53

Laboratory organization

53

Laboratory organization

53

Laboratory organization

53

Laboratory organization

53.1 Laboratory testing

Lothar Thomas

53.1.1 Harmonization of laboratory results

The objective of laboratory result harmonization means that results are comparable irrespective of the method of determination used and the performing laboratory. Harmonization of test results comprises consideration of pre analytical, analytical, post analytical and medical aspects /1/.

Pre analytical considerations

Pre analytical considerations include terminology of the laboratory request form, instructions for preparation of the patient, sample collection handling, and transportation of the sample to the laboratory. The responsibility for the pre analytical phase in part rests with the attending physician/nurse in the hospital and/or the physician/nurse in the private medical office setting and in part falls within the realm of the laboratory.

Analytical considerations

These aspects include calibration, traceability to a reference system, commutability of reference materials (refer to Section 50.5 – Quality assurance using control samples) used in a traceability scheme (refer to Section 50.3 – Standardization – traceability), and specificity of the measurement of the molecule of interest. For many biomarkers, multiple methods for measurement exist, with a long series of manufacturers developing and marketing assays. Laboratories and physicians willingly accept that existing variability and the different medical statements associated with it /2/.

Post analytical considerations

These considerations include units, reference intervals, cutoffs, nomenclature.

Medical considerations

Aspects of harmonization during the medical phase consist of result assessment. This step is mainly carried out by the treating physician and in some cases also in communication with the laboratory. The clinical laboratory result is assessed as part of either a transverse or a longitudinal assessment. Biological influence factors to be considered include age, gender, race, physical activity and pregnancy. An important aspect immediately upon creation of the laboratory report is to filter out critical, abnormal values to be immediately communicated by the clinician.

Literatur

1. Miller WG, Tate JR, Barth JH, Jones GRD. Harmonization: the sample, the measurement, and the report. Ann Lab Med 2014; 34: 187–97.

2. Bossuyt P. Laboratory measurement’s contribution to the replication and application crisis in clinical research. Clin Chem 2019; 65 (12): 1479–80.

53.2 Pre analytical phase

Lothar Thomas

The pre analytical phase consists of /12/:

  • Decision oriented order of the physician
  • Patient preparation; it depends on the type of specimen to be collected and the analyte to be tested
  • Collection of various samples
  • Sample transport
  • Preprocessing of the sample based on the requested analysis
  • Storage and, if necessary, long term storage of the sample
  • Preparation of the specimen prior to analysis, if necessary
  • Being aware of, and considering, biological influence factors and interference factors.

It is important during the pre analytical phase to be aware of biological influence factors and interference factors that may interfere with the clinical laboratory result.

53.2.1 Pre analytical variables

Biological influence factor

The biological influence factor has in vivo and in vitro influence on the result of a laboratory test. Variable biological influence factors are differentiated from invariable ones /3/:

  • Variable biological influence factors include nutrition, fasting, alcohol, body weight, muscle mass, physical activity, posture, climate, altitude, diurnal rhythm, and therapeutic drugs
  • Invariable biological influence factors are gender, age, race, and hereditary factors.

Interference factor

After collection and analysis of the sample, interference factors lead to a result that does not correspond to the in vivo level of the analyte. The interference factor causes a systemic error of measurement caused by a sample component which does not by itself produce a signal in the measuring system. Among the intrinsic factors, differentiation is made between /3/:

  • Interference factors which change the concentration or activity of the analyte itself such as the release of LD from erythrocytes to the serum due to hemolysis
  • Interference factors which are not identical to the analyte but interfere with the analysis reaction such as hemoglobinemia, bilirubinemia, hyperlipoproteinemia
  • Internal substances such as anticoagulants, monoclonal antibodies, therapeutic drugs and their metabolites, and infusion solutions.

External substances admixed with the sample include anticoagulants (heparin, citrate, EDTA), serum separators, bacteria, yeasts and detergent residues.

53.2.2 Request form

The laboratory offers a multitude of tests to the physician. The laboratory assists the ordering physician in his/her diagnostic approach by implementation of a medical decision oriented request form in place of a menu of individual tests. Based on the symptoms and the disease in question, the physician can, thus, request the right tests in place of a menu of individual tests in a timely manner. This is possible by the adequate grouping of tests for liver disease, hyperthyroidism or diabetes mellitus. Many laboratories have created decision oriented request forms for diseases in question and follow up tests.

It is important that the request form (hardcopy or electronic document) includes the following information for the laboratory:

  • Identification of the ordering physician
  • Patient identification
  • Date and time of sample collection.

Further information may be necessary for special laboratory analyses and functional tests:

  • Weight, fasting state, pregnancy, therapeutic drugs, infection with multi-resistant pathogens
  • In endocrinological analysis: oral contraceptives, date of last day of menstrual cycle, gestational week, diuretics and antihypertensives
  • In drug monitoring: date of last ingestion
  • In blood group serology: date of last transfusion, known antibodies, immunoprophylaxis, pre maternity medical care
  • In microbiological diagnostics: type of specimen, site and time of sample collection, ongoing antibiotics therapy, suspected sepsis, known or unknown pathogen.

Reflective testing

Reflective testing is a practice in which a laboratory, when assessing laboratory findings, adds on one or several further investigations either to help establish a diagnosis or to assist patient management. This practice presupposes a general agreement between the clinician and the laboratory physician/clinical biochemist and long years of experience of the laboratory physician. According to a study /4/, reflective testing was welcomed in the hospitals if the following parameters were involved: free triiodothyronine (86%), γ-glutamyltransferase (78%), lipid profile (59%), thyroid peroxidase antibodies (63%), pituitary hormones (58%), troponin (55%), serum electrophoresis (68%), pregnancy (30%) and prostate specific antigen (45%).

53.2.3 Blood specimen collection

Equipment

Prior to blood collection, the following items should be prepared on the tray ready for the person collecting the specimen /56/:

  • Blood collection set, e.g. butterfly device with polyvinyl chloride (PVC) tubing (1.40 × 300 mm) with Luer adapter and needles of different sizes such as 21 G (0.80 mm × 19 mm), 23 G (0.60 mm × 19 mm) and 25 G (0.50 mm × 19 mm)
  • Disinfectant, gauze pads and compression bandage
  • Collection tubes made of plastic or glass with or without anticoagulant, with or without serum separator. The collection tubes are provided with adhesive labels showing the patient’s name, gender,and the order number.

Venipuncture procedure

Gloves should be worn and universal precautions observed when handling and processing all specimens. Before blood collection, the patient is asked for his/her name and age. Blood collection for monitoring should always be performed at the same time of day, ideally between 7:00 and 8:00 in the morning. The patient’s last food or liquid intake should have been between 6:00 and 7:00 the evening before. If possible, the blood should be collected during the drug free interval (i.e., before morning medication). The patient should be in the supine position and the site of collection should always be located in the same vessel area, usually the peripheral arm vein /4/. There is no significant difference between the results from phlebotomy in the left vs. right arm /7/. Refer to Tab. 53.2-1 – Venipuncture technique.

Tube additives

2 mg EDTA (disodium or dipotassium salt) per mL of blood. EDTA blood is used, for example, for complete blood count, the Coombs test and hemoglobin electrophoresis. Since EDTA forms complexes with bivalent ions which, in many cases, participate as reactants in the determination of enzymes or substrates, EDTA plasma is used for clinical chemistry analyses only if enzymes with a proteolytic effect on the analyte are to be inhibited (e.g., as in the case of specimens for the determination of renin and ACTH).

Heparin (NH4+, Li, Na, K salt), 25 U per mL of blood.

Heparinized plasma is used for tests in which even mild hemolysis may cause interference.

Fluoride (sodium salt), 2 mg per mL of blood. This additive results in the inhibition of both coagulation and glycolysis; it is used in tubes for blood glucose determination.

Citrate (sodium salt), 3.8% or 0.11 molar solution. Mixtures consisting of 1 part of sodium citrate and 9 parts of whole blood are employed for coagulation tests. For the erythrocyte sedimentation rate (ESR) determination, 1 part of sodium citrate and 4 parts of whole blood are collected in the syringe

53.2.3.1 Influence factors

Posture

During the transition from a supine to an upright position, the shift in body water from the vascular to the interstitial compartment is approximately 8% /8/.

A 3–8% increase in proteins, protein bound substances and corpuscles is observed if blood is collected with the patient in a sitting position or in a supine position after a preceding upright position rather than having the patient assume a supine position for a period of at least 10 min. prior to the blood collection. Parameters which are affected by increases that are bigger than the analytical imprecision include, for example, hemoglobin, RBC count, WBC count, hematocrit, total protein, cholesterol, albumin, immunoglobulins and calcium. In patients with edema, the changes are even more pronounced than in healthy individuals. Standing in an upright position with the arms hanging down causes more hemoconcentration than keeping an arm elevated at the level of the atria /9/.

Via circulatory changes, the transition from a supine to an upright position leads to an increase in norepinephrine, aldosterone and renin by factor two or more; the increase in epinephrine is lower.

Needle size

Needle sizes 21 G and 23 G do not show significant differences regarding 15 enzymes, substrates and electrolytes. For the 25 G needle, increased variability for K+ compared to a 23 G needle was observed /9/.

Duration of venous compression

Venous compression has the same effect as the postural change from a horizontal to a vertical position. All high molecular weight substances and blood cells increase (e.g., given a 3-min. compression period, total protein rises by up to 20%). The problem of hemoconcentration becomes markedly evident if the compression is applied to arms which are edematous. Short compression periods of up to 1 min. cause but insignificant changes.

Last food intake

The intake of food causes an increase in glucose, phosphate and bilirubin, a more pronounced increase in ALT and K+ as well as a moderate to minimal rise in uric acid, protein, calcium and cholesterol. The extent of fat intake determines the triglyceride level. Individuals who are blood group 0 Lewis positive show a marked increase in alkaline phosphatase (ALP) after a fatty meal.

For practical, diagnostic purposes, a light, low-fat breakfast has no significant impact on the concentration of many blood substances. On the other hand, important prerequisites include adherence to a 12-h fast prior to obtaining a blood specimen for the evaluation of the fat metabolism and intake of a diet rich in carbohydrates for several days prior to a glucose challenge test /3/.

Physical exertion

Temporary fluid shifts from the intravascular compartment to the interstitial space lead to hemoconcentration with a rise in proteins, protein bound substances and blood cells. A rise in muscle enzymes such as CK, AST, LD does not occur until hours later, especially in untrained individuals, due to exertion-induced cell damage.

Refer to Chapter 51 – Effect of physical exercise on laboratory test results.

