3 – Acute metabolic changes following alcohol consumption.

Ethanol uptake and resorption

Food intake prior to alcohol consumption reduces alcohol resorption in the gastrointestinal tract. The effect of alcohol is accelerated following the intake of warm alcoholic beverages (mulled wine, grog, hot punch) and by a combination of alcohol and sugar (liqueur) and a combination of alcohol and carbon dioxide (champagne, long drink).

Following resorption in the gastrointestinal tract, alcohol is transported to the liver via the portal vein and metabolized in the cytoplasm and the smooth endoplasmic reticulum of the approximately 300 × 109 hepatocytes. The consumption of 0.5 liters of lager beer and a corn schnapps (together 22 g of alcohol) in a fasting state results in a blood alcohol concentration of 0.44 ‰ that is degraded again after 2–3½ hours.

Ethanol and drugs

Chronic alcohol consumption induces the synthesis of microsomal alcohol-oxidizing enzymes /2/. The activity of the most important enzyme of alcohol oxidation, cytochrome P-4502E1, for example, increases 5–10 fold. The induction of this enzyme results in alcohol tolerance and also affects drug metabolization (Fig. 18.6-4 – Biochemical effects of ethanol). For instance, the clearance of meprobamate, pentobarbital, propranolol, antipyrine, diazepam, warfarin and rifampicin is increased in individuals with chronic alcohol consumption. Cytochrome P-4502E1 metabolizes numerous exogenous substances and drugs to toxic metabolites. This also applies to acetaminophen that competes with alcohol for cytochrome P-4502E1 if the alcoholic takes acetaminophen to reduce his/her alcohol-related symptoms after alcohol consumption. Stimulated by alcohol, the cytochrome P-4502E1 activity is still high in this situation, but blood alcohol concentration is already low again. Hence, there is no major competition between acetaminophen and alcohol, and acetaminophen is very rapidly metabolized forming toxic radicals. Since alcohol also inhibits the synthesis of reduced glutathione, an insufficient amount of radical scavengers is available. The intake of 2.5–4 g of acetaminophen may lead to toxic liver injury caused by the free radicals.

Contrary to chronic alcohol consumption, occasional drinking does not cause increased stimulation of the cytochrome P-4502E1 activity, and drugs such as methadone compete with alcohol for enzyme-induced degradation. This is what drug addicts make us of. If alcohol is consumed prior to methadone abuse, de methylation of methadone in the liver is reduced and its concentration in the brain remains elevated for a longer period of time. An enhanced effect is also achieved by combining the intake of alcohol and barbiturates or tranquilizers.

Alcohol abuse

Alcoholism is the most serious addiction problem in Europe and North America. The annual per capita consumption of pure alcohol is 5–10 L in many countries.

In the USA, 9% of the adults and 13% of those who drink meet the criteria for an alcohol-use disorder /20/. Approximately 10% of men drink daily, 21% have an average of 60 drinks (14 g of ethanol) or more per month and 9% have 5 or more drinks on one occasion or at least once a week. The others are occasional drinkers. Approximately 3% of women drink alcohol daily, 6% have at least 60 drinks per month and 3% have at least 5 drinks on one occasion. Low to moderate alcohol consumption (up to 30 g/day in men and up to 15 g/day in women), as practiced by many individuals, does not represent a risk in healthy individuals. Alcohol-associated disorders, alcohol addiction and mortality increase with increasing alcohol consumption.

A distinction is made between /21/:

  • Risky alcohol consumption; it describes the amount of alcohol associated with a clear risk of health implications. Such a situation exists at a daily consumption of more than 30 g of pure alcohol (1.0 liters of beer) for men and more than 20 g for women.
  • Harmful alcohol consumption; these individuals present with physical (alcoholic hepatitis) or psychic (depressive episodes) disorders
  • Alcohol addiction; the following criteria apply to these individuals: compulsory alcohol consumption, reduced ability to control the handling of alcohol, development of alcohol tolerance, physical withdrawal syndrome, progressing neglect, continued consumption.

Alcohol abuse has the following risk factors /22/:

  • Chronic pancreatitis in 1–4% of the cases
  • Injury of the gastrointestinal mucosa (resorption disorder, hemorrhage)
  • Alcoholic liver disease (fatty liver disease, hepatitis, cirrhosis). The risk of liver cirrhosis is low in alcohol consumption of up to 40 g/day, 6-fold elevated at 60 g/day, 14-fold elevated at 80 g/day and more than 50 fold at higher amounts /23/.
  • Bacterial infections due to dysfunction of the immune system; they are an important cause of increased susceptibility of the alcoholic to disease.

Diseases induced by chronic alcohol abuse are listed in Tab. 18.6-4 – Diseases induced by chronic alcohol abuse. According to forensic investigations, the incidence of fatal alcohol intoxication is up to 7%. Alcohol concentrations of 2.5–5 ‰ and 2.5–3.5 g of alcohol per kg of body weight are measured /24/.

Moderate daily drinking (10–12 g of alcohol/day) is said to reduce the risk of cardiovascular disease. The risk factor is 0.65 in men compared to those who do not drink alcohol regularly /25/. Causes are thought to be: increase in HDL cholesterol, reduced predisposition for thrombosis, positive effects on sensitivity to stress and stress management. In contrast, episodes of drinking large amounts of alcohol significantly increase the risk of acute myocardial infarction.

Table 18.6-2 Standard drink (USA)

Drink

Pure alcohol

5 fl oz (142 mL) wine, 12% alcohol

14 g

5 fl oz (142 mL) sparkling wine, 12% alcohol

14 g

12 fl oz (423 mL) regular beer, 5% alcohol

14 g

1,5 fl oz (43 mL) distilled spirits, 40% alcohol

14 g

National Institute on Alcohol Abuse and Alcoholism (USA)

1 fluid ounze; fl oz (USA) = 28.4 mL; 1 standard drink corresponds to 14 g of alcohol

Table 18.6-3 Acute metabolic changes following alcohol consumption

  • Hypoglycemia because ethanol inhibits hepatic gluconeogenesis if the blood alcohol concentration is ≥ 0.45 ‰
  • Increase in osmolal gap (see Section 8.5.5.2 – Osmotic gap) because alcohol increases the osmolal gap
  • Increase in anion gap (see Section 5.5 – Ketone bodies) because alcohol leads to ketoacidosis
  • Lactic acidosis because in alcoholic ketoacidosis lactate uptake by the hepatocytes is inhibited by ketone bodies.

Table 18.6-4 Diseases induced by chronic alcohol abuse /26/

Organ system

Disease

Liver

Fatty liver, hepatitis, cirrhosis

Nervous system

Blackouts, tremor, dementia, encephalopathy (Wernicke)

Cardiovascular

Hypertension, arrhythmia

Respiratory tract

Tuberculosis, pneumonia, rib fracture

Lipid metabolism

Hypertriglyceridemia

Gonads

Men: erectile dysfunction, loss of libido, reduced sperm function

Women: menstrual irregularity, subfertility, loss of secondary sexual characteristics

Endocrinium

Pseudo-Cushing syndrome, hypoglycemia

Fetus

Fetal alcohol syndrome

Table 18.6-5 Laboratory investigations of alcohol consumption

Clinical and laboratory findings

Ethanol

The blood ethanol concentration does not provide any information as to whether occasional drinking or chronic alcohol abuse is involved. Many alcoholics do not consume any alcohol 24 hours before their visit to the doctor and, therefore, their symptoms cannot be correlated with alcohol. Elevated blood alcohol concentrations suggest recent drinking; the presence of alcohol tolerance indicating chronic alcohol abuse can be detected by associating the alcohol level per mille and the clinical findings. According to recommendations of the US National Council on Alcoholism (NCA), individuals are to be considered alcoholics if the following blood alcohol levels are measured:

  • Above 1.5 ‰ without gross evidence of intoxication
  • Above 3 ‰ any time or above 1 ‰ in a routine physical.

Alcoholization of an individual is classified based on the blood alcohol concentration as follows: 0–0.5 ‰ no alcoholization; > 0.5–1.5 ‰ mild intoxication; > 1.5–2.5 ‰ moderate intoxication; > 2.5–3.5 ‰ severe intoxication; > 3.5 ‰ very severe intoxication.

Gamma-glutamyl transferase (GGT) /26/

Increases in GGT vary significantly between individuals depending on the amount of alcohol consumed and the duration of drinking. In chronic alcoholics, the daily intake of 40 g of alcohol results in elevated GGT. In individuals who have not been drinking, the daily consumption of least 60 g of alcohol for 5 weeks is necessary to achieve the same effect. GGT is rarely elevated in individuals under 30 years of age and has a lower sensitivity in women than in men as a marker of alcohol abuse. Elevated levels and progressive accumulation of GGT in the population are associated with increasing alcohol consumption. Elevated GGT is measured in 20% of men and 15% of women consuming approximately 40 g of alcohol per day, and in 40–50% of men and 30% of women consuming more than 60 g of alcohol per day. Approximately 75% of alcohol-dependent individuals have elevated GGT; diagnostic sensitivity is 60–90%. In individuals who occasionally consume large amounts of alcohol without being chronic drinkers, GGT has a diagnostic sensitivity of 20–50%. GGT is also a marker for chronic alcohol abuse if alcohol intake involves large quantities. A problem of GGT is its low diagnostic specificity of 55–100% depending on the relevant study. This is because many drugs and liver diseases also lead to elevated GGT (see Section 1.9 – Gamma-glutamyl transferase (GGT)).

The half-life of GGT is 14–26 days; after alcohol abstinence, the increased activity returns to levels within the reference interval after 4–5 weeks.