Diagnostic measures

Diagnostic measures exert effects on clinical laboratory results in many ways. Palpation of the prostate prior to blood collection results in elevated acid phosphatase. During a glucose challenge test, the concentrations of K+, phosphate and magnesium rise. Intramuscular injections of drugs (e.g., benzodiazepine, dolantin, pentazocine, chlorpromazine, lidocaine, phenobarbital, promethazine) may lead to an increase in CK and myoglobin. Surgical procedures raise the level of acute phase proteins and thus also increase the erythrocyte sedimentation rate.

Timing of blood specimen collection

Significant diurnal variation of the iron concentration is measured with a maximum in the afternoon, of cortisol, epinephrine and norepinephrine with maximal levels in the morning and of renin, aldosterone, growth hormone and parathyroid hormone with maximal levels late in the night /7/.

Blood collection from central venous catheters

The following procedure is recommended to clear the catheter from substances interfering with the analytes to be determined /10/: flush the catheter with 5 mL of physiologic saline, take and then discard 3 mL of blood. Then take EDTA blood for CBC count, citrate plasma for coagulation tests and serum or heparinized plasma for clinical chemical analyses.

Hemolysis

Hemolysis is one of the frequently occurring problems in the clinical laboratory and an important reason for rejecting samples. When taking the blood hemolysis can occur intravascularly due to excessively long compression and extravascularly due to aspiration that is too strong (blood collection using a high volume syringe) or as a result of aspirating para venous blood after inaccurate puncture of the vein. Hemolysis causes elevated K+ levels and an increase in the activity of LD, ALT, AST and acid phosphatase. Hemolysisis defined as concentration of free hemoglobin of approximately 5–10 mg/L in serum or plasma. Hemolysis is visible to the naked eye if the hemoglobin concentration exceeds 300 mg/L.

Extravascular hemolysis can be recognized by determining the haptoglobin concentration. In the case of limiting visible, intravascular hemolysis, the haptoglobin level is low or no longer measurable.

About 95% of laboratories adopt the alert limits recommended by the manufacturer /11/. The Centers of Clinical and Laboratory Standard Institution (CSLI) guidelines states that an assay’s instructions for use (IFU) should document /12/:

  • The concentration of both hemoglobin and analyte tested, as well as the bias observed
  • Interference testing should be done a two medical decision levels of the analyte and performed up to 1,000 mg/dL.

A study investing the information provided by the manufacturers documented the following findings across all IFUs /13/:

  • The medical decision limit of ± 10% of the CSLI recommendation was only documented in 16 out of 28 cases
  • Only 1 manufacturer routinely tested up to 1,000 mg/dL of hemoglobin before claiming no significant interference
  • Manufacturers rarely state that they have tested for interference at 2 different analyte concentrations. This is of concern because hemolysis interference is not always independent of analyte concentration.

Refer to Tab. 53.2-2 – Increasing and decreasing effects of hemolysis on laboratory results.

Exogenous contamination

The specimen may be contaminated with detergents, phosphate and iron mainly because of inadequately cleaned containers.

Infusion solutions

Inadequately flushed indwelling venous catheters may be the cause of contamination with gelatin (interferes with the determination of protein), dextran, glucose, electrolytes, especially K+, cardiac glycosides and lipids.

Plasma versus serum

K+, phosphate, LD and acid phosphatase are significantly higher in serum than in heparinized plasma. The reason for this is the release of K+ and enzymes from the red blood cells and the platelets during coagulation /14/. Total protein is lower in serum since fibrinogen is absent. In EDTA plasma, the following enzymes are inhibited: ALP, acid phosphatase and leucine aminopeptidase. EDTA can induce pseudo-thrombocytopenia. Glucose values are comparable between serum and plasma /15/.

Capillary blood versus venous blood

Concentrations and activities of substances in capillary blood yields results that are more difficult to reproduce than those obtained in venous blood because the composition of capillary blood shows larger variations of the parameters than venous blood; in addition, errors in blood collection by skin puncture are higher than in venipuncture. Capillary blood samples provide clinically more relevant results than samples obtained by venipuncture in respect of components that are strongly influenced by muscle metabolism such as blood gases, lactate and glucose (both in the glucose tolerance test as well as the diurnal profile).

Glucose concentrations are higher in capillary blood than in venous blood while total protein, calcium and K+ are lower.

For interferences during the pre analytical phase refer to:

53.2.4 Urine collection

Qualitative analyses involve mainly the use of random specimens, while 24 h urine collections are employed for quantitative analyses /19/. The 24 h collection represents the reference method for stable analyte quantification (protein to creatinine ratio is an alternative) /17/. The patient requires precise instructions for the collection of both specimen types. Tab. 53.2-4 – Collection of midstream urine specimen in women for a bacterial culture lists the instructions given to women for the collection of midstream urine. The instructions for the collection of a 24 h urine specimen can be found in Section 12.3.2.3 – Procedure for urine collection.

53.2.5 Specimen transport

The transport of specimens must be carried out in such a manner that the analysis results after the transport are the same as those immediately after the collection of the samples /1819/.

Transport containers

Thermos bottle: suitable for transporting the sample in ice water or at 37 °C. For transport at 37 °C, the thermos bottle must be filled with water heated to 40 °C.

Cooler: suitable especially for transport of urine samples (e.g., to the microbiological laboratory).

Styrofoam box: should hold at least 5 kg of dry ice. The samples must already be deep-frozen before they are placed in the box with dry ice. Test tubes need to be frozen in an upright position.

Cooling aids

Ice water: ice cubes mixed with a small amount of water are filled into a cooling bag; such a bag then maintains temperatures between 1 and 4 °C for a few hours.

Freezing mixtures: ice and water-free calcium chloride: temperatures as low as –50 °C are reached.

Ice/table salt mixture: temperatures as low as –21 °C are reached.

Dry ice: has a temperature of –78 °C.

Liquid nitrogen: samples are cooled at –196 °C. Transport in a safety Dewar container.

Packaging

Cartridges: they should be able to withstand temperatures as low as –80 °C, be able to repeatedly undergo autoclaving, be impermeable to light and, for mailing purposes, contain an inner layer of absorbable material.

Various bags and pouches: should be made out of material that is autoclavable, be tear resistant, water resistant on the inside as well as the outside and have an absorption capacity of at least 25 mL.

Sample container

Tubes: screw top with a sealing ring, evacuated tubes with tightly fitting rubber stoppers. Tubes and serum separators contained within (e.g., separator gel) must neither release substances into the sample nor absorb substances from the sample (trace elements). The tubes must be: gas-tight to as low as –80 °C, especially for CO2 (dry ice), mechanically stable, colorless, clear as glass in order to detect hemolysis, turbidities, discolorations, precipitates, fibrin clots, gel formation and bacterial contamination. Items required for labeling: irreversible temperature indicator stickers, labels containing a field for patient data, time of the sample collection, address of the laboratory physician, any information in regard to functional tests, additives and alerts.

Bottles or containers with a threaded lid: with an approximately 100 mL volume capacity for random urine specimens; with a 2-liter volume capacity and made out of light-impermeable material for urine collections.

Slides: only slides with a frosted labeling area should be used. A pencil must be used for writing on the frosted end of the slide. Drying is required prior to mailing (in slide cases), otherwise cytolysis occurs.

Transport media: are required for infectious material and swabs which are to be examined bacteriologically. These media do not contain nutrients. They prevent drying and reduce oxygen and the concentration of immunoglobulins and antibiotics by diffusion. In the case of swabs, the transport medium must be covered with two gauze pads. Attention must be paid to the shelf life. Special transport medium for Bordetella pertussis must be used.

Blood culture bottles: must be transported at 37 °C (thermos bottle with a plastic insert, filled with warm water).

Special tubes for lymphocyte enrichment: evacuated citrated and/or heparinized tubes with tissue culture medium and separator gel.

Common errors during sample transport

  • Whole blood in dry ice: hemolysis
  • Specimen swabs without transport medium: reduced quantity of specimen available for analysis
  • Container into which the agar coated stick is immersed contains residual urine: yields falsely elevated bacterial counts due to self inoculation
  • Inadequate identification
  • Excessively long transport period: lack of accuracy in coagulation, differential blood counts
  • Preparation of aliquots of urine samples fails to take into account insoluble components at the bottom of the container
  • Preparation of aliquots of serum samples fails to take into account precipitated monoclonal immunoglobulins or lipids located on top of the liquid phase.

Blood sample transportation by pneumatic transportation systems (PTSs) are increasingly used in hospitals, but whether PTSs in general are safe for transportation of blood samples the existing literature gives no clear information /20/.

53.2.6 Specimen processing

The use of plasma vs. serum for the determination of substances in the liquid phase of the blood offers the advantage that the analysis can be performed earlier since no waiting period is required for the blood to coagulate. For the preparation of serum, the waiting period includes the coagulation time and clot retraction time. Usually, this process is completed after 30 minutes. The process of coagulation and retraction is delayed if the blood sample is placed in a refrigerator after sampling or if for blood sampling plastic tubes are used without coagulation activators such as beads with a rough surface. This results in gel formation in the serum. Therefore, specimen containers should generally be used with additives facilitating both coagulation and centrifugation (e.g., synthetic granules). Hemolysis is commonly observed especially in transparent polystyrene tubes.

Centrifugation

According to the following formula, the centrifugal force (Frot) can be calculated based on the rotation speed of the centrifuge (rpm) in combination with the distance between the bottom of the centrifuge tube and the axis r of the centrifuge /21/:

Frot = r × ( rpm ) 2 × 11.18 m/s 2 1,000

The factor 11.18 m/s2 is derived from gravity (g). In the same centrifuge, the centrifugal force is characterized by a reciprocal ratio of the gravity (g)-number to centrifugation time i.e., by doubling the centrifugation time, the g-number can be reduced to half and vice versa. Whole blood is centrifuged at 2,000 × g for 10 min., higher g-numbers using the same or a longer centrifugation time may cause hemolysis. In uncooled centrifuges during the run, the operating temperature should not exceed the ambient temperature by more than 15 °C, but under no circumstances should it be higher than 37 °C. The serum yield is approximately 30% without and 40% with centrifugation enhancing additives. If whole blood is centrifuged, the yield of plasma is higher than that of serum. Free fibrin clots, that form physiologically in the coagulating whole blood as well as in plasma due to inadequate mixing of the specimen with the anticoagulant, cannot precipitate as sediment because of their low specific weight.

The separation of serum from the blood clot should be completed not later than 2 h after blood collection.