Aminotransferases /26/

Acute drinking with an alcohol intake of 3–4 g/kg of body weight, corresponding to a blood alcohol level of 3–5 ‰, can lead to a transient increase in ALT and AST within 24–48 hours. An AST/ALT ratio above 1.5–2 indicates alcohol-induced liver injury. In chronic alcohol abuse, the aminotransferases are elevated with a diagnostic sensitivity of 15–40% (ALT) and 25–60% (AST). As a rule, the increase is 2–4-fold the upper reference interval value.

Mean corpuscular volume (MCV) of the erythrocytes /26/

The height of the MCV value is correlated with the frequency of drinking and the amount of alcohol intake. However, the occurrence of macrocytosis presupposes a daily intake of 60 g of alcohol for at least 1 month. A disadvantage of the MCV value is its low diagnostic sensitivity of 40–50%; its diagnostic specificity, however, is high. A normal MCV value implies a 80–90% exclusion of chronic alcohol abuse.

The positive predictive value of MCV > 94 fL for chronic alcohol abuse is 34.2% in men and women and 66.7% in men alone /26/. The negative predictive value of MCV < 95 fL for exclusion of chronic alcohol abuse is 94.2% in men and women and 92.0% in men alone /26/. All anemic alcoholics are macrocytic. The causes of elevated MCV due to chronic alcohol abuse are based on the fact that alcohol inhibits intestinal absorption and renal tubular reabsorption of folate and impairs the folate-dependent intermediary metabolism of C1 residues. C1 residues, such as formyl groups and hydroxy methyl groups, come from various metabolic processes, bind to tetrahydrofolate and are passed on to suitable acceptors for the synthesis of substances (see Chapter 13 – Homocysteine, vitamin B12, folates, vitamin B6, choline, betaine).

Ethyl glucuronide

See Section 18.6.8.3 – Ethyl glucuronide

Carbohydrate deficient transferrin

See Section 18.6.8.4 – Carbohydrate deficient transferrin.

Table 18.6-6 Diseases and conditions that may lead to false-positive CDT findings /15/

Clinical and laboratory findings

CDG syndrome

CDG syndrome (Congenital Disorder of Glycosylation) is one of the causes of high CDT concentrations without alcohol abuse. It is a genetically determined generalized disorder involving the synthesis of the carbohydrate structure of glycoproteins. Patients show abnormalities already after birth. A marked coincidence in the (incomplete) carbohydrate structures of glycoproteins in serum (Tf, α1-antitrypsin and haptoglobin β-chain) of patients with CDG syndrome or chronic alcohol abuse has been described.

Genetic transferrin (Tf) variants /4/

The allele frequencies of the Tf subtypes TfC, TfD and TfB vary among populations of different ethnicities. Black Africans, African Americans and Australian aborigines carry a high frequency of the D allele besides the wild-type TfC. Exact measurement of D variants of CDT is difficult, however, because di- and trisialylated Tf may coelute with the tetrasialylated D peak in HPLC or comigrate in capillary zone electrophoresis (CZE). Algorithms have been proposed to correct for the presence of these isoforms. In the presence of isoforms, %CDT is quantified too low by CDT immunoassay. The CDT immunoassay/CDT ratio in the wild-type Tf is about 2, while it is only about 1 in the B heterozygous and D heterozygous genotypes in CZE. Therefore, it is important to use only chromatographic or electrophoretic procedures for determining CDT in forensic investigations.

Chronic active hepatitis

The diagnostic value of CDT is reduced in advanced-stage liver disease.

Iron deficiency

Tf and CDT concentrations are elevated in the presence of iron deficiency. The values decline under iron substitution. Iron deficiency reduces the diagnostic specificity of CDT in normal alcohol consumption.

Hemochromatosis

Iron overload reduces the diagnostic sensitivity of CDT in alcohol abuse. There is a significant correlation between liver iron content and reciprocal concentration of CDT in alcohol abuse and between Tf and CDT in normal alcohol consumption. Iron clearance in hemochromatosis patients leads to an increase in CDT concentration.

Catabolic metabolism

CDT is significantly elevated in catabolic metabolism situations that are based on psychiatric disorders.

Anti-epileptic drugs

A trend toward elevated absolute and %CDT concentrations has been determined in women. However, it is unclear whether this trend is directly induced by anti-epileptic drugs or results from low Tf concentrations under anti-epileptic drugs.

Table 18.6-7 Equations for calculating amounts of alcohol and alcohol consumption

1)

Amount of alcohol in drinks (g/dL) = Volume (%) × 0.79

2)

Consumed alcohol (g) = Volume (%) × 0.79 × volume (dL)

3)

Blood alcohol (‰) = Amount of alcohol (g)/kg × 0.7

4)

Consumed amount of alcohol (g) = Body weight × 0.7 × blood alcohol (‰)

5)

Blood alcohol concentration (g/kg or ‰) = Serum alcohol concentration (mmol/L) × MW of alcohol (46) × 10–3 × serum density (1.026 g/kg) × water distribution coefficient between serum and blood (1.20). Simplified:

  • Blood alcohol (‰) = Serum alcohol (mmol/L) × 37.36 × 10–3
  • Serum alcohol (mmol/L) = Blood alcohol (‰)/37.36 × 10–3

BW, body weight; MW, molar weight in Dalton (Da). Conversion factors: mg/dL × 0.2171 = mmol/L

mmol/L × 4.61 = mg/dL

Table 18.7-1 Reference intervals for ceruloplasmin

Children /3/

  • Cord blood

0.05–0.33

  • 1st day – 4 months

0.15–0.56

  • 5–6 months

0.26–0.83

  • 7–18 months

0.31–0.91

  • 18–36 months

0.32–0.90

  • 4–9 yrs

0.26–0.46

  • 10–12 yrs

0.25–0.45

  • 13–19 yrs

0.22–0.50

  • 13–19 yrs

0.15–0.37

Adults /34/

0.22–0.40

0.25–0.60

0.27–0.66

(oral contraceptives)

0.30–0.50
(> 50 years, estrogen intake);
up to 1.30 g/L (pregnant women)

Data expressed in g/L

Table 18.7-2 Common ATP7B mutations /5/

Population

DNA nucleotide
change

Protein amino
acid change

Exon

Frequency

(%)

Other common
mutations

Asian

c.2333G>T

p.Arg778 Leu

8

30–50

c.2871delC

European

c.3207C>A

p.His1096 Gln

14

35–45

c.2299insG

c.1934T>G

India

c.813C>A

p.Cys271 Stop

2

About 20

c.3305T>C

c. 2975C>T

Arabia

c.4196A>G

p.Gln1399 Arg

21

About 30

Table 18.7-3 Prevalence of findings in Wilson’s disease patients /9/

Assay

Prevalence (%)

Free Cu in serum above 250 μg/L

86.6

Ceruloplasmin below 0.2 g/L

88.2

Cu excretion in urine ≥ 100 μg (1.6 μmol)/24 h

87.1

Kayser-Fleischer corneal rings

92.7

Cu concentration in the liver ≥ 250 μg/g dry weight

66.3

Histological signs of chronic liver disease

73.0

Table 18.7-4 Diagnosis of Wilson’s disease according to the AASLD practice guidelines /1/

Clinical and laboratory findings

Serum ceruloplasmin (Cp) concentration

The AASLD recommends the determination of Cp in patients between 3 and 55 years of age with liver disease of uncertain cause. A Cp concentration < 0.20 g/L may suggest WD and must be verified by further analysis. In a study /9/ involving 2867 patients with liver disease, low Cp concentrations were found in 5.9%, but only 1 patient had Wilson’s disease. In another study /14/, it was shown that in cases where liver disease was suspected, clinicians took a “shotgun” approach and ordered the determination of Cp together with markers of infectious hepatitis and autoimmune hepatitis, even for patients > 55 years of age. For instance, Cp measurements in 5023 patients yielded 424 patients with a Cp concentration < 0.20 g/L as well as 8 patients with and 416 patients without Wilson’s disease. The positive predictive value of Cp was 8.4% and the false-positive rate was 98.1%. The analyses show that a rational order approach is necessary in suspected Wilson’s disease considering the fact that low Cp levels are found in heterozygous Cp deficiency, conditions associated with renal Cp losses, exudative gastroenteropathy (including celiac disease), in the presence of massive burns due to losses via the skin, in severe hepatic insufficiency, Menkes disease and hereditary aceruloplasminemia.

Since Cp is an acute phase protein, patients with WD can also have normal concentrations in the presence of inflammation and infection. Moreover, the intake of oral contraceptives and the presence of pregnancy must be taken into account.

Low-normal Cp levels are found in 5% of patients with WD during the chronic, clinically inapparent stage and in 15% of those with hepatic damage. In fulminant liver failure, Cp concentrations can be higher than during the preceding asymptomatic stage.

Serum concentration Of Copper /1/

In serum, more than 90% of Cu is bound to Cp. Although WD is associated with Cu overload, the plasma Cu concentration is reduced according to the decreased Cp concentration. During the chronic, clinically inapparent stage, Cu concentration is usually < 60 μg/dL (11.8 μmol/L). However, in the acute hepatitic type of manifestation and in the presence of hemolysis, levels are higher and may be high-normal despite low Cp concentrations /2/.

Free Cu: the non-Cp-bound Cu is a diagnostic criterion. Normal concentration of free Cu is < 150 μg/L and > 250 μg/L in WD. The free Cu is calculated based on Cu concentration (μg/L) and Cp concentration (mg/L). The amount of Cu bound per mg of Cp is 3.15 μg. Accordingly, the concentration of free Cu is the difference between the serum Cu concentration and 3.15-fold the Cp concentration. Example: Cp = 200 mg/L, Cu = 830 μg/L. Free Cu (μg/L) = 890 – 3.15 × 200 = 260. It is important to correctly follow the Cu and Cp determination method because otherwise negative values will be obtained.