Centrifugation at 200–250 × g for 10 min. is recommended for preparing platelet-rich plasma for light transmission and aggregometry. The P-selectin expression at this centrifugation speed is activated in 11–15% of platelets in comparison to standard centrifugation (2,000 × g for 10 min). At the standard centrifugation speed approximately 50% of platelets from EDTA and citrate plasma are activated. Platelet activation defined by P-selectin expression increases with increased centrifugation speed /22/.

Visual assessment of the blood sample

If whole blood samples are used, attention must be paid to the presence of interfering blood clots. Such clots point to the partial coagulation of blood and lead to incorrect complete blood count and coagulation test results. Serum and plasma must be checked for hemolysis, lipemia and bilirubinemia. Hemolysis is visible if the hemoglobin concentration is 300 mg/L or higher.

Clarification of lipemic serum samples

Use of fluorinated hydrocarbons (Freon). In these hydrocarbons, the hydrogen atoms are substituted by the halogens chlorine and fluorine.

Procedure: serum and/or plasma are mixed in a glass tube with Freon at a ratio of 1 : 1. Freon settles below the serum or plasma; rotating the tube by 180° for 3 min. brings the Freon into close contact with the serum. This is followed by centrifugation at 3,000 × g for 6 min. The supernatant is the clarified serum while the intermediate layer contains precipitated lipoproteins and the layer underneath it excess clarification agent. Clarification can be repeated several times without affecting enzyme activities or substrates.

Polyethylene precipitation for the detection of serum macroanalytes

Polyethylene glycol (PEG) is a polymer of the basic unit -CH2-CH2-O-. The phase can be liquid or solid depending in the chain length and is soluble in water /23/. PEG precipitation is a crude and non specific technique which separates proteins by virtue of their solubility. PEG acts as an inert solvent sponge, reducing solvent availability. With increasing concentration of PEG, the effective protein concentration is increased until solubility is exceeded and precipitation occurs. When applied to serum, PEG precipitation is relatively specific for immunoglobulins and their complexes.

Procedure: one part of serum is mixed with one part of PEG 6000, allowed to react at room temperature for 10 min. and then centrifuged at 2,000 × g for 10 minutes. After centrifugation, the macro analytes are in the sediment. The analyte concentration in the untreated serum and in the supernatant is measured. The difference between serum and supernatant corresponds to the macro analyte concentration.

Indication /24/: detection of macro-AST, macro-CK, macro-ALP and macro-prolactin.

53.2.7 Specimen storage

Samples need to be stored either because the analysis cannot be conducted immediately or for the purpose of retesting the sample after the analysis, should the need arise. For the latter purpose, samples can be stored without being affected by significant alterations, given the following recommendations:

  • Serum or heparinized plasma for the determination of enzymes and substrates for one week at 4 °C
  • Platelet poor citrated plasma for global plasma based hemostasis tests for up to 8 h at 20 °C (room temperature); exceptions refer to Section 16.9.4 – Plasma and blood storage
  • EDTA blood for the determination of the complete blood count (without a differential, but including a platelet count) for up to 24 h at 20 °C (room temperature).

53.2.7.1 Short time storage

The storage time is dependent on the analyte to be determined /2425/.

Enzymes

Enzymes can usually be stored for up to 5 days at refrigerator temperature without leading to a decrease by more than 10%. Exceptions are the LD whose activity declines because of the cold lability of LD-4 and LD-5, and acid phosphatase which only remains stable if the sample is acidified.

Substrates

Substrates can usually be stored for up to 6 days at refrigerator temperature without any essential changes in concentration. Exceptions are the triglycerides from which glycerol is cleaved by endogenous lipase. However, the concentration remains unchanged if the analysis method determines total glycerol.

Storage at room temperature results in a decrease in phosphate, uric acid and creatinine if creatinine determination is based on the Jaffé reaction. Bilirubin is destroyed if exposed to daylight during storage. Glucose should be stored only after deproteinization of the blood sample or if a stabilizer is added.

Plasma proteins, immunoglobulins and specific antibodies (infection serology)

Storage for up to 1 week at refrigerator temperature; regular mailing (2 days) is possible if temperatures of 25 °C are not exceeded.

Hormones and tumor markers

Steroid hormones are relatively stable for a period of up to 3 days if stored at room temperature; in general, the same applies to tumor markers. Peptide hormones should be deep-frozen if the analysis is not performed on the same day. Especially unstable hormones are: ACTH, renin, vasoactive intestinal peptides, insulin, growth hormone and calcitonin (for details, refer to the respective sections).

Coagulation

The platelet-poor plasma should not be older than 8 h, starting from the time the blood was collected, for the determination of prothrombin time and aPTT. The activity of clotting factors should be determined within 3 h; up to that point, plasma must be stored at 4 °C (Section 16.9 – Pre analytics and methodology of plasma-based hemostasis tests).

Complete blood count (without a differential)

The EDTA blood should not be older than 24 h.

Differential blood count

The smear should be prepared within 5 h of the blood collection. If hematology analyzers are used for blood cell differentiation, samples should be not older than 8 h.

53.2.7.2 Long-term storage of serum and plasma

Storage temperatures lower than –20 °C are required. Snap freezing is important for maintaining the structure of proteins. If liquid nitrogen is unavailable, snap freezing can effected within a cold block. The latter is a metal block containing indentations for the sample tubes. The cold block and the tubes should be cooled to a temperature lower than –20 °C. The sample is pipetted into the cooled tubes that are situated within the cold block. The cold block is then immediately placed into the freezer. This approach guarantees that the sample freezes immediately after being pipetted into the deep frozen tubes.

The process of thawing must occur very slowly, either overnight at refrigerant temperature or in a water bath under constant agitation. Not infrequently, concentration gradients develop during thawing; therefore, the sample must be mixed well prior to analysis. Attention must be paid to sediments at the bottom of the tube (e.g., caused by cryoglobulins, monoclonal immunoglobulins or cryofibrinogen). If necessary, such sediments must be redissolved by heating the sample.

Mailing

Reports concerning the stability of enzymes and substrates during mailing vary /26/. In general, changes within a 2-day period are not greater than ± 10%.

53.2.8 Specimen collection and transport for the determination of cell free DNA

Cell free DNA from maternal plasma is used for noninvasive prenatal testing. To maintain a high fetal DNA fraction, care must be taken after blood collection to prevent dilution of the sample by DNA from maternal white blood cells. The sample is collected into K2-EDTA tubes, stored at 4 °C and processed for plasma preparation within 6 h. First centrifugation is performed at 2,500 × g for 10 min., the plasma is fractionated from blood cells and centrifuged at 15,500 × g for 10 min. for debris removal. The supernatant plasma is stored at temperatures as low as –70 °C until analysis is performed. Cell free DNA remains stable for up to 7 days at room temperature if Streck blood collection tubes are used /27/.

53.2.9 Storage and transport of microbiological specimens

Storage times not exceeding two to four hours are the prerequisite for diagnostic accuracy /28/. If the samples arrive at the laboratory after longer storage times, parts of the microorganisms may have died. Moreover, the pathogens are detected too late and specific antibiotics treatment is delayed. Even if smears applied into gel containing transport tubes are transported in specific transport containers, sensitive microorganisms such as anaerobe pathogens may be lost.

Examples of the loss caused by time dependent storage and transport are shown in Tab. 53.2-5 – Loss of pathologic bacteria in comparison to colonizing bacteria due to the storage and transport of samples.

Samples must be transported in compliance with the regulations of the dangerous goods law (European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR), Annex A, margin number 2650). For prerequisites under the dangerous goods law, please refer to Ref. /29/.

53.2.10 Pre analytical uncertainty

Pre analytical uncertainty is attributable to variations in blood specimen collection and sample processing until analysis. Errors occur in up to 0.5% of laboratory analyses /30/. Most of these errors are pre analytical mistakes.

Qualitative pre analytical uncertainty

These errors include missing identification of the patient, use of the wrong blood tubes for blood sample collection, and ordering of the wrong test /6/. In a study /31/, 15% of the pre analytical errors were due to missing requests and 1 in 100 to 200 samples was inadequately labeled, resulting in sample collection from the wrong patient in 1 in 1,300–2,000 cases. In a further study /32/, pre analytical errors were detected in 7.4% of 52,669 evaluated samples. This included missed samples (45.4%), hemolyzed samples (36.2%), coagulated samples (10%), and incorrect sample volume (2.8%).

Quantifiable pre analytical uncertainty

Quantifiable pre analytical uncertainty is due to variation in current laboratory practice such as the use of different blood tubes, variation in clotting time, and variation in centrifugal force and storage time /6/. In a study /6/ no significant systemic differences were found between successive venipunctures. However, statistically significant mean differences were seen between serum separation tubes containing silica clot activator and rapid serum tubes containing a thrombin based medical clotting agent for 7 of 15 analytes.

The pre analytical standard deviations (SDs) for LD, glucose and K+ were significantly higher than the SDs for measurement repeatability.

According to a different study /33/, the pre analytical uncertainty in blood collections at a 3-hour interval and subsequent 4-hour transport of the samples was:

  • Approximately 13–16% for K+, cholesterol and albumin. The major uncertainty factor for cholesterol was biological variation, whereas those for albumin and K+ were sample collection and pretreatment.
  • For free thyroxin (FT4), thyrotropin (TSH) and C-reactive protein (CPR): 20%, 42% and 125%, respectively, largely due to their biological variation
  • Below 10% for hemoglobin, RBC count and MCV
  • For the platelet and leukocyte counts 24% and 31%, respectively and for the reticulocyte fraction 41%.

References

1. Miller WG, Tate JR, Barth JH, Jones GRD. Harmonization: the sample, the measurement, and the report. Ann Lab Med 2014; 34: 187–97.

2. Thomas L. Blood collection and transport of blood samples. Important preanalytic steps towards the clinical laboratory result. Dt Aerztebl 1991; 88: B2226–32.

3. Keller H, Guder WG, Hansert E, Stamm D. Biological influence factors and interference factors in clinical chemistry. J Clin Chem Clin Biochem 1985; 23: 3–6.

4. Darby D, Kelly AM. Reflective testing – what do our service users think? Ann Clin Biochem 2006; 43: 361–8.

5. Clinical and Laboratory Standards Institute (CLSI) Procedures for the Collection of Diagnostic Blood Specimens, Approved Guideline, 7th ed., CLSI document GP41-ED7 Wayne PA, USA, 2007

6 Von Meyer A, Cadamuro J, Streichert T, Gurr E, Fiedler GM, Leichtle A, et al. Standard-Arbeitsanleitung zur peripher venösen Blutentnahme für die labormedizinische Diagnostik. J Lab Med 2017; 41 (6): 333–40.

7. Sylte MS, Wentzel-Larsen T, Bolann BJ. Estimation of the preanalytical uncertainty for 15 clinical chemistry serum analytes. Clin Chem 2010; 56: 1329–35.

8. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig LM, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD Initiative. Clin Chem 2003; 49: 1–6.

9. Emerson JE, Ngo G, Emerson SS. Screening for interference in immunoassays. Clin Chem 2003; 49: 1163–9.

10. Odum L, Drenck NE. Blood sampling for biochemical analysis from central venous catheters: minimizing the volume of discarded blood. Clin Chem Lab Med 2002; 40: 152–5.

11. Aslan B, Stemp J, Catomeris P, Allen L, Kerekes R Gun-Munro J. Clinical chemistry sample interferences reporting patterns in Ontario laboratories. (abstract) Clin Chem 2012; 58: A23–4.

12. Clinical and Laboratory Standards Institute (CLSI) Interference testing in clinical chemistry. Approved Guideline, 2nd ed. Wayne, PA.,Clinical Laboratory Standards Institute; 2005. CLSI document EP7-A2.

13. Farrell CJL, Carter AC. Hemolysis interference: are laboratories getting the information they need? Clin Chem 2016; 62 (9): 1274–6.

14. Narayanan S, Guder WG. Pre-analytical variables and their impact on the quality of laboratory results. J Lab Med 2002; 26: 263–6.

15. Miles RR, Roberts RF, Putnam AR, Roberts WL. Comparison of serum and heparinized plasma samples for measurement of chemistry analytes. Clin Chem 2004; 50: 1704–5.

16. Kouri T, Fogazzi G, Gant V, Hallander H, Hofmann G, Guder WG. European Urinalysis Guidelines. Scand J Clin Lab Invest 2000; 60: Suppl 231.

17. Lippi G, McBride KB, Behulova D, Bowen RA, Church S, Delanghe J, et al. Preanalytical quality improvement: in quality we trust. Clin Chem Lab Med 2013; 51: 229–41.

18. Seuffer R. Sample transport. In Thomas L, ed. Clinical laboratory diagnostics. Frankfurt, TH-Books 2000: 1493–4.

19. European Committee for Standardization (CEN). EN 829. Transport packages for medical and biological specimens. Brüssel, 1996.

20. Nybo M, Lund ME, Titlestad K, Maegaard CU. Blood sample transportation by pneumatic transportation systems: a systematic literature review. Clin Chem 2018; 64 (5): 782–90.

21. Lauritzen M. Quantities and units for centrifugation in the clinical laboratory. Clin Chim Acta 1992; 205: S5–S15.

22. Söderström AC, Nybo M, Nielsen C, Vinholt PJ. The effect of centrifugation speed on pre-analytical platelet activation. Clin Chem Lab Med 2016; 54 (12): 1913–20.

23. Fahie-Wilson M, Halsall D. Polyethylene glycol precipitation: proceed with care. Ann Clin Biochem 2008; 45: 233–5.

24. Davidson DF, Watson DJM. Macroenzyme detection by polyethylene glycol precipitation. Ann Clin Biochem 2003; 40: 514–20.

25. Heins M, Heil W, Withold W. Storage of serum or whole blood samples. Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995; 33: 231–8.

26. Koch CD, Burkhardt H, Wepler R, Blersch H. Einfluss von thermischer und mechanischer Belastung auf 17 klinisch-chemische Parameter. Lab Med 1983; 7: 23–8.

27. Wong D, Moturi S, Angkachatchai V, Mueller R, De Santis G, van den Boom D, et al. Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing. Clin Biochem 2013; 46: 1099–1104.

28. Mauch H. Bedeutung von Probenlagerung und -transport für eine schnelle mikrobiologische Diagnostik unter Berücksichtigung ökonomischer Aspekte und Patienteninteressen. J Lab Med 2009; 33: 188–93.

29. Thurm V, Tschäpe H. Gefahrgutrechtliche Voraussetzungen für den Versand von diagnostischen Proben, Bakterienkulturen u.a. infektiösen Materialien. Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz 2001; 44: 823–8.

30. Sylte MS, Wentzel-Larsen T, Bolann BJ. Estimation of the preanalytical uncertainty for 15 clinical chemistry serum analytes. Clin Chem 2010; 56: 1329–35.

31. Wallin O, Söderberg J, van Guelpen B, Stenlund H, Grankvist K, Brulin C. Preanalytical venous sampling practices demand improvement – a survey of test-request management, test-tube labelling and information search procedures. Clin Chim Acta 2008; 391: 91–7.

32. Romero A, Cobos A, Lopez-Leon A, Ortega G, Munoz M. Preanalytical mistakes in samples from primary care patients. Clin Chem Lab Med 2009; 47: 1549–52.

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36. Bowen RAR, Sattayapiwat A, Gounden V, Remaley AT. Blood collection tube-related alterations in analyte concentrations in quality control material and serum specimens. Clin Biochem 2014; 47: 150–7.

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53.3 Analytical phase and evaluation of the results

Lothar Thomas

53.3.1 Analysis

The steps to the generation of a laboratory result include the determination of the analyte, the plausibility control and the medical assessment. The entire process for obtaining a laboratory result, beginning with the sampling and ending with the delivery of a laboratory result is controlled by a system of quality assurance.

The analytical process consists of four sections /1/:

53.3.1.1 Method of determination

In laboratory medicine, the measurement accuracy is defined by trueness and precision. The determination of analytes in specimens is described in instructions for use (IFU). The IFUs are based on biological, chemical and physical criteria as well as mathematical principles. According to their quality, methods are classified into routine, reference and standardized reference methods. See also Chapter 50 – Standardization and quality assurance of quantitative determinations.

Quantitative method of determination

The objective is to determine the number of molecules, ions, atoms, free DNA particles or events. The result is specified as molar concentration (mmol/L) or mass concentration (mg/L or mg/dL) of a substance. Most of the analytes in laboratory medicine to be quantitatively determined are detected indirectly (e.g., by means of activity measurements, conductivity measurements or ligand binding) since they occur in the specimen together with many other components. The method is influenced by these components (matrix effect) and quantification is therefore only possible in relation to calibrators.

Direct quantitative analyses that can be considered to be true (accurate) without being related to a calibrator (e.g., the cell count in a counting chamber) are less frequent in laboratory medicine.

Qualitative analysis

Depending on the detection limit of the method used, the result is specified as positive (detectable) or as negative (undetectable).

53.3.1.2 Quality assurance

Trueness and precision, which define measurement accuracy, are the key aspects in assuring the quality of results and are continuously monitored and managed in laboratory practice.

The ratio of biological to analytical variation is an indicator of the performance of a method of determination. At a ratio of 2, the inherent analytical variation gives reason of about 12% of the total observed variation /2/.

For objectively defining analytical performance the desirable goal for analytical variation should be less than or equal to one-half of the within-subject biological variation of the analyte (≤ 0.50 CV) and minimum goal should be ≤ 0.75 CV /3/.

Refer to Chapter 50 – Standardization and quality assurance of quantitative determinations.

53.3.1.3 Calibration and control materials

A prerequisite for obtaining high quality laboratory results is the employment of suitable calibrators and control materials.

Calibrator

The concentration of the analyte to be determined is usually obtained as a relative measurement (i.e., the measurement signal of the specimen is compared to the signal and the known concentration of a reference material). Reference materials are used for the calibration of methods. The composition of calibrators is related to standardized reference materials.

The following reference materials are distinguished:

  • Primary (definitive) standard material: the exact quantity of a defined substance was weighed or determined by a reference method
  • Secondary standard: the concentration of the analyte was determined analytically and not by weighing. The standard sample is a secondary standard that usually contains the same components as patient samples.

Controls

Control samples resemble the samples of patients as closely as possible regarding the composition and concentration of the analytes and should also contain all of the other components that are usually present in patient samples /4/.

Control samples for internal laboratory quality control are employed for evaluating the precision and trueness of a method:

  • Precision control: the concentration of the analyte to be determined should be close to the clinical decision limit normal/abnormal. Precision control samples should be run in parallel to the patient series. The cutoff limits for the precision of a method are defined in the guidelines of the national quality control associations.
  • Trueness control: the assigned value is determined by accredited reference laboratories which are independent of the control sample manufacturer. The accuracy control sample must be run simultaneously day-to-day with the patient samples. The cutoff values for the accuracy of a method are defined in the guidelines of national quality control associations.

53.3.2 Analytical assessment

The analytical assessment of a laboratory result includes quality control and plausibility control.

Intra individual variation (CVi)

The CVi describes how tightly repeated measurements over time are distributed around a homeostatic set-point for an individual.

Interindividual variation (CVg)

The CVg reflects differences between individuals within a group.

Index of individuality

The index of individuality describes the ratio of CVi to CVg. Values less than 0.6 being considered to be highly individual or tightly distributed around a set-point. For serum creatinine the index of individuality is 0.47 which is highly individual. The index for cystatin C is 1.64 and less individual. A patient with a significant change in serum creatinine will fall in the reference intervall. However, an abnormal result of cystatin C is more likely to fall outside the reference interval.

Plausibility control

The analytical part in the determination of a laboratory result is monitored by quality control. However, quality control only detects analytical errors, but not pre analytical ones. The plausibility control involves the assessment of the pattern of the analytical results of a given patient prior to release of the laboratory report to the clinician /1/. Plausibility control includes extreme value control, constellation control (pattern of results) and preliminary result control.

Alert threshold results

Alert threshold results should be used to identify critical risk results. The objective is to assess whether the results are compatible with the specimen (serum, urine, extravascular fluid), the age, the gender, and the habitual reactions (e.g., smoking, habitual drunkard) of the patient. Alert threshold results, also referred to as critical limits, are usually defined as the maximum and minimum values compatible with life based on medical experience /5/. Such results must be immediately communicated by telephone if they are confirmed by repeat measurement from the same sample. Moreover, they should be communicated by a skilled and experienced member of the laboratory personnel. For critical quantitative limits refer to:

No national or international consensus has been reached regarding critical limits. In addition, the extreme values will increasingly extend into abnormal intervals due to medical progress /6/.

Constellation control

A constellation control (or pattern of results) evaluates the results of a sample, against one another /7/. A constellation is implausible if medical or methodological inconsistencies occur in physiologically interdependent analytes. This is the case, for instance, if the Rule of Three applied in the complete blood count give an implausible result in a hematologically healthy individual with normal RBC count.