Copper/caeruloplasmin ratio (μmol/g) /19/: 5.05–8.09. Values are 2.5th and 97.5th percentiles. Patients without WD or copper deficiency.

Urinary excretion of COPPER

The Cu excretion in 24 h urine reflects the Cu not bound to Cp in the circulation. For the analysis of the entire urine collection, the creatinine and the urine volume must be determined. Normally, urinary excretion of Cu is < 40 μg (0.63 μmol)/24 h in healthy individuals. The amount of Cu is to be determined using atomic absorption spectrometry. An excretion of > 100 μg (1.6 μmol)/24 h is found in patients with Wilson’s disease, who are clinically symptomatic; however, 16–23% of the patients have lower values /1/. Therefore, many laboratories have defined 40 μg (0.63 μmol)/24 h as the threshold value. In cases with a fulminant hepatitic course, 10-fold higher excretion rates are measured. It is important to collect the urine samples in Cu-free containers. Cu excretions > 100 μg (1.6 μmol)/24 h are also observed in cholestatic and autoimmune liver diseases.

D-penicillamine challenge /15/: this is a confirmatory assay performed in children with Cu excretion below 100 μg (1.6 μmol)/24 h. First, the basal Cu excretion in 24 h urine is determined. This is followed by oral administration of 500 mg D-penicillamine before the next 24 h urine collection and again by oral administration of the same dose at half of the collection period after 12 hours. Urinary Cu excretion above 1600 μg (25 μmol)/24 h points to Wilson’s disease /1/. Diagnostic sensitivity for Wilson’s disease is below 100% /1/. In adults /16/, the assay is performed with various modifications. If the assay is performed as in children, a 20-fold increase in Cu excretion is considered an indication of Wilson’s disease.

concentration OF COPPER in liver TIssue  /1/

A Cu concentration in the liver parenchyma > 250 μg/g of dry weight is considered a biochemical proof of Wilson’s disease. Cu content of the liver is normally < 50 μg/g of liver dry weight; in untreated Wilson’s disease, it is 250–3000 μg/g of dry weight. However, such values can also be exceeded in the case of cholestatic liver disease such as primary biliary cirrhosis. In patients with Indian childhood cirrhosis, values of up to 6000 μg/g of dry weight are reached. The main problem in Cu determination is the inhomogeneous distribution of Cu in the liver in later stages of Wilson’s disease. In extreme cases, the liver consists of cirrhotic lesions that do not contain any Cu. Further analyses should be performed if Cu concentrations are 70–250 μg/g of dry weight.

Kayser-Fleischer corneal rings (KFC ring)

The green-brown rings in the Descemet’s membrane of the cornea are detected by means of a slit lamp. KFC rings are not detectable in 27–73% of patients at the time of clinical presentation of the isolated hepatic type of Wilson’s disease, but most patients who show neuropsychiatric symptoms, have the KFC ring /17/. Patients with cholestatic liver disease can also have the KFC ring.

Genetic analysis

The affected gene ATP7B encodes the protein ATP7B for Cu-transport. More than 500 different mutations across almost the entire encoded region of ATP7B are known. However, most of these mutations are rare and only found in individual patients. Moreover, many patients have compound heterozygosity, where each of the two characteristics of a carrier is affected by a different mutation (e.g., M769V combined with H1069Q). This means that two different multiplex PCR and DNA strip technologies are to be employed in the lab analysis. Thus, it is possible to detect the four Europe-wide common mutations M769V, W779X, H1069Q and P1134P-fs in a commercially available assay /18/.

Table 18.7-5 Scoring systems for Wilson’s disease diagnosis, developed in Leipzig in 2001 /1/

Score system I

Points

Score system II

Points

Kayser Fleischer rings present

2

Liver copper

Neurologic symptoms

> 5 × ULN (> 250 μg/g)

2

Severe

2

50–250 μg/g

1

Mild

1

< 50 μg/g (normal)

–1

Absent

0

Rhodanine positive granules

1

Ceruloplasmin (serum)

Urinary copper

> 0.2 g/L (normal)

0

> 2 × ULN

2

0.1–0.2 g/L

1

1–2 × ULN

1

< 0.1 g/L

2

Normal

0

Hemolytic anemia

Normal but > 5 × ULN after D-pen

2

Present

1

Mutation analysis

Absent

0

On both chromosomes detected

4

On one chromosome detected

1

No mutation detected

0

Evaluation in score system I or score system II: 4 or more points = diagnosis established;

3 points = diagnosis possible, more tests are needed; 2 or less points = diagnosis very unlikely

* absence of acute hepatitis, ULN, upper level of normal

Table 18.7-6 Diseases that may be associated with Cp deficiency

Clinical and laboratory findings

Wilson’s disease /120/

Wilson’s disease is a hereditary copper metabolism dysfunction with an autosomal recessive mode of inheritance. Prevalence of individuals afflicted worldwide is estimated at 1 : 5000 to 1 : 30,000, while that of heterozygous carriers is estimated to be 1 : 90. Afflicted individuals are always homozygous – both mother and father carry a Wilson’s disease gene.

Patients with liver cirrhosis, neurological manifestations and Kayser-Fleischer corneal rings correspond to the classic type of Wilson’s disease and are easy to diagnose. Patients present between 5 and 40 years of age. However, half of Wilson’s disease patients with liver involvement represent a diagnostic challenge because they do not even fulfill two of the above-mentioned three criteria /21/. In such cases, diagnosis is based on the clinical symptoms of liver disease, neurological symptoms and the classification of laboratory findings.

Hepatic presentation: patients with hepatic Wilson’s disease usually present in late childhood or during adolescence. They can be asymptomatic, where only the liver is enlarged and aminotransferases are elevated, or are afflicted with acute hepatitis, fulminant liver failure, chronic progressive hepatitis or macro nodular-type liver cirrhosis. Coombs-negative hemolytic anemia and acute liver failure can also be present. Some patients undergo transient episodes of jaundice due to hemolyses. Autoimmune hepatitis is primarily suspected in children in many cases. Most patients with acute liver failure show a characteristic spectrum of findings (Tab. 18.7-7 – Findings in patients with Wilson’s disease and acute liver failure).

Neurological presentation: clinical manifestations start on average in the second to third decade of life. Manifestations include motion abnormalities with dystonia similar to Parkinson’s disease, hypertonicity and rigidity, either chorea-like or pseudo sclerotic, tremor and dysarthria.

Nutritional Cu deficiency

Nutritional Cu deficiency causes hypochromic microcytic anemia comparable to that seen in iron-deficient anemia. Underlying causes may include, for example, malabsorption or prolonged parenteral nutrition. While nutritional Cu deficiency is rare in man, it is common in sheep in certain regions.

Menkes disease /21/

Mutations in ATP7A gene on chromosome Xq 13.3 are typically detected in Menkes disease, an X-linked recessive disorder. The absorbed Cu is not released into the circulation and Cu is accumulated in the red enterocytes. This results in complete Cu deficiency. This condition is also referred to as Menkes disease. Menkes disease is a multisystemic lethal disease with neurodegenerative symptoms and connective tissue manifestations. The normal ATP7A gene encodes the synthesis of the intracellular Cu efflux exporter ATP7A (Cu-transporting alpha polypeptide). See also Section 10.4 – Copper (Cu).

Clinically, Menkes disease presents in males at 2–3 months of age with loss of previously obtained developmental milestones and the onset of mental retardation, seizures, feeding problems, loose skin, loose joints, facial dysmorphism and abnormal hair. Usually, these children do not live past the age of 3 years.

Laboratory findings: low serum concentrations of Cu and Cp.

Hereditary Cp deficiency /3/

Hereditary Cp deficiency is an autosomal recessive disorder and has only been described in a few families to date. There is no Cu overload of the body. The defect is due to a mutation of the Cp-encoding gene on chromosome 3q25. Patients may present with blepharospasm, retinal degeneration, diabetes mellitus or dementia.

Laboratory findings: usually serum concentrations of Cp are below 0.02 g/L, Cu below 90 μg/L (1.4 μmol/L), urinary Cu excretion is below 50 μg (0.8 μmol)/24 h, Cu in hepatic tissue is below 50 μg/g of dry weight.

MEDNIK syndrome

MEDNIK represents the syndromic constellation of mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia. Mutations in the gene AP1S1 disturb the systemic metabolism of Cu by pertubing Cu ATPase trafficking, resulting in hepatic Cu accumulation as in WD and low levels of Cu and Cp in serum /5/.

Huppke-Brendl syndrome

Homozygous or compound heterozygous mutations in the gene SLC33A1 which encodes the acetylCoA transporter AT-1 causes low serum Cu and Cp levels. Huppke-Brendl syndrome is a lethal autosomal recessive disorder of congenital cataracts, hearing loss, and severe developmental delay /5/.

Aceruloplasminemia

Aceruloplasminemia is a neurodegenerative disorder with brain iron accumulation. It may be confused with WD due to very low or absent Cp levels in patients who may present with symptoms and brain imaging findings suggestive of WD /5/.

Table 18.7-7 Findings in patients with Wilson’s disease and acute liver failure /1/

Coombs negative hemolytic anemia

Coagulation defect that cannot be corrected by vitamin K administration

Fast progression of renal insufficiency

Relatively moderate increase in aminotransferases (markedly below 2000 U/L) in relation to acute hepatitides caused by hepatotropic viruses

Normal or subnormal alkaline phosphatase

Men women ratio 2 : 1

Table 18.8-1 Reference intervals for haptoglobin and hemopexin

Type-independent /4/*

Type-dependent /5/*

12 months

0.02–3.0

Adults

10 yrs

0.08–1.72

Hp 1-1

0.57–2.27

0.27–1.83

Hp 2–1

0.44–1.83

16 yrs

0.17–2.13

Hp 2–2

0.38–1.50

0.38–2.05

Hx /6/

0.50–1.15

25 yrs

0.34–2.27

 

0.49–2.18

 

50 yrs

0.47–2.46

 

0.59–2.37

 

70 yrs

0.46–2.66

 

0.65–2.60

 

Data expressed in g/L. * Values are the 5th and 95th percentiles.

Table 18.8-2 Assessment of haptoglobin (Hp) in combination with C-reactive protein (CRP)

Hp
(g/L)

CRP
(mg/L)

Clinical significance

< 0.2

≤ 5

Hemolytic reaction, hepatocellular disease

< 0.2

> 5

Hemolysis and acute phase response

> 0.2

> 5

Acute phase response, possibly mild hemolysis

> 0.2

≤ 5

Normal

Table 18.8-3 Investigations on the diagnosis and monitoring of hemolytic anemias

Investigation

Reticulocyte count

Reticulocytosis is found to occur in hemolytic anemias and even in cases of compensated hemolysis. In the presence of an acute hemolytic episode, the reticulocyte count rises within 24 hours. The positive predictive value of reticulocytosis for the presence of a hemolytic reaction is only 38% compared to decreases in Hp level to ≤ 0.20 g/L /14/.

Haptoglobin

After the occurrence of intravascular hemolysis, Hp concentration declines rapidly since the half-life of the Hp/Hb complex is only 8 min.

Hemopexin

A reduction in Hx is only measurable in the presence of free heme in serum. The half-life is 7–8 hours; therefore, significant decreases take several hours to occur.

Hemoglobin (Hb)

Plasma contains less than 20 mg of Hb/L. The transport capacity of Hp is exhausted as soon as an amount of about 15 g of Hb (2 fold the daily Hb turnover) is liberated from erythrocytes; free Hb occurs and is excreted renally. Above a concentration of 0.3 g/L, free Hb is visible to the naked eye; at a concentration of more than 0.5 g/L, plasma has a yellow-reddish appearance (not in myoglobinemia). Severe hemolytic transfusion reactions always cause a visible red discoloration of the serum and lead to hemoglobinuria.

Methemalbumin

This substance represents heme bound to albumin. It occurs in the event of pronounced intravascular hemolysis, is detectable at the earliest 5 hours after the hemolytic episode and remains positive for about 24 hours. Presence of methemalbumin is associated with a coffee-brown appearance of serum and is detected in the Schumm test /7/.

Hemosiderin

Analysis of urine for hemosiderin is a good screening test for detection of chronic hemolytic anemia or recent acute hemolytic episode. The test shows small blue clumps or intracellular granules in cells of the urinary sediment stained with Prussian blue. These changes are detectable 2–5 days after the acute hemolytic event.

Bilirubin

Clinically, mild jaundice is one of the most important symptoms indicating the presence of hemolytic disease. This also applies to transfusion reactions due to antibodies which cause an early reaction (e.g., anti-D, -c, -S, -Fya, -K). An elevated concentration is usually not found until the hemolysis rate exceeds 5% (normal hemolysis rate is approximately 1%). Rises in bilirubin level occur after 6–12 hours. With the exception of transfusion-related immediate-type reactions (anti-A, anti-B), total bilirubin rarely exceeds a level of 5 mg/dL (85 μmol/L).

Lactate dehydrogenase

Elevated activities are found mainly during the course of hemolytic episodes and in hemolytic transfusion reactions. Elevated levels also usually occur in megaloblastic anemias.

Direct Coombs test, indirect Coombs test, cold agglutinin screening test, hemolysins

The direct Coombs test detects erythrocyte-bound alloantibodies (e.g., in a transfusion reaction and the presence of autoantibodies). In a study /17/ involving patients with immune hemolysis, the underlying causes were: warm antibodies in 78%, cold agglutinins in 15%, drug-induced antibodies in about 2% and bithermal hemolysins in almost 1%. In autoimmune hemolytic anemia, the direct Coombs test remains negative in 1–6% of the cases. False-positive findings are due to in vitro complement activation (e.g., if the blood sample is stored at low temperature; this is not the case if EDTA blood is used). Detection of free antibodies by means of a positive indirect Coombs test points to severe hemolysis /17/.

Peripheral blood smear

Microspherocytes (hereditary spherocytosis), elliptocytes (hereditary elliptocytosis), target cells (thalassemia, HbC disease), sickle cells (sickle cell anemia), basophilic stippling, Howell-Jolly bodies (thalassemia, sickle cell anemia).

Heinz body test

Positive in the presence of the following enzyme defects: G-6-PD deficiency, glutathione reductase deficiency as well as in hemoglobinopathy and toxically induced poisoning with oxidants.

Osmotic resistance

Decreased: spherocytosis, antibody-induced hemolytic anemia

Increased: target cells, hypochromic erythrocytes.

Autohemolysis test

Type I: hereditary spherocytosis, G-6-PD deficiency

Type II: pyruvate kinase deficiency, autoimmune hemolytic anemia.

Determination of CD16b

Absent or decreased in paroxysmal nocturnal cold hemoglobinuria.

DL antibodies

Donath-Landsteiner antibodies lead to paroxysmal cold hemoglobinuria.

HEMOGLOBIN electrophoresis

Qualitative and quantitative hemoglobinopathies (e.g., thalassemia, sickle cell anemia).

Erythrocyte enzymes

G-6-PD deficiency is the most frequently encountered defect, pyruvate kinase deficiency is the second most common defect, followed by glucose-phosphate isomerase defect.

Carbonic Anhydrase Immunoreactivity

Mild hemolysis occurs physiologically in neonates,but more severe forms can lead to life-threatening anemia. Carbonic anhydrase immunoreactivity (CAI) is increased in neonates with raised serum bilirubin (> 7.3 mg/dL; 125 μmol/L) including those qualifying for phototherapy. Newborns with serum bilirubin < 7.3 mg/dL (125 μmol/L) and CRP > 20 mg/L produce urine with strong CAI immunoreactivity. CAI is a direct biomarker of intravascular hemolysis in newborn infants. CAI is determined using an ELISA with primary goat anti-human CAI polyclonal antibody (R&D biosystems, Novus) /24/.

Table 18.8-4 Classification of hemolytic anemias /18/

Hereditary hemolytic anemias

Membrane defects: spherocytosis, elliptocytosis, stomatocytosis, acanthocytosis

Enzyme defects (with permanent hemolysis), e.g., G-6-PD deficiency, pyruvate kinase deficiency

Enzyme defects (with hemolytic episodes), e.g. favism, primaquine-induced hemolysis in conjunction with G-6-PD deficiency

Hemoglobinopathies: due to

  • Excess of physiologic hemoglobins such as HbA2, HbF in thalassemias
  • Production of abnormal hemoglobins with an aggregation tendency, HbC, HbS (sickle cell anemia)
  • Production of unstable hemoglobins (positive Heinz body test).

Acquired hemolytic anemias

Autoimmune hemolytic anemia: warm autoantibodies (e.g., drug- or infection-induced), cold autoantibodies (e.g.; infection-induced), bithermal hemolysins

Hemolytic transfusion reaction

Toxic hemolytic anemias: heavy metals, oxidants

Mechanically induced hemolytic anemias: march hemoglobinuria, micro-angiopathies, disseminated intravascular coagulation, hemolytic uremic syndrome, membrane defects (acanthocytosis in the presence of liver diseases)

Infection-induced hemolytic anemias: malaria, neuraminidase-induced ones (e.g., in bacterial infection)

Ineffective erythropoiesis: vitamin B12 deficiency, folate deficiency, myelodysplastic syndrome

Paroxysmal nocturnal hemoglobinuria.

Table 18.8-5 Haptoglobin in hemolysis and diseases associated with various haptoglobin haplotypes

Clinical and laboratory findings

Intravascular hemolysis

For the detection of hemolytic reactions and their differentiation from non-hemolytic disorders, Hp concentrations ≤ 0.2 g/L have a diagnostic sensitivity of 83% and a diagnostic specificity of 96% /14/. Daily hemolysis of 1% of the blood in addition to physiological destruction of senescent erythrocytes, corresponding to hemolysis of about 40 mL of red blood cells or 15 g of Hb, is sufficient to saturate the Hp transport capacity with Hb, resulting in complete clearance of Hp from the plasma. This stage is reached, for example, if in intravascular hemolysis the erythrocyte life-span decreases to below 55% (about 60 days) /19/. Under these circumstances, Hp is no longer measurable in serum.

Intravascular hemolysis accounts for approximately 10–20% of total erythrocyte destruction /19/. Most of the process is extravascular and primarily takes place within the spleen. The intravascular/extravascular ratio varies considerably in hemolytic reactions. Hp is low in immunohemolytic, microangiopathic, mechanical (cardiac valve replacement), drug-induced (G-6-PD deficiency) and infection-induced (malaria) hemolyses.

Some hereditary chronic hemolytic anemias as well as megaloblastic anemia and hypersplenism only show decreased Hp concentrations during a hemolytic crisis.

HAptoglobin-associated diseases – Generally

Numerous diseases are associated with the HP alleles. It has been reported, for example, that individuals with the Hp 1-1 phenotype are preferably susceptible to chronic hepatitis C, liver cirrhosis and cardiovascular disease. Those with the Hp 2-1 phenotype are more prone to ovarian cancer and esophageal cancer. The Hp 2-2 phenotype is often associated with essential atherosclerotic hypertension, gastric cancer and lung carcinoma /6/. Other publications list even more associations /20/. Three recent examples are described in the following.

– Fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. It is characterized by obesity, dyslipidemia, diabetes mellitus and insulin resistance. In the industrialized countries, NAFLD is the essential cause of abnormal liver function and hepatic fibrosis. In an investigation /21/, the incidence of NAFLD was higher in Hp 2-2 phenotype carriers (odds ratio 11.7) than in carriers of the other two phenotypes, and ferritin concentrations were also higher. Ferritin concentrations were positively correlated with ALT activities. It is assumed that elevated ferritin concentrations released due to increased hepatocyte injury are a risk factor for insulin resistance and influence NAFLD progression.

– Diabetes mellitus

The risk of myocardial infarction is several times higher in patients with diabetes mellitus and the Hp 2-2 phenotype than in those with the Hp 1-1 phenotype, as shown in the Strong Heart Study /22/. This is thought to be caused by the elevated HbA1c value in diabetics. The binding capacity of this glycoprotein to Hp of Hp 2-2 phenotypes and, thus, its anti oxidative function, is smaller than in Hp 1-1 genotypes. Unbound free Hb increases oxidative stress in individuals of the Hp 2-2 phenotype and consequently also the rate of myocardial infarction.

– Celiac disease

Up to 1% of the European and North-American population suffer from celiac disease. Celiac disease is a T-cell mediated, chronic inflammatory autoimmune disease induced by wheat gluten or barley and rye proteins. The HLA antigens DQ2 or DQ8 seem to influence the development of the disease in 50% of the patients. The Hp haplotype also plays a role. It has been shown, for example, that phenotypes Hp 1-1, Hp 2-1 and Hp 2-2 had severe malabsorption with the following incidences: 5.6%, 49.1% and 45.3% /15/.

Erythrocytes undergo progressive deleterious morphological and biochemical changes during storage. Transfusion of packed red blood cells (PRBCs) that have been stored for prolonged intervals increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion, but also by high concentrations of hemoglobin and heme that are associated with the transfusion of PRBCs stored for 35–40 days. Treatment with haptoglobin or hemopexin improved the survival rate and attenuated heme induced inflammation /23/.

Table 18.8-6 Causes of a divergent behavior pattern between Hp and Hx

Hp

Hx

Cause

n

Mild hemolytic condition

n

Thalassemia (occasional finding), hemorrhagic pancreatitis, hemorrhage into body cavities, masked reduction in Hp (e.g., due to an inflammatory reaction).

Nephrotic syndrome

n, normal; , increased; , decreased

Table 18.9-1 Physicochemical and biological properties of the Ig classes /123/

Properties

IgG

IgA

IgM

IgD

IgE

Molecular
weight (kDa)

150

160

971

175

190

Carbohydrate
content (%)

3

7

10

9

13

Molecular
formula

γ2 κ2

γ2 λ2

α2 κ2

α2 λ2

2 κ2)5

2 λ2)5

δ2 κ2

δ2 λ2

ε2 κ2

ε2 λ2

Synthesis rate

33

65

6–7

0.4

0.016

Antibody valence

2

2

5

2

2

H-chain domains

4

4

5

4

5

H-chain isotopes

4

2

2 (?)

H-chain allotypes

25

2

1

Serum level (g/L)

7–16

0.7–
5.0

0.4–
2.5

0.04–
0.40

0.2*

Half-life (days)

7–21

6

5

2.8

2.5

Complement
activation**

+

+++

Agglutination
capability

+

±

+++

Placental transfer

+

Fc receptor binding to

  • Macrophages

+

+

±

  • Neutrophil
    granulocytes

+

+

Opsonization

+

+++

Hypersensitivity reaction

Type I

+++

Type III

+

+

* Values expressed in mg/L; ** classic pathway; synthesis rate in mg/kg body weight per day

Table 18.9-2 IgG, IgA, IgM reference intervals in serum/plasma /1/

Age

IgG

IgA

IgM

Newborn

6.6–
17.5

0.01–
0.06

0.06–
0.21

0.06–
0.21

Infants/
children

1 month

3.9–
10.5

2 months

2.5–
6.8

3 months

2.0–
5.5

0.1–
0.34

0.17–
0.66

0.17–
0.66

4 months

2.0–
5.4

5 months

2.2–
6.0

6 months

2.6–
6.9

0.08–
0.6

0.26–
1.0

0.26–
1.0

7 months

2.9–
7.7

8 months

3.2–
8.4

9 months

3.3–
8.8

0.11–
0.8

0.33–
1.3

0.33–
1.3

10 months

3.5–
9.1

11 months

3.5–
9.3

12 months

3.6–
9.5

0.14–
0.9

0.37–
1.4

0.40–
1.5

2 yrs

4.7–
12.3

0.21–
1.5

0.41–
1.6

0.47–
1.8

4 years

5.4–
13.4

0.30–
1.9

0.43–
1.6

0.52–
1.9

6 yrs

5.9–
14.3

0.38–
2.2

0.45–
1.7

0.56–
2.1

8 yrs

6.3–
15.0

0.46–
2.5

0.47–
1.8

0.60–
2.2

10 yrs

6.7–
15.3

0.52–
2.7

0.48–
1.8

0.62–
2.3

12 yrs

7.0–
15.5

0.58–
2.9

0.49–
1.8

0.65–
2.4

14 yrs

7.1–
15.6

0.63–
3.0

0.50–
1.8

0.66–
2.5

16 yrs

7.2–
15.6

0.67–
3.1

0.50–
1.9

0.68–
2.6

18 yrs

7.3–
15.5

0.70–
3.2

0.51–
1.9

0.68–
2.6

Adults

7.0–
16.0

0.70–
5.0

0.40–
2.3

0.40–
2.8

Data expressed in g/L; values expressed as 5th and 95th percentiles.

Table 18.9-3 Laboratory findings in secondary immunoglobulin deficiency

Clinical and laboratory findings

Malignant tumor

Solid tumors do not cause significant reductions in Ig until they have reached a far advanced or terminal stage.

Lymphocytic leukemia

In children with acute lymphocytic leukemia, Ig concentrations are significantly lower in 30% of cases than in a group of age-matched healthy children. Up to 50% of patients with chronic lymphocytic leukemia show a decrease in Ig, particularly in IgA and IgM.

Thymoma

About 5–10% of patients develop antibody deficiency that persists even after tumor resection. The Ig are reduced to about one half of the levels of the lower reference interval value.

Multiple myeloma, Waldenström’s macroglobulinemia

Approximately 65% of patients with multiple myeloma have antibody deficiencies in the presence of an M-gradient detected by serum protein electrophoresis. The underlying cause for this are suppression mechanisms possibly exerted in part by light chains since the suppression of polyclonal Ig production is especially pronounced in the presence of a light chain myeloma. Another cause, especially in patients with IgG myeloma, is accelerated Ig catabolism. In patients with Waldenström’s macroglobulinemia, the concentration of polyclonally produced Ig is less decreased than in those with multiple myeloma.

Iatrogenic causes

The most predominant causes are therapies with corticosteroids and cytostatic drugs such as cyclophosphamide, methotrexate, azathioprine, 6-mercaptopurine, as well as radiation therapy.

Immunosuppressive therapy following transplantation

During the first month after transplantation, there is an aplastic phase with marked granulocytopenia and lymphopenia as well as Ig reduction. Approximately from the 3rd month (after normalization of granulocytes and T lymphocytes), the antibody production is activated, often with an excessive rise in IgM, followed by IgG and finally by IgA.

Protein-losing enteropathy (e.g., due to intestinal lymphangiectasia)

Nonselective loss of plasma proteins into the intestine. This affects all Ig equally. The immunoreactivity is not impaired until lymphocytes move into the intestine at an increased rate and, hence, lymphopenia develops.

Nephrotic syndrome

Mostly IgG is renally lost, IgM may be increased. The decrease in Ig rarely leads to clinical manifestations.

Burns

In the presence of extensive burns involving large areas of the skin, major losses of Ig, lymphocytes and granulocytes occur. The antibody production and cellular immune response may be strongly impaired.

Viral infections (e.g., measles, rubella, EBV infections)

The impaired immune defense observed in the case of measles is present for about 6 weeks and affects the cellular as well as the humoral immune response; Ig may be low. A decrease in Ig has been described during the course of rubella infections. EB viruses may cause symptoms of a variable immunodeficiency syndrome.

Table 18.9-4 Pattern of IgG, IgA, IgM in liver diseases /10/

Clinical and laboratory findings

Hepatitis A

Marked increase in IgM with the highest levels during the first week of the illness. Decline in IgM during the 2nd week and normalization after 4 weeks. IgG rises about 2 weeks after the onset of the disease, reaches a maximum during the 3rd to 4th week, and normalizes by the 8th week.

Hepatitis B, C

Increases in IgM and IgG at the onset of the disease. IgM usually shows lower concentrations than those seen in hepatitis A. Normalization of IgM and IgG tends to be slower than in hepatitis A. IgG normalizes within 6 months. An increase in IgA suggests preceding liver damage.

Chronic persistent hepatitis

Develops from acute viral hepatitis in the case of viral persistence. A continuous mild to moderate increase in IgM may be present besides increased IgG.

Chronic active hepatitis B, C, autoimmune hepatitis

An increase in IgG during the course of hepatitis, especially if combined with a decline in IgM, indicates the transition to a chronic course of the disease. It is characterized by an almost exclusive increase in IgG. Antinuclear antibodies and antibodies to smooth muscles are often positive. Autoimmune hepatitis may be associated with an excessive increase in IgG, which can be noticed by a γ-globulin fraction > 40% and a pseudo M-gradient on serum protein electrophoresis.