Rules of Three: Red blood cell count (nL) × 3 = hemoglobin (g/dL) × 3 = hematocrit (%)

Tendency control

Tendency control checks whether an actual result agrees with the preliminary result of a patient, while taking into consideration changes in diagnostic and therapeutic regiments /4/. Trend control plays an important role, for example, in therapy monitoring, e.g., anticoagulation

Average of daily mean, average of normals

The plausibility control of serial results or of averages of the daily mean of the results of an analyte is based on comparing:

  • The distribution of the analytical results from a given day with another day
  • The actual frequency distribution of an analyte in comparison to the frequency distribution over a long period of time by the same laboratory in a comparable group of patients or healthy people. The tests must have been determined by the same laboratory in a comparable cohort of patients.

Systemic deviations in the trueness of an analysis method are detected by the average of daily mean.

53.3.3 Medical assessment

The analytical result reported by the laboratory becomes a clinical finding through the medical assessment by the physician. The interpretation of a clinical finding is based on the weighting of the laboratory result in relation to clinical findings of the patient. The basic requirements for medical assessment include: medical experience as well as knowledge of the patient, the disease in question, the course of the illness and the results of other medical examinations /8/. The framework for laboratory testing in medical decision making is shown in Fig. 53.3-1 – Framework for laboratory testing in medical decision making.

Medical reliability of laboratory test results

To perform test dependent medical decisions the physician must know the medical reliability of laboratory tests.

For this purpose, it is important to know /68/:

  • How the laboratory result converts prevalence to a predictive value for a certain disease, by demonstrating, for example, that serum lipase activity above 400 U/L is associated with acute pancreatitis in more than 95% of cases. The physician usually obtains this knowledge based on his/her own experience, exchange of know-how with colleagues or advanced medical training.
  • The threshold probability of a laboratory test. In many cases, clinical decision making is problematic due to a gray area between the upper reference interval value for a laboratory test and its actual value determined in the event of disease. The physician has no clear threshold to aid the decision as to whether to pursue a presumptive diagnosis or even initiate treatment. For instance, the upper reference interval value of lipase is 100 U/L, but acute pancreatitis is not to be expected in patients with acute upper abdominal pain unless the lipase concentration exceeds the threshold of 400 U/L. Usually, a low threshold is assumed within the gray area if the patient will benefit from treatment and side effects are low. The assessment of the threshold probability requires close cooperation between the physician, who knows the patient’s clinical symptoms, and the laboratory physician, who knows the biological influence and the interference factors of the laboratory tests.

53.3.3.1 Approaches to medical assessment

Medical assessment comprises a transverse approach and a longitudinal approach /5/.

Transverse assessment

Transverse assessment is understood to be the comparison between the patient’s test result and the reference interval, a therapeutic interval or a threshold value. For an assessment of the patient’s test result based on a reference interval, the deviations from the true value must be small in order for the medical reliability to be high. This necessitates high precision of the method of determination. Important biological influence factors that exert an effect on the test result must also be considered in a transverse assessment.

Longitudinal assessment

In longitudinal assessment, the test result and previously obtained values of a patient are compared. The variance of laboratory results obtained in longitudinal monitoring of a patient is significantly smaller than that in a reference population of gender and age matched individuals. Prerequisites for longitudinal assessment include:

  • No change in the method of determination nor in the laboratory where the analysis is performed
  • Same conditions for collection, storage, and transport of the specimen
  • Consideration of biological influence factors
  • The method of determination must be subject to controls [i.e., be precise and true (accurate)].

Important biological influence factors to be considered when interpreting laboratory results are listed in:

53.3.4 Validity of laboratory results for medical decisions

Laboratory results support the physician in his or her clinical decision making processes and, ideally, to differentiate between sick and healthy individuals. However, an analytically reliable laboratory result does not guarantee that it is useful for diagnosis or differential diagnosis nor for monitoring or the assessment of the outcome of therapy. The utility, relevance or reliability of a laboratory result for such medical information is also referred to as the validity of a clinical laboratory result. Against this background, it is important to know the predictive value of a clinical laboratory result for a disease. This necessitates knowledge of the terms listed in Tab. 53.3-7 – Terms characterizing the conclusiveness of laboratory findings.

Diagnostic (clinical) sensitivity and specificity

The diagnostic sensitivity and specificity of a quantitative laboratory test, and thus its clinical utility, depend on the definition of a decision threshold in the distributions of healthy and diseased populations. This is accomplished by establishing frequency distribution curves for a healthy population and a population with the specific disease

Refer to Fig. 53.3-2 – Frequency distribution curves of healthy and diseased populations.

The decision threshold can be defined according to the following two approaches:

  • Selection of an optimal diagnostic sensitivity. The diagnostic specificity is thus defined, as well, and is determined by the frequency distribution curve of healthy individuals
  • Selection of an optimal diagnostic specificity. For many laboratory tests, this is the 97.5th percentile of the healthy reference population. The diagnostic sensitivity is thus also defined and is determined by the frequency distribution curve of diseased individuals.

It is evident from Fig. 53.3-2– Frequency distribution curves of healthy and diseased populations that the degree of overlap determines the clinical relevance (validity) of a test. Diagnostic uncertainty is present in individuals with a result that falls within the overlapping interval; this may result in erroneous classification which cannot be prevented by shifting the decision threshold to higher or lower values but only by minimizing the overlapping interval.

The analytical imprecision of a laboratory test has an important impact on the size of the overlapping interval and thus the diagnostic sensitivity and specificity of the test. If the imprecision increases, the overlapping interval widens and the number of false-positive and/or false-negative results rises.

Methods such as the ROC analysis and the calculation of the likelihood ratio are employed for optimizing the diagnostic sensitivity and specificity and optimal distinction between overlapping frequency distribution curves.

Predictive value

The physicians’ task of managing a patient’s problem is composed of a series of medical decisions. The result of a laboratory test changes the pretest probability of disease to the post test probability (predictive value) of disease /8/. The calculation of the predictive value of disease depends on the prevalence of the disease in the physicians office and is based on the clinicians’ experience. The prevalence of disease, includes the ratio between diseased and healthy individuals in the physicians office. The significance of prevalence for the predictive value is shown in Tab. 53.3-8 – Dependence of positive predictivity on prevalence of disease and clinical sensitivity and specificity of the test.

The predictive value combines the diagnostic sensitivity and diagnostic specificity of a test with disease prevalence. The positive predictive value of a test (PVpos) is distinguished from the negative predictive value (PVneg). The number of true-positives and false-positives is dependent on the prevalence of disease and on the diagnostic sensitivity and specificity of the test.

Based on the equations, it can be concluded that the positive predictive value rises with the disease prevalence and the diagnostic sensitivity and specificity of a test. The positive predictive value is always low in the case of a low prevalence, even if the test has high diagnostic sensitivity and specificity (Tab. 53.3-8– Dependence of positive predictivity on prevalence of disease and clinical sensitivity and specificity of the test).

The negative predictive value rises with the number of individuals that are not affected by the disease (100 minus prevalence). Given a very low disease prevalence, the negative predictive value will always be high, even if the diagnostic sensitivity and specificity of the test are poor or its imprecision is high.

Other methods of assessment of the utility of clinical laboratory results are diagnostic efficiency, likelihood ratio and odds ratio /8/.

For calculations of diagnostic sensitivity, specificity and predictive values refer to:

Diagnostic efficiency

The diagnostic efficiency of a test describes the ratio of the correct results to all results of a test. It depends on the diagnostic sensitivity and specificity of the test and the disease prevalence (Tab. 53.3-12 – Calculation of the diagnostic efficiency of a laboratory test).

Likelihood ratio

The ratio between the likelihood of a test result occuring in diagnosed persons versus healthy persons is defined as the likelihood ratio (LR). The LR is disease specific and corresponds to the ratio between the diagnostic sensitivity and nonspecificty of the diagnostic laboratory test: true positivity in diagnosed persons versus false positivity in healthy persons. The likelihood ratio is related to a certain diagnosis. However, many laboratory tests relate to various diagnosis. If the diagnosis in suspicion is open to various diseases, a test result should help with the differential diagnosis. The provision of LRs by the laboratory would put the test result into the context of one or several possible diagnoses and would increase the impact of laboratory diagnostics /26/. Positive likelihood ratio is the ratio of sensitivity and 1-specificity.

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4. CEN/TC 140 N8. Working draft on quality assurance in laboratory medicine: terminology for quality and application of classification, counting and measuring systems.

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15. Krintus M, Sypniewska G, Kuligowska-Prusinska M. Effect of second and third generation oral contraceptives on C-reactive protein, lipids and apolipoproteins in young, non-obese, non-smoking apparently healthy women. Clin Biochem 2010; 43: 626–8.

16. Römer T. Medical eligibility for contraception in women at increased risk. Dtsch Arztebl Int 2019; 116: 764–74.

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18. Ritchie RF, Ledue TB, Craig WJ. Patient hydration: a major source of laboratory uncertainty. Clin Chem Lab Med 2007; 45: 158–66.

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53.4 Cost accounting

Gudrun Hintereder

The task of cost accounting is to collect, apportion and allocate the costs incurred in a manufacturing process Its objective is to achieve controlled historical costing and/or decision oriented future costing /1/.

Examples of standard term definitions providing the foundation for cost accounting are listed in Tab. 53.4-1 – Important business management terms. These definitions are required to utilize cost accounting as an aid for decision-making.

The terms return and costs are used in cost accounting; proceeds and expenditure are terms relating to financial/commercial law accounting. The cost of personnel placement and the cost of goods are described as input; as output the product, work result, goods and services.

53.4.1 Full cost accounting in the laboratory

The objective of full cost accounting is to determine the actual or planned costs of a cost object (goods, service, product). In addition, the economic efficiency of the production process can be controlled and profit and loss accounting can be performed. The main point of criticism of full cost accounting is the fact the costs are apportioned to the cost objects independently of the cause (especially the cost of output quantity). This is also referred to as overhead apportioning.

Despite decades of vehement criticism is full cost accounting, performed in many variations, is still the most common accounting method in Germany. The method of full cost accounting incorporates all overhead costs accrued for production. Absorption provides a view whether a given product makes a positive or negative contribution to the operative result of a business taking into account all attributable overhead costs. Each product should make its contribution to attributably covering the overhead costs, unless strategic control stipulates that certain products with a high positive contribution to the operative result are to compensate for other products with a negative contribution and, thus, business success can be accounted for based on the sum of total output.