Chronic destructive cholangitis, primary biliary cirrhosis

Mainly women ≥ 35 years of age are affected. Characteristic findings include a relatively strong IgM elevation in the Ig pattern, elevated alkaline phosphatase and detection of mitochondrial antibodies.

Liver cirrhosis

IgG, IgA and IgM elevations are common. Conclusions concerning the pathogenesis are possible. A relatively marked IgG increase points to posthepatitic cirrhosis.

Alcohol-induced liver disease

Fatty liver, hepatitis, fibrosis and cirrhosis represent histopathological manifestation forms of alcohol-induced, chronic liver damage. Development of alcoholic cirrhosis depends on the duration of alcohol abuse and the amount of alcohol consumed daily. Concentrations of all three Ig classes rise as the liver tissue is increasingly undergoing parenchymal transformation from chronic hepatitis to fibrosis and finally to cirrhosis. However, the Ig classes (and especially IgA) do not differ significantly between groups of patients with and without ongoing alcohol abuse.

Table 18.9-5 IgE increase in non-atopic diseases

Clinical and laboratory findings

Parasitic infection

IgE concentrations that may exceed 10-fold the upper reference interval value are found in parasitic infections with ascarides, echinococci, oxyuris, toxocara canis, schistosoma, necator americanus, fasciola hepatica, ancylostoma and trichinae as well as in intestinal capillariasis and intestinal myiasis /12/. In principle, helminthic infections (e.g., those involving nematodes, cestodes and trematodes) cause IgE elevations while protozoal infections do not (refer to Chapter 44). IgE levels decline after successful antiparasitic therapy.

AIDS, Wiskott-Aldrich syndrome, Nezelof syndrome, non hodgkin lymphoma

IgE synthesis is controlled by Th2 cells. Diseases with T cell dysfunction can, therefore, be associated with a rise in synthesis of IgE and elevated serum IgE concentrations. B-cell defects, on the other hand, cause decreased IgE levels; for instance, non hodgkin lymphomas of the B-cell lineage are associated with decreased serum IgE concentrations while those of T-cell lineage show increased serum levels.

Malignant tumor

Malignant tumors in tissues with a high content of IgE-producing B cells may be associated with elevated IgE levels. Such malignancies include, for example, tumors of the ear-nose-throat region, bronchopulmonary system, gastrointestinal tract, mesenteric lymph nodes and testes.

Hyper-IgE syndrome /13/

The hyper-IgE syndrome, also known as Job syndrome, is a very rare primary immunodeficiency.

Clinical manifestation: approximately 80% of the patients have moderately severe to severe eczema that is typically itchy and lichenified. Moreover, characteristic manifestations include chronic skin infections in the form of boils and abscesses, mainly due to S. aureus. Other characteristic symptoms include excessive joint flexibility and a typical facies with prominent forehead, mild prognathism, broad nose, coarse complexion and hypertelorism. Abnormalities develop with increasing age. Occurrence of hyper-IgE syndrome has also been described in conjunction with non hodgkin lymphomas of high-grade malignancy /14/.

Laboratory findings: elevated IgE; diagnostic values (mIU/mL) indicating hyper-IgE syndrome in dependence of patient age: 15 (1–28 days), 150 (1–6 months), 250 (7–12 months), 650 (1–3 years), 1250 (4–6 years), 3300 (1–10 years), 2400 (11–14 years), 2000 (> 15 years). Eosinophilia with cell count > 0.7 × 109/L. IgG, IgA and IgM are normal.

Table 18.9-6 Causes of oligoclonal IgG increase in serum /15/

Clinical and laboratory findings

Antibodies to blood group antigens

Certain blood group antigens such as A1 or IH provoke the production of antibodies with limited heterogeneity. These are natural antibodies of the IgM class.

Viral infection

In many cases, an oligoclonal Ig increase consisting of antibodies to viral surface antigens occurs in the course of acute viral infections.

Immunosuppressive therapy

Oligoclonal Ig patterns occur during the first few weeks after organ transplantation. They indicate the beginning recovery of Ig synthesis under immunosuppressive therapy.

Autoimmune disease, parasitic infection

Constant stimulation of the immune system by an unchanged antigen spectrum causes an increasing selection of B-cell clones and a selective production of antibodies with an oligoclonal Ig pattern.

Mucosal infection

Immune reactions on mucous membranes (e.g., those of the genitourinary tract) may lead to an oligoclonal Ig increase.

Disease of the central nervous system (CNS)

Local, humoral immune response in the CNS causes a limited production of antibody populations, especially of the IgG class (oligoclonal IgG) because of the limited presence of immunocompetent B cells. An oligoclonal antibody pattern, detectable by means of isoelectric focusing in cerebrospinal fluid but not in serum, is a sign of localized Ig synthesis. It is of diagnostic value in certain CNS diseases (e.g., in multiple sclerosis) (refer to Section 46).

Immune complexes

Immune complexes in high concentrations such as rheumatoid factors may electrophoretically cause an oligoclonal band pattern (refer to Section 18.3.5.1).

Table 18.10-1 Properties of the IgG subclasses /12/

Properties

IgG1

IgG2

IgG3

IgG4

Molecular
weight (kDa)

146

146

170

146

Amino acids in
hinge region

15

12

62

12

Disulfide bonds
of the H-chains

2

4

11

2

Mean g/L in serum
(adults)

6.98

3.8

0.51

0.56

Relative
abundance (%)

60

32

4

4

Half-life (days)

21

21

7–21

21

Kappa/lambda ratio

2.4

1.1

1.4

8.0

Gm allotypes

4

1

13

2

Complement
(C1q) binding

++

+

+++

-

Placental transfer

++++

++

++++

+++

Antibody response
to proteins

++

+/-

++

++

Antibody response
to polysccharides

+

+++

+/-

+/-

Antibody response
to allergens

+

-

++

Fc receptor binding

Macrophages, PMN

++

+

±

Mast cells

+

Reaction with SPA

+

+

+

PMN, polymorphonuclear neutrophils; SPA, Staphylococcus aureus protein A

Table 18.10-2 IgG subclass reference intervals /34/

Age
(years)

IgG1

IgG2

IgG3

IgG4

0.5–1

1.5–
7.9

0.36–
1.4

0.09–
0.86

0.006–
0.46

1.4–
6.2

0.41–
1.3

0.11–
0.85

Up to
0.008

1–1.5

3.2–
9.2

0.26–
1.5

0.12–
0.88

0.007–
0.37

1.7–
6.5

0.40–
1.4

0.12–
0.87

0.12–
0.87

1.5–2

2.6–
7.8

0.42–
2.2

0.11–
0.97

0.017–
0.75

2.2–
7.2

0.50–
1.8

0.14–
0.91

Up to
0.41

2–3

2.7–
9.4

0.44–
1.9

0.09–
6.3

0.023–
0.59

2.4–
7.8

0.55–
2.0

0.15–
0.93

Up to
0.69

3–4

2.8–
13.7

0.44–
3.0

0.13–
0.84

0.05–
1.14

2.7–
8.1

0.65–
2.2

0.16–
0.96

0.01–
0.94

4–6

3.8–
11.7

0.73–
2.9

0.13–
0.75

0.013–
1.57

3.0–
8.4

0.70–
2.6

0.17–
0.97

0.02–
1.20

6–9

4.2–
9.9

0.63–
3.5

0.17–
0.88

0.01–
1.20

3.5–
9.1

0.85–
3.3

0.20–
1.04

0.03–
1.60

9–12

3.6–
11.2

0.89–
3.6

0.23–
0.83

0.052–
1.56

3.7–
9.3

1.0–
4.0

0.22–
1.09

0.04–
1.90

12–18

3.9–
10.0

1.02–
4.5

0.12–
1.02

0.061–
1.86

3.7–
9.1

1.1–
4.9

0.24–
1.16

0.05–
2.0

> 18 /4/

4.1–
10.1

1.7–
7.9

0.11–
0.85

0.03–
2.0

3.82–9
.29

2.42–
7.0

0.22–
1.76

0.04–
0.87

Data expressed in g/L. Values are the 2.5th and 97.5th percentiles. The reference intervals in the top rows are provided by Siemens, those in the bottom rows are provided by The Binding Site.

Table 18.10-3 Diseases commonly associated with IgG subclass deficiency

Recurrent bacterial infections:

  • Otitis
  • Pneumonia
  • Sinobronchial syndrome
  • Meningitis

Bronchiectasis

Intrinsic bronchial asthma

Therapy-resistant bronchial asthma

IgA deficiency

Therapy-resistant seizure disorder

Chronic intestinal disease

Autoimmune disease

HIV infection

Table 18.10-4 IgG subclass deficiency associated disease

Clinical and laboratory findings

IgG1 subclass deficiency

Since this subclass accounts for the highest proportion of total IgG, patients with IgG1 deficiency always also suffer from total IgG deficiency. In many cases, total IgG deficiency is, therefore, taken into consideration within the scope of immunodeficiency syndrome in patients with primary antibody deficiency or common variable immunodeficiency syndrome (CVID) /6/. Since IgG1 accounts for about 65% of total IgG, IgG1 deficiency leads to the electrophoretic finding of hypogammaglobulinemia. Reduction in IgG1 is usually accompanied by decreased IgG2 and IgG3. Low concentrations of IgG1 and IgG2 are detectable in patients with variable immunodeficiency syndrome and in secondary immunodeficiencies as seen in patients with nephrotic syndrome /14/.

IgG2 subclass deficiency

IgG2 deficiency occurs either isolated or in conjunction with IgA or IgG4 deficiency. Some of the patients with IgG2 deficiency show an increased susceptibility to infections with encapsulated bacteria such as H. influenzae type B, Streptococcus pneumoniae and other respiratory infection pathogens /726/. However, IgG2 deficiency does not predispose patients to recurrent respiratory infections. Autoimmune diseases and autoimmune cytopenias are also detected in conjunction with IgG2 deficiency.

IgG2 + IgG4 subclass deficiency

About one third of the children with low or non detectable IgG4 levels are also afflicted with IgG2 deficiency.

IgG2 + IgA subclass deficiency

In one fifth of the patients with IgA deficiency, a deficiency in IgG2 is also detectable. Patients with this immunodeficiency are prone to development of sepsis involving encapsulated bacteria. Patients with IgA deficiency are also at risk for infections; although these patients have normal IgG2 concentrations, their immune response to pneumococcal polysaccarides is inadequate /27/.

IgG3 subclass deficiency

Basically, IgG3 deficiency occurs isolated or in conjunction with IgG1 deficiency. IgG3 is significant in the immune response to respiratory viruses and Moraxella catarrhalis. IgG3 are thought to be the most effective virus-neutralizing antibodies. In a study involving 6580 patients with recurrent or severe infections, 4.8% had IgG3 deficiency, while in 60% it was isolated and in 36% it was combined with a decrease in IgG1 /28/. Essential clinical diagnoses were: recurrent infections of the upper airways, diarrhea and bronchial asthma.

IgG3 + IgG1 subclass deficiency

The infections that are associated with this combined immunodeficiency are more serious in nature and are often combined with obstructive pulmonary disease that at times leads to the development of bronchiectasis.

IgG4 subclass deficiency

Selective IgG4 deficiency has little clinical relevance since it is detectable in 5.6% of healthy individuals /14/. In patients with infections, IgG4 deficiency is combined with another IgG subclass deficiency, especially IgG2 and/or a defect in the polysaccharide-specific immune response /15/.

Table 18.10-5 Increases in IgG subclasses

Clinical and laboratory findings

HIV infection

Polyclonal increase, preferentially in antibodies of the IgG1 and IgG3 subclasses.

Allergic alveolitis

Massive increase in allergen-specific IgG2 antibodies.

Frequent bee stings

An IgG4 dominated immune response has been recorded in frequently stung beekeepers.

Immunotherapy in inhalative allergy

During 5 years of monitoring, patients with grass pollen allergy and allergy caused by D. pteronyssimus show a 50% increase in IgG4 compared to the initial concentration /29/. This does not apply to untreated allergy sufferers. The increase in IgG4 in these patients is caused by immunomodulation under treatment. A continuous increase in the allergen-specific IgG4/IgG1 ratio has also been recorded.

Alcohol-induced sclerosing pancreatitis

Patients have IgG4 concentrations of 1.36–11.5 g/L as compared to 0.15–1.28 g/L in healthy individuals /30/. This does not apply to non-alcoholic pancreatitis and Sjögren’s syndrome.

Autoimmune pancreatitis (AIP) /31/

AIP is a special type of chronic pancreatitis. In 30% of the cases, patients also show other manifestations of autoimmune disease. AIP is thought to be the pancreatic manifestation of a clinicopathological entity of IgG4-related disease. Elevated concentrations of total IgG (≥ 18 g/L) and IgG4 (> 135 mg/dL) are considered a diagnostic criterion of AIP with a diagnostic sensitivity of 42–70% and 73–90%, respectively /32/. However, the sensitivity of IgG4 in different populations varies worldwide. For example, diagnostic sensitivities were 73.3% in a Korean study, 53.8% in a Spanish study and about 20% in French and German studies.

Table 18.11-1 Types of cryoglobulins

Type

Composition

I

Only monoclonal immunoglobulins (Ig)

II

Mixture of monoclonal Ig, usually IgM type κ, and polyclonal IgG

III

Mixture of polyclonal Ig, usually IgM, and polyclonal IgG

Table 18.11-2 Measurement and differentiation of cryoglobulins /1/

1.

10 mL of blood is collected into a pre warmed EDTA tube and a pre warmed serum tube.

2.

Allow the blood to clot in a water bath at 37 °C.

3.

Serum is obtained by centrifugation at 37°; possibly, the centrifuge is warmed up by running for 30 min. without samples.

4.

Plasma and serum are aspirated using pre-warmed pipettes and separately transferred into tubes for determination of the cryocrit.

5.

The tubes are incubated at 4 °C for up to 7 days.

6.

Both tubes are centrifuged at 4 °C for 15 min.

7.

The cryocrits are evaluated. The cryocrit in the plasma tube minus cryocrit in serum tube represents the proportion of cryofibrinogen.

8.

For differentiation of the cryoglobulins: the serum is removed from the precipitate in the centrifugation tube. Precipitate is washed three times with ice-cold 0.9% NaCl and then resuspended in a small volume of NaCl of 37 °C.

9.

Protein determination is performed as well as immunofixation electrophoresis or a Western blot.

Table 18.11-3 Cryoglobulinemias at the Sheffield Protein Reference Unit /9/

Type

Cases

%

Concentration (g/L)

Mean

Median

Interval

I

27

14.4

4.1

2.1

0.05–25

II

42

22.3

2.5

0.95

0.13–15

III

119

63.3

0.72

0.48

0.04–2

Cryo

5

 

2.6

0.79

0.15–7.9

Cryo, cryofibrinogen

Table 18.11-4 Diseases and conditions associated with mixed cryoglobulinemia

Clinical and laboratory findings

Mixed cryoglobulinemia – Hepatitis C (HCV) /16/

Approximately 90% of the patients with type II mixed cryoglobulinemia have HCV infection. The cryoglobulin contains an IgM kappa rheumatoid factor with anti-idiotypic activity. Cryoglobulins of type III are seen more rarely. The prevalence of cryoglobulinemia in HCV patients is 25–30%, but only 10–15% of these patients show symptoms that vary in severity, ranging from mild to life-threatening. Cryoglobulinemia leads to vasculitis that affects small- and medium-sized vessels of the skin, kidneys and peripheral nerves. HCV core protein and immunoglobulins are homogeneously distributed in the vessel walls. The pathophysiological pathway is as follows:

When incorporating HCV particles and HCV core protein via toll-like receptors, dendritic cells release B-cell activating factor (BAFF). BAFF acts as a survival signal for marginal zone B-cells and VH1-69+B1 cells and maintains strong clonal expansion. The cells synthesize large amounts of IgM with rheumatoid factor (RF) activity. IgM-RF molecules bind HCV particles forming cold-precipitable, multi molecular immune complexes. These, in turn, bind C1q, thus mediating binding to the C1q receptors in the vessel wall. Finally, this leads to complement activation /20/.

– Purpura

Purpura is the most common symptom and occurs in 80–90% of the patients. Palpable lesions appear on the lower legs, buttocks and trunk. In many cases, they are preceded by paresthesia and pain.

– Raynaud phenomenon

Affects about one third of the patients. It involves hands, feet, lips, external ear and nose. In severe cases, patients develop necrosis and digital gangrene.

– Nephropathy

Up to 40% of the patients develop nephropathy. The most common type is membranoproliferative glomerulonephritis. This acute nephritic syndrome is associated with proteinuria, hematuria and hypertension.

– Peripheral neuropathy /9/

Up to 60% of the patients have motor sensory axonopathy. It is caused by impaired blood flow through the vasa nervorum due to deposition of cryoglobulins.

– Arthralgia

Approximately 70–80% of the patients have arthralgia, 20% have myalgia or fibromyalgia.

– Sjögren syndrome

Approximately 5–61% of the patients with Sjögren syndrome have mixed cryoglobulinemia and half of these have chronic HCV infection.

Table 18.12-1 Reference intervals for β2-microglobulin

Serum, plasma

0.8–2.4 mg/L (< 60 years) /2/

≤ 3.0 mg/L (> 60 years) /2/

0.7–1.8 mg/L* /3/

Cord blood

2.5–4.5 mg/L /4/

Random urine

≤ 200 μg/g creatinine /5/

≤ 300 μg/L /5/

≤ 200 μg/L* /3/

Clearance

0.03–0.12 mL/min /6/

24-hour urine

33–363 μg /6/

* Particle-enhanced immunoassay; values are the 2.5th and 97.5th percentiles

Table 18.12-2 β2-microglobulin (β2-M) in various diseases

Clinical and laboratory findings

Malignant lymphomas – Chronic lymphocytic leukemia (CLL)

In CLL, the increase in β2-M is stage-dependent. For instance, in a study /8/ involving untreated patients, the serum concentration was 2.0–2.8 mg/L during stage 0, 2.7–5.3 mg/L during stages I, II and 4.4–16.9 mg/L during stages III, IV. In the presence of stable disease during stage 0 or stage I, so-called low-grade lymphomas, to which CLL belongs, are associated with β2-M concentrations that usually do not exceed 5 mg/L. The β2-M concentration prior to start of therapy is a prognostic indicator. It was reported that the complete remission rate was 71% if the levels were < 3.0 mg/L and only 36% if the levels were higher /9/.

– Non Hodgkin lymphoma (NHL)

In a multicenter study /10/ on patients with lymphomas (with the exception of Burkitt lymphoma), 36% of the untreated patients in stages I, II and 61.8% of those in stages III, IV had a serum β2-M > 3.0 mg/L. In patients with NHL known to be of poor prognosis, those with β2-M concentrations < 4.0 mg/L had a significantly longer survival time than those with levels higher than that. In 9.8% of stage I and stage II patients as well as in 20.7% of the patients in stages III, IV, who were in remission, the β2-M concentration was > 3.0 mg/L.