The change in managerial and cost accounting general regulations require a reorientation of classical cost accounting. The increasing degree of automation has led to an increase in the fraction of equipment costs; the increasing significance of indirectly productive departments, such as electronical data processing has caused an increase in overhead costs. Therefore, strategic and/or strategy-based cost accounting should be performed in addition to classical cost accounting /5/. For instance, activity-based accounting was developed in response to the continuous increase in overhead costs and increasing business process orientation and is conceived as full cost accounting /1/.

Both industrial enterprises and hospitals are generally recommended to use general, combined cost accounting systems /6/. Cost center accounting assigns the type of costs to the cost object.

Historically and methodically, a distinction is made between various different systems of cost accounting, often with overlapping contents, and their manifestations. Contribution costing is useful for short-term decisions and full cost accounting is useful for short-term to medium term decisions /7/.

Hospitals are required by legal obligation to perform full cost accounting. In addition, the development and sources of the operating result must be traceable /3/.

The results of full cost accounting provide the basis of internal transfer pricing of general in-house service providers in direct costing.

The types of costs relevant in a laboratory comprise the direct costs and the overhead costs. The cost source data usually emenates from financial accounting and operating statistics or from cost accounting.

Direct costs include, for example, personnel costs, material costs and other costs which incur directly with the provision of the activity and can be allocated to the cost object without key accounting Personnel costs comprise all costs in connection with the production factor manpower. The main personnel cost categories are wages, salaries, statutory social security contributions, fringe benefits and other personnel costs. They are collected in payroll accounting /1/. The costs for further education and training, for example, are also part of the personnel costs of a production unit. In this case, the actual costs and the opportunity costs (for the deployment of manpower at an alternative site) must be taken into account.

Material costs comprise all costs incurred in connection with the technical provision of a laboratory finding. A distinction must be made in this context between (direct) manufacturing and material costs and (indirect) manufacturing and material overhead costs straightly connected with the value creation process.

Overhead costs or indirect costs cannot be allocated directly to a cost object (e.g. vendable product or service). Indirect costs are, for example, the costs for consumables such as power, water, heat, as well as lease, EDP, departments, building services and management costs. They are also known as infrastructure costs. Infrastructure costs are apportioned because they cannot be allocated directly. Various distribution or apportionment keys are available for this purpose.

The direct costs together with the indirect costs make up the total costs of a product in cost object accounting. Both direct costs and indirect costs are terms from full cost accounting /8/. The total of direct costs and infrastructure costs serves as the basis for full cost accounting.

53.4.2 Cost and activity based data; internal point value

For internal transfer pricing, an internal point value (IPV) is determined for the laboratory activity or service (e.g., expressed in the reported result). Based on the IPV, the costs for the rendered activity or product can be allocated and accounted directly to the customer. The IPV allows to compare the effectiveness between different service providers. The amount of points for a reported result reveals the complexity of its required production process and is specifically defined in medical fee schedule (MS).

The internal point value (IPV) is an individual dimension of a service provider and is bases on the ratio between costs and rendered activity expressed as the sum of points (IPV = cost/activity). Since the total costs serve as the basis for calculation, the individual internal point value depends directly on the amount of fixed and variable costs.

Variable costs dependent on synergy effects and economies of scale. Performs the lab a higher amount of analytics which are underrated in the MFS the lab’s efficacy and effectiveness can be lower than producing a high number of analyses that are realistically rated or overrated in the MPS.

The calculation and comparison of internal point values must take into account all differences regarding data access. Differences can arise in the total number of points, based on data from the hospital information system (HIS) and a selection retrieved from the laboratory information system (LIS). This may be due to different scoring in the MFS (by points) and the Uniform Assessment Standard by Euro values (EBM).

Tab. 53.4-2 – Calculation of the internal point value shows the development of the internal point value (IPV) as a time series, not taking the infrastructure costs into account. The IPV increased by 4.1% in 2010. This higher cost/activity ratio can (IPV)might have been caused by a longer adjustment period of new personnel or by the centralization of expensive activities with a lower cost/return ratio in the MFS.

53.4.3 Costing – cause related

The above-mentioned calculation of the internal point value refers to the total costs and total activities of the laboratory. Further breakdown of the costs and activities is advisable if the laboratory renders the activity at different locations or is responsible for additional ranges of tasks such as Point of Care Testing (POCT). Direct costs for personnel and material can be broken down and related to the relevant activities (Tab. 53.4-3 – Allocation of costs to different locations).

53.4.4 Direct costing and location-based internal point value

If the task range-based basic data for the costs and activities are known, an individual calculation of the internal point value can be performed. The result can be used to compare the activities of individual task ranges or locations and, if deviations are detected, schemes of improvement can be initiated /8/. The example in Tab. 53.4-4 – Location based calculation of the internal point value (IPV) shows the direct location-based costs without taking the overhead costs into account, as well as the relation to the point value defined in the MFS. The continuous increase in total IPV is due, among other things, to an disproportionate increase in the point value at laboratory B and also to the higher expenditure for the POCT task (Tab. 53.4-4).

53.4.5 Contribution costing

Break even analysis or contribution costing is a method to determine the operating result of a business using the profit contributions of manufactured products. Contribution costing is based on the separation of fixed and variable costs or direct and indirect costs. Thus, the cost structure of the activity provision and the controllability of the costs are demonstrated. The profit contribution is obtained by subtracting the costs from the return.

A distinction is made between (single level/direct) contribution costing and multi-step/indirect contribution costing (fixed-charge coverage calculation). Single-step contribution costing adds up the individual profit contributions (if several products are produced) and subtracts the total fixed costs from the result.

Multistep contribution costing attempts a further breakdown of the fixed cost block to allocate the costs to the causing departments /10/. Due to its structure and high volume of fixed costs a multi-step contribution costing is recommended in a hospital. If adequately structured, the contribution of a department to business success can be shown. The development can be quantified in respect of utilization, proceeds and costs, as well as strengths and weaknesses of individual subsections /3/.

Total calculation based on full cost accounting and the short-term performance review with partial absorption costing calculations complement each other and provide the information necessary to influence inefficient processes in time. Moreover, the obtained information supports a make-or-buy decision regarding a product.

Contribution costing is based on data of the operational accounting system. For better representation, activities are real timely considered on an accrual basis (i.e., the time of provision of an activity and not the time of invoicing is relevant for internal contribution costing). This approach is based on the commercial prudence principle /11/. In addition, risk structure compensation can be included in contribution costing. Risk structure compensation is a fee that can be applied to direct proceeds and is used, within the scope of business strategy, for risk management or reserve to compensate extraordinary investment or loss. The following example provides an explanation hereof /10/ (Tab. 53.4-5 – Example of single-level contribution costing and Tab. 53.4-6 – Example of single level contribution costing).

An increase in production up to full capacity reduces the mean fixed costs per item. If the price demanded on the market can only be realized up to a certain amount, an increase in production would lead to a fall in prices. A second market, export or second brand, must be established to reach better utilization. In this case, the price can be below the average costs as long as a positive contribution is added. A cause of a lower price can be a reduction of fixed costs due to increase in production. As described above, organizations use different numbers of levels for (indirect) contribution costing to optimally reflect the operating result. The example below shows the three-level contribution costing of a hospital, related to previous time periods and the earned activity in MFS points.

Profit contribution I = direct proceeds (use fees and outpatient department proceeds) – directly assignable costs (i.e. excluding infrastructure costs/indirect costs/overhead costs)

Profit contribution II = profit contribution I + credits from internal transfer pricing (ITP) + charges from ITP + charges from 3.5% risk structure fee

Profit contribution III = profit contribution II – apportioned infrastructure costs

The result of profit contribution III provides the basis for internal point value calculation. This internal point value is used for cross-disciplinary internal transfer pricing and allows a comparison of internal and external activities/services. A difficulty for public service providers to show a clear discrimination of services in research and teaching, which are not included in infrastructure costs.

53.4.5.1 Contribution costing I

A first comparison with the previous year shows an increase in IPV in profit contribution costing step I due to increased material costs. Since, in the calculation of the internal point value, the denominator is the number of activities in MFS points, the IPV decreases in the further comparison between 2009 and 2008 due to the increased level of efficiency as documented by the increase in MFS points and an increase in proceeds. The effects becomes apparent only if the increase in material costs does not exceed the difference in proceeds in the comparison with the previous year; prerequisite is a concurrent increase in efficiency. Further comparison between 2010 and the previous year 2009 shows an increase in IPV, due, for example, to insourcing of expensive or complex activities, which cannot be compensated by an increase in proceeds (Tab. 53.4-7 – Calculation of profit contribution I).

Profit contribution I = total of direct proceeds (use fees and outpatient department proceeds) – directly assignable costs (manpower costs and material costs).

In a pure proceeds analysis where direct proceeds are related to the activities/services rendered, an increase in proceeds can be shown. Interestingly, the example shows an increase in the key performance indicator proceeds per MFS point despite an increased IPV in profit contribution I. Thus, an improvement in return can be concluded (Tab. 53.4-8 – Key indicator for profit contribution I per point).

53.4.5.2 Contribution costing II

In the second level of contribution costing, the result of contribution costing I is applied against the internal credits and charges. Contribution costing I describes all costs after externally generated proceeds; the figure is a negative number.

The sender-related credits in profit contribution II are calculated from the total of all activities/services rendered for SHI (statutory health insurance) inpatients. The activities/services are represented in the total of achieved MFS points which add up to the current credit, when multiplied with the internal point value defined for the basic year. In internal transfer pricing, the thus calculated amount tis invoiced to the sender and credited to the laboratory.

The charges for the medical service providers arise from the total of all activities/services for SHI inpatients of internal private outpatient departments and for activities/services for patients of all external senders, provided these activities/services for private health insured in- and outpatients. Both the credits and charges for the medical service providers directly dependent on the internal point value defined for the laboratory activity.

For internal coverage, other deductions can be claimed in profit contribution II, (e.g., a risk structure fee) (Tab. 53.4-9 – Calculation of profit contribution II).

This results in:

Profit contribution II = profit contribution I + credits from internal transfer pricing (ITP) + debits from ITP + debits from an internal risk structure fee

The size of credit of the medical service providers (e.g., internal inpatient departments as senders) is determined based on the requested scope of activity and the defined IPV for the laboratory. The IPV is variable and can depend on business policy. The debits for the medical providers (e.g., from outpatient departments as senders) are determined based on the requested scope of activity for SHI (statutory health insurance) and PHI (private health insurance) outpatient activities and the defined IPV.

53.4.5.3 Contribution costing III

In this example, contribution costing III is obtained from the result for profit contribution II after overhead costs, such as infrastructure costs and management costs.

Infrastructure costs are overhead costs apportioned to the users. For the distribution of the overhead costs to different cost units, an apportionment key is used which reflects consumption as closely as possible. This apportionment key can, for example, relate to the number of personnel employed in an institution in full-time equivalent (FTE) or to manpower costs (MC) in Euros (€). Moreover, it can relate to the performed MFS points. In the example shown below for the calculation of the infrastructure costs for the different laboratory service providers, the apportionment key is related to the FTE (Tab. 53.4-10 – Calculation of profit contribution III).

Profit contribution III = profit contribution II – apportioned infrastructure costs

The relation of profit contribution III to the different locations is interesting because of the different personnel inventory at the task locations Central laboratory A, laboratory B and POCT. This is described in Tab. 53.4-11 – Profit contribution III – Fractions of Central Laboratory A, Laboratory B and POCT below for manpower costs and number of personnel and relation to the MFS points attained in each case (1 MFS point = 0.0582873 €). The site specific rendered activity/service in MFS points also shows the relevant fraction of apportion of infrastructure costs (= profit contribution III) (Tab. 53.4-11 – Profit contribution III – Fractions of Central Laboratory A, Laboratory B and POCT).

This result is used to calculate the internal point value related to the different locations: central laboratory A and laboratory B and leads to the actual IPV of the laboratory, integrating the total costs and proceeds.

The thus defined site specific IPV can be applied in internal transfer pricing and ideally supports the regulation and controlling of costs and services.

For further activity controlling, this calculation can also be applied to specific day times or shifts. The detailed defined point value precisely reflects the expenditure and proceeds in the particular laboratory. It can also be used to control activities/services in consideration of higher costs at defined time periods.

53.4.6 Full cost accounting – location based internal point value

Contribution costing III is the result after all proceeds and costs incurred. A positive result represents the profit of the specific location; a negative result reflects inefficient procedures and, thus, a loss regarding the operative business result /11/.

Tab. 53.4-11 – Profit contribution III – Fractions of Central Laboratory A, Laboratory B and POCT lists the amount of profit contribution III per FTE and per MFS point, related to the different locations: central laboratory A and laboratory B. Tab. 53.4-12 – Location-based IPV calculation in consideration of profit contribution III integrates profit contribution III per MFS point into the location-based IPV.

Multi-step contribution costing of a non-profit center organization provides the basis for the definition of the internal point value considered in internal transfer pricing. Since the contribution costing result includes all profits and expenditures the laboratory activities can be adjusted to zero based on the defined internal point value in non-profit center organizations. In this situation the credits from internal transfer pricing and the proceeds and charges from internal transfer pricing and costs nullify each other out (i.e. the IPV selected for the ITP must ensure that the total of all positive contributions equals the total of all costs incurred). This can be achieved by adjustment in the calculation of contribution costing II because the variables included in contributions costing I and III are defined.

Depending on the business philosophy and policy, the laboratory can be calculated to zero or, for example, to a positive result. A positive result extends the financial scope and allows options for further development.

The absolute profit contributions enable an internal comparison between departments and analyses and thus indicate starting points for action; relative changes allow internal comparison and reflect improvement or deterioration of performance.

Therefore, in a non profit center organization, the internal point value defined through contribution costing is used for cross disciplinary internal allocation of the laboratory activity in the internal transfer pricing system (ITPS). The IPV also allows an internal and external benchmark. Thus, the definition of the internal point value depends on the structure of the organization. The height of the internal point value in a non-profit center organization is dependent on the philosophy and policy.

The internal point value base on the total proceeds and total expenditures of the previous year and define the cots for the laboratory activities for the following time period.

In a non-profit center organization, profits are only indicated by proceeds in profit contribution costing I. Internal proceeds cannot be compared with those of a typical business because internal transfer pricing is considered as a credit. Since all costs of a laboratory are allocated by internal transfer pricing, the laboratory will always be efficient (i.e., even inefficient work would be rewarded in transferring idle costs). The laboratory might not care about its own costs in this case. Therefore, for internal controlling, a budget is created to limit the expenses. Budgets often have the problem that they are fixed and, thus, may leave no room for new activities and technologies, internal centralization and consolidation or external insourcing of laboratory activities. Therefore, budgets must be adjusted to changes.

Competition and increasing cost awareness necessitate cost-effective orientation of the laboratory processes to facilitate internal fusion and consolidation or prevent outsourcing /89/.

References

1. Wöhe G. Einführung in die allgemeine Betriebswirtschaftslehre 52: 358, 843 ff, 1077, 1149. München 2005.

2. Varian HR. Grundzüge der Mikroökonomie 306: 417. München 2011.

3. Haubrock M, Schär W. Betriebswirtschaft und Management in der Gesundheitswirtschaft 395–401, 654. Bern 2009.

4. No author: Deckungsbeitrag: https://de.wikipedia.org/wiki/Kosten-_und_Leistungsrechnung > Abrufdatum: 06.08.2020.

5. Coenenberg A G. Kostenrechnung 40 ff. Stuttgart 2009.

6. Lauterbach, W, Schrappe M (eds). Gesundheitsökonomie, Qualitätsmanagement und Evidence-based-Medicine 242. Stuttgart 2010.

7. No author: Deckungsbeitragsrechnung: https://de.wikipedia.org/wiki/Deckungsbeitrag > Abrufdatum: 17.05.2020.

8. Oberender P. Wachstumsmarkt Gesundheit. Stuttgart 2010.

9. Hintereder G. Steigerung der Qualität und Wirtschaftlichkeit durch umfassende Prozessoptimierung eines dezentralen Routinelabors, Master Thesis zur Erlangung des akademischen Grades MBA, 2007.

10. No author: Deckungsbeitragsrechnung: https://de.wikipedia.org/wiki/Deckungsbeitrag, Stand > Abrufdatum: 17.05.2020.

11. No author: Vollkostenrechnung: https://de.wikipedia.org/wiki/Vollkostenrechnung, Stand > Abrufdatum: 26.08.2019.

 

Further important references

  • Travers EM, Delahunty DC, Hunter LL, McClatchey KD, Rudar JM. Basic cost accounting for clinical services. Approved guideline. Clinical and Laboratory Standards Institute 1998; Vol 18, No 14.
  • Hindriks FR, Bosman A, Rademaker PF. The significance of indirect costs – application to clinical laboratory test economics using computer facilities. J Automatic Chemistry 1989; 11: 174–8.
  • Rinker G. Cost accounting applied to the clinical laboratory. Clin Lab Sci 1995; 8: 339–42.
  • Hernandez JS. Cost effectiveness of laboratory testing. Archives of Pathology & Laboratory Medicine 2003; 127: 440–5.
  • Ninci A, Ocakacon R. How much do lab tests cost? Analysis of Lacor Hospital Laboratory Services. Health Policy and Development 2004; 2: 144–50.

Table 53.2-1 Venipuncture technique

  • Look for a suitable vein (venous compression 10 cm above the antecubital fossa)
  • Decompress, disinfect (70% isopropanol, 70–80% ethanol)
  • Compress (30–50 mm Hg)
  • Make the skin taut over the puncture site by pulling it with the thumb of the free hand
  • Direct a 21G needle towards the vein while maintaining an angle of 30° and pointing the tip of the needle down
  • Do not puncture the vein deeper than the venous diameter
  • With the plunger create only such negative pressure as to allow the blood to flow freely into the syringe (twist the needle when the blood flow stops)
  • Decompress
  • After removal of the needle, stop the blood flow by applying pressure; do not let the patient bend the arm (since this interferes with the wound closure)

Table 53.2-2 Increasing (+) and decreasing (–) effects of hemolysis on laboratory results /40/

Parameter

Bias

Cause

ACTH

(–)

Proteolysis

APTT

(–)

Prothrombin release

AT

(–)

Analytical interference

AST

(+)

Release from cells

ALT

(+)

Release from cells

Albumin

(–)

Dilution

ALP

(–)

Analytical interference

Bilirubin

(–)

Analytical interference

Calcitonin

(+)

Proteolysis

Chloride

(–)

Dilution

Cortisol

(–)

Analytical interference

CK

(+)

Analytical interference

Creatinine

(+)

Analytical interference

D-dimers

(+)

Prothrombin release

Fibrinogen

(–)

Prothrombin release

Folate

(+)

Release from cells

GGT

(–)

Analytical interference

Gastrin

(–)

Proteolysis

Glucagon

(–)

Proteolysis

Glucose

(–)

Dilution

Haptoglobin

(–)

Analytical interference

Homocysteine

(–)

Analytical interference

Insulin

(–)

Proteolysis

Iron

(+)

Release from cells

Potassium

(+)

Release from cells

LD

(+)

Release from cells

Lipase

(+)

Analytical interference

Magnesium

(+)

Release from cells

Sodium

(–)

Dilution

Parathyroid hormone

(–)

Proteolysis

Phosphate

(+)

Release from cells

PSA

(+)

Analytical interference

Thromboplastin time

(–)

Prothrombin release

Urea

(+)

Release from cells

Testosterone

(–)

Analytical interference

Troponin I

(+)

Analytical interference

Troponin T

(–)

Analytical interference

Vitamin B12

(–)

Analytical interference

The bias was put in parentheses ( ) because, in many cases, low free hemoglobin concentration have no effect on the clinical assessment.

Table 53.2-3 Interferences to be considered during the pre analytical phase

Interference

Blood collection in glass or plastic tubes

The blood collection in glass or plastic tubes in the determination of enzymes, substrates, complete blood cell count and coagulation screening assays does not cause any alterations that might lead to results outside the reference interval in healthy individuals. The plasma free hemoglobin concentration, however, is significantly lower in glass tubes (0.16 ± 0.16 g/L) than in plastic tubes (0.25 ± 0.21 g/L) /29/. Slight differences are obtained in the determination of the analytes FT4, progesterone, prolactin, PSA and PAPP-A, but no effects are found in AFP, androstenedione, hCG, testosterone, insulin, IGF-I, PTH, CA-125 and ACTH assays /30/. Tube related differences in quality control material are caused by non inert additives /31/.

Serum vs. plasma

Differences in serum and plasma levels have been described for albumin, ALP, Ca2+, CO2, chloride, CK, glucose, LD, phosphate, K+ and total protein. However, it is only the differences in phosphate, K+, Ca2+ and total protein levels that can influence clinical decision making /32/.

Central venous catheter (CVC) vs. venous blood collection /34/

No significant differences between venous blood collection and blood collected by CVC have been detected regarding the markers of renal function and bone metabolism, urea, creatinine, phosphate, calcium, magnesium and ALP. However, this does not apply to intact PTH, where the concentrations in venous blood were 144 (59–273) μg/L and those in blood collected by CVC were 229 (87–360) μg/L. The difference results from the site of the CVC immediately below the thyroid veins.

EDTA plasma

Samples containing potassium EDTA as an anticoagulant give falsely elevated K+ and falsely low Ca2+, magnesium and ALP.

Hemolysis

Hemolysis is defined as the degradation of erythrocytes and the release of intracellular components into the surrounding (extracellular) fluid. Hemolysis may occur in vivo or in vitro. The upper cutoff limit of free hemoglobin (fHb) is 20 mg/L in plasma and 50 mg/L in serum. Values> 300 mg/L (18.8 μmol/L) can be visually detected by showing a pink tinge in serum or plasma. A fHb concentration above 500 mg/L is easily detectable and corresponds to 0.2–0.3% of erythrocytes/plasma volume in addition to physiological hemolysis at normal Hb concentration /34/. Visually detectable hemolysis is the upper limit at which many analyses may become subject to interference. In the presence of fHb ≥ 2.5 g/L, clinical chemical test results change as follows /35/: ALP –18%; AST +35%; bilirubin –12%; CK +15%; GGT –22%; K+ +14%; LD +149%; SP +13%. Falsely low test results are found when using immunoturbidimetric methods, for example for lipase or plasma protein determination if fHb concentrations above 2 g/L. Many laboratories apply a hemolysis index offered by analytical platforms, where absorbance at 570 nm triggers a signal indicating hemolysis. A hemolysis index of 100 is equivalent to a known fHb concentration of 1 g/L. The occurrence of hemolytic samples in the laboratory is approximately 3%. In a prospective study /36/ performed to identify the causes of hemolysis during venipuncture, the odds ratio for hemolysis was 19.5 (95% confidence interval (CI) 5.6–67.4) if the tourniquet time exceeded 1 minute. This applied to 99 in 333 blood sampling events. The hemolysis index is not applicable in the presence of hyperlipidemia, hyperbilirubinemia and in lyophilized quality control samples /37/. This is due to overlapping absorption spectra (Fig. 16.9-1 – Absorbance spectra of triglycerides, bilirubin and hemoglobin).

Hyperbilirubinemia

Bilirubin causes spectral interference as well as chemical interference /38/.

  • Spectral interference: bilirubin absorbs light of wavelengths between 340 and 500 nm, thus causing high background absorbance. In assays using acidic reagents, absorbance of direct bilirubin shifts into the UV range. At alkaline pH, bilirubin is oxidized and loses part of its absorbance.
  • Chemical interference: bilirubin, a reductant, is oxidized to biliverdin resulting in a decrease in absorbance. In oxidase-peroxidase-catalyzed reactions, H2O2 is formed and consumed in the presence of bilirubin. The decrease in H2O2 is dependent on the bilirubin concentration. Oxidase-peroxidase-catalyzed reactions are seen in assays for the determination of glucose, uric acid, cholesterol and triglycerides. The results of these assays are falsely low in the presence of bilirubin.

Hyperlipidemia

Hyperlipidemia causes spectral interference and a volume displacement effect.

  • Spectral interference: lipoprotein particles scatter light, with the scattering depending on the size and number of the particles. The highest scattering is from chylomicrons and VLDL. There may be an increasing or decreasing influence on the measurement result, depending on the blank value correction applied during photometry.
  • Volume displacement effect: the increased proportion of lipoprotein particles leads to a reduction in volume (and falsely increased concentration) of the water soluble substances of the serum. Lipophilic substances (including drugs) bind to lipoprotein particles and do not participate in the analytical reaction, resulting in falsely low concentrations.

Ascorbic acid

Ascorbic acid interferes with methodologies involving redox reactions. For instance, in a study /39/, a serum ascorbic acid concentration of 12 mmol/L caused the following increases: Na+ 43%, K+ 58%, calcium 103% and creatinine 26%. The values for chloride, total bilirubin and uric acid decreased by 33%, 63% and 83%, respectively

Bacterial contamination during blood collection

Contamination of venous blood cultures with bacteria colonizing the skin is relatively common during blood collection from peripheral veins. Contamination among 1460 samples was 8.8%. When venepuncture was achieved on the same sites as days before, contamination was significantly higher /40/.

Table 53.2-4 Collection of midstream urine specimen in women for a bacterial culture

  • Four gauze pads saturated with soap, four dry gauze pads, gloves, sterile container
  • Put on gloves and position yourself in front of the toilet bowel with legs apart, spread the labia with one hand
  • 4 × Using the gauze pads with soap, wipe the genital area from front to back (with the free hand)
  • 4 × Using the dry gauze pads, wipe from front to back
  • Pick up the urine container in one hand
  • Void urine into toilet bowl
  • Place urine container briefly into the stream of urine
  • Void the remainder of the urine into the toilet bowl

Table 53.2-5 Loss of pathologic bacteria (%) in comparison to colonizing bacteria due to the storage and transport of samples /28/

Time

Tempe-
rature

S.
pneum-
oniae

H.
influen-
zae

S.
aureus

Colonizing
bacteria

2–5 h

RT

– 46%

– 30%

Neisseria sp. + 33%

24–48 h

RT

– 25%

– 40%

Not determined

24 h

RT

– 51%

– 65%

– 9%

E. coli +> 400%

24 h

4 °C

– 39%

– 42%

– 47%

E. coli – 31%

48 h

RT

– 95%

– 74%

– 29%

Heavy growth, not evaluable

48 h

4 °C

– 56%

– 53%

– 49%

E. coli – 31%

RT, room temperature; –, not determined

Table 53.3-1 Analytical performance chracteristics of tests /125/

Criterion

Note

Accuracy

Agreement between measured concentration and the true concentration in the sample

Analytical sensitivity

Limit of detection: lowest concentration that can be reliably identified as being qualitatively present

Analytical specificity

Assessment of the effects of common interfering substances, such as hemoglobin, bilirubin, and lipids

Analytical reliability

The analytical reliability of a method depends on the criteria for accuracy, precision, specificity and detectability.

Bias

The bias of measurement is the difference between the expectation of the results of measurement and the true value of the measured quantity.

Carry over

Transfer of material from one analysis to the next. A carry over effect may be related to a sample or may occur independently.

Detection limit

The lowest result of a measurement by a given measurement procedure that can be accepted with a stated confidence level as being different from the value of the quantity obtained on a blank material .

Drift

The drift of an assay describes the degree of the time dependent systematic deviation in the values of the same specimen within the same test run or in different test runs.

Linearity

For a method, linearity is present within an interval where the expectation value and the actual value differ but randomly.

Precision

Agreement between repeated independent measurements of the same sample. The following terms are differentiated:

  • Within run precision: indicator for the closeness between measurement results if the method is repeatedly applied. This term is used synonymously with intraserial (or intraassay) precision
  • Reproducibility: indicator for the closeness between measurement results under comparable conditions (e.g., inter laboratory precision)
  • Between run precision: indicator for the closeness between measurement results if determinations are conducted using the same material in the same laboratory in consecutive test runs. If the test runs are conducted on consecutive days, this term is referred to as day-to-day (or inter assay) precision.

Reference interval

An interval, between upper and lower limits of test results that includes a defined proportion (usually 95%) of a reference group of (usually) healthy people

Cutoff

Level of biomarkers that is used for the categorization of test results e.g., as positive or negative

Robustness

The capability of an assay to reliably determine the analyte under consideration although the procedural guidelines are not closely adhered to.

Analytical stability

Ability of a system, when kept under specific conditions, to maintain a stated property value within specified limits for a specified period of time .

Table 53.3-2 Adult and pediatric quantitative limits of laboratory results that need urgent notification of the physician /5/

Parameter

Result

Note

Activated
partial
thrombo-
plastin time
(APTT)

≥ 75 sec.

Deficiency or inactivity of factors VIII, IX, XI, with risk of hemorrhage. In patients receiving heparin therapy there is a risk of hemorrhage due to prolonged aPTT to > 2.5-fold the upper reference limit.

Albumin

< 1.5 g/dL

Associated with ascites and edema.

Amino-
transferases

> 1,000 U/L

Notification depends on the patient population of the clinic or practice in question.

Ammonia

≥ 100 μg/dL
(59 μmol/L)

Risk of hepatic encephalopathy. Comatose states do not usually occur unless levels exceed 300 μg/dL (176 μmol/L).

Anion gap

≥ 20 mmol/L

Indicative of ketoacidosis or lactic acidosis, uremia, salicylate intoxication, poisoning from methanol or ethylene glycol.

Inorganic
phosphate

≤ 1.0 mg/dL
(0.32 mmol/L)

Muscle weakness, muscle pain, central nervous system symptoms such as disorientation, confusion, convulsions, coma, respiratory insufficiency with metabolic acidosis.

≥ 9.0 mg/dL
(2.9 mmol/L)

Occurs in acute tumor lysis syndrome and in terminal renal insufficiency.

Antithrombin
(AT)

≤ 50%

There is substantial inhibitor deficiency, which in those with elevated procoagulant activity poses a high risk of thromboembolic complications.

Ethanol

≥ 3.5 g/L
(76 mmol/L)

Blood alcohol concentrations of 3–4 g/L can be fatal, even in those who are not simultaneously using medical products.

≈ 4,0 ‰

Bilirubin

≥ 15 mg/dL
(257 μmol/L)

Hepatobiliary disease caused mainly by hepatotropic viruses and thus of infectious origin with risk of contagion.

Calcium, total

≤ 6.6 mg/dL
(1.65 mmol/L)

The ionized calcium concentration is within a range that may lead to hypocalcemic tetany.

Ca2+

≤ 3.1 mg/dL
(0.78 mmol/L)

≥ 14 mg/dL
(3.5 mmol/L)

Risk of hypercalcemic crisis associated with symptoms such as volume depletion, metabolic encephalopathy and gastrointestinal symptoms.

Ca2+

≥ 6.3 mg/dL
(1.6 mmol/L)

Chloride

≤ 75 mmol/L

Indicative of substantial metabolic alkalosis.

≥ 125 mmol/L

Indicative of massive primary metabolic acidosis or pseudohyperchloremia in the case of bromide intoxication.

Creatinine

≥ 7.4 mg/dL
(653 μmol/L)

Acute renal failure, e.g. in multiple organ failure or sepsis.

Creatine kinase

> 1,000 U/L

Notification depends on the patient population of the clinic or practice in question.

D-dimers

positive

In disseminated intravascular coagulation (DIC), detection of D-dimers is indicative of phase II (decompensated activation of the hemostasis system) or phase III (full-blown DIC).

Digoxin

> 2,0 μg/L
(2.56 nmol/L)

Non