– Hodgkin’s lymphoma

The β2-M concentration rises with the stage. Approximately 5–32% of patients in stages I, II and 43–71% of those in stages III, VI had β2-M concentrations > 3.0 mg/L. During complete remission, only 3.8% of patients in stages I, II and 18.2% of those in stages III, IV had levels > 3.0 mg/L /10/. In children with hodgkin’s lymphoma, the β2-M concentration, the LD activity and the ESR correlate with the lymphoma mass. At the time of diagnosis, the β2-M concentration was elevated in 60% of the children (above 2.8 mg/L). Children with B-cell symptoms had higher concentrations than those without these symptoms. Concentrations returned to within the reference interval after successful therapy /11/.

– Multiple myeloma

In patients with multiple myeloma, β2-M is a prognostic indicator. For instance, patients with a serum level < 3 mg/L had a mean survival time of 64 months, while in those with a concentration of 3–5 mg/L it was 29 months, and in those with a concentration of more than 5 mg/L it was 11 months. Patients with a myeloma of plasmocytoid cell types 1 and 2 as well as Marschalko type generally had levels < 5 mg/L. The smoldering myeloma was associated with concentrations < 3 mg/L /12/.

Renal disease – Glomerulopathy

If the GFR is reduced to below 80 [mL × min–1 × (1.73 m2)–1], the upper serum reference interval value of β2-M may be exceeded /13/. Especially in children, the β2-M is an acceptable criterion for assessing GFR since the concentration does not depend on the muscle mass. Overall, levels that are within the reference interval rule out a reduction in GFR to below 60 [mL × min–1 × (1.73 m2)–1]. Elevated β2-M concentrations must be interpreted with care and all other conditions that may cause a rise in β2-M must be ruled out. A statistically significant correlation exists between the increase in serum β2-M concentration and the decrease in GFR /13/. The relationship between β2-M and serum creatinine as well as the inulin clearance is shown in Tab. 18.12-3 – Relationship between β2-M and serum creatinine as well as inulin clearance.

– Tubulointerstitial disease

Based on the serum β2-M concentration, it is not possible to differentiate glomerular from tubular renal diseases. Instead, this is possible by determining the β2-M excretion in the urine. A good criterion, especially in children, is the measurement of the fractional excretion (FE) of β2-M excretion (FE-β2-M), which is obtained by simultaneously measuring the inulin or creatinine clearance. In a study /14/, patients with tubular kidney damage always had a higher FE-β2-M than those with glomerular damage, regardless of the GFR. Also, in the case of decreased GFR, additional tubular damage was reliably distinguishable. The determination of urinary α1-microglobulin has proven to be superior to β2-M for diagnosis of tubulo-interstitial diseases (refer to Section 12.9.6.8.3 – α1-microglobulinuria).

– Tubular damage due to the heavy metals cadmium and mercury

Acute and chronic cadmium and mercury poisoning leads to necrosis of the proximal tubular cells. A β2-M excretion > 200 μg/g creatinine in spontaneously voided urine is the earliest measurable sign of tubular damage due to the listed heavy metals /4/. Elevated β2-M excretion in exposed individuals is only an indication of heavy metal-induced damage, which must be confirmed by the measurement of the heavy metals themselves. Serum cadmium concentrations > 10 μg/L and a urinary excretion > 5 μg/g creatinine in a random urine sample indicate accumulation of this heavy metal. Corresponding concentrations for mercury in the blood are > 10 μg/L and in urine > 40 μg/L. Chronic lead intoxication does not cause proximal tubular damage and β2-M excretion is normal.

– Pyelonephritis during pregnancy /18/

A β2-M excretion > 300 μg/L occurs in all pregnant women with infections of the upper urinary tract (i.e., during pyelonephritis with concomitant tubulo-interstitial damage). As part of the differential diagnosis in cases involving flank pain of uncertain origin and the presence of a dilated caliceal system within the renal pelvis, pyelonephritis can be ruled out if the β2-M excretion is normal.

– Chronic kidney disease (CKD) /15/

In patients with CKD the concentration of β2-M in serum increases progressively, in parallel to the decrease in glomerular filtration rate. In patients with end-stage renal disease (ESRD) serum β2-M levels are usually in the range of 20–50 mg/L, but levels higher than 100 mg/l can be observed in exceptional cases. Enhanced production of β2-M in response to diverse factors as inflammation, acidosis, calcitriol treatment, and dialysis technique modality explain the highly variable levels of serum β2-M from one ESRD patient to the other.

– β2-M amyloidosis (dialysis-related amyloidosis) /15/

The dialysis-related amyloidosis is a dramatic painful complication of ESRD and generally made in patients treated by dialysis for 6–10 years. Occurrence of β2-M amyloidosis has never been reported in non uremic patients, whose serum β2-M levels rarely exceed 5 mg/L. The main symptoms and signs are the carpal tunnel syndrome and chronic arthralgias, with eventual occurrence of destructive arthropathies and spondylarthropathies. Long-standing increase in serum β2-M concentration are a prerequisite for the formation of β2-M amyloid fibrils. Factors other than high circulating β2-M levels are post translational modifications by partial proteolysis of the β2-M molecule, advanced glycation end-products or advanced oxidation protein products. A cleavage product form of β2-M that has a deletion of lysine at position 58 on the molecule ΔK58-β2-M and behaves differently from normal β2-M was suggested that it could play a role in β2-M amyloid fibrillogenesis. The prevalence and severity of dialysis-related amyloidosis appear to have decreased in the last 30 years.

– Cytomegalovirus infection following kidney transplantation

In kidney transplant recipients, the determination of β2-M facilitates detection of Cytomegalovirus infection. In 11 of 13 cases, a more than 3-fold increase in urinary excretion of β2-M was found to occur during the observation period /16/. The increase in urinary excretion of β2-M precedes the detection of the Cytomegalovirus-immediate early antigen (CMV-EA).

– Function following kidney transplantation

After transplantation and the onset of functional capability in the kidney transplant, the serum β2-M concentration normalizes within a few days. Even in primarily nonfunctional kidneys, whose function is not adequate until about 30 days after transplantation, a marked decline in the serum β2-M occurs within the first 2–4 days /17/. The clinical diagnosis of a transplant rejection episode is already 2–7 days before preceded by a marked rise in β2-M, which often occurs 1–3 days prior to the increase in serum creatinine /6/. The value of β2-M determinations for the detection of kidney transplant rejection episodes has been disputed by other authors /16/.

Monitoring in HIV infection

In 462 HIV-positive men, the course of infection was monitored over a period of 3 years. During this time, 26% of the patients developed clinical AIDS symptoms and 19% developed symptoms of the AIDS-related complex (ARC). The β2-M concentration proved to be a very good parameter for assessing disease progression. 75% of HIV-positive patients with β2-M concentrations > 5.0 mg/L developed AIDS, while the rate was 28% in those with levels of 3.1–5.0 mg/L and 7% in those with levels of < 3.0 mg/L /19/.

Allogenic bone marrow transplantation

The serum β2-M concentration is a good parameter for detection of acute and chronic rejection episodes. In addition, in these patients, the reactivation of an infection with Herpes simplex, Varicella zoster virus or Cytomegalovirus is also associated with an elevation in β2-M concentration /20/.

Chronic pulmonary disease is a common complication in pre term infants and plays an important role in morbidity and mortality both in the newborn nursery and after discharge from hospital. It is assumed that chronic pulmonary disease is caused by inflammatory response to a lung trauma. Moreover, chronic pulmonary disease often occurs in cases of chorioamnionitis infection. Elevated urinary β2-M excretion above 10 × 104 μg/g creatinine, collected at the age of 0 or 2 days, is considered a predictor of incipient chronic pulmonary disease /21/.

Fetal infections with Cytomegalovirus or Toxoplasma gondii are diagnosed with a diagnostic sensitivity of 93.3% based on β2-M concentrations in the fetal serum above 5 mg/L.

Table 18.12-3 Relationship between β2-M and serum creatinine /23/ as well as inulin clearance /24/

1.

Serum creatinine (x) in relation to serum β2-M (y)

y (mg/L) = 3.713 times × (mg/dL) – 2.049

y (mg/L) = 0.042 times × (μmol/L) – 2.049

2.

Serum β2-M (y) in relation to GFR (x), related to inulin clearance

log y (mg/L) = –0.89 times log × (mL/min) + 2.0

Figure 18.3-1 Zone electrophoresis on cellulose acetate sheet. The protein fractions and the proteins contained in the fractions are shown.

Serum protein electrophoresis Prealbumin Albumin α 1 -Lipoprotein (HDL) α 1 -Glycoprotein α 1 -Antitrypsin α 2 -Macroglobulin Haptoglobin Pre-β-Lipoprotein T ransferrin β-Lipoprotein Complement IgA IgM IgG α 1 α 2 βγ

Figure 18.3-2 Separation of serum proteins on agarose gel electrophoresis. Alb, albumin; αLP, α-lipoprotein; α1-AT, α1-antitrypsin; Om, orosomucoid; Gc, Gc globulin; α2M, α2-macroglobulin, Hp2-1, haptoglobin type 2-1; β-LP, β-lipoprotein; Hpx, hemopexin; C3, ceruloplasmin; Tf, transferrin; Fg, fibrinogen.

ALB β 1 β 2 αLP GC CIG IgM Hx C3 Hp 2-1 Tf Fg IgA CRP IgG Alb a 1 a 2 βLp α 1 AT α 2 M α 1 - GP γ

Figure 18.3-3 Scans of serum electrophoretograms on cellulose acetate supporting medium in typical dysproteinemias /2/.

Chronic inflammation Acute inflammation Liver cirrhosis Nephrotic syndrome Monoclonal gammopathy Antibody deficiencysyndrome

Figure 18.4-1 Albumin structure and functional domains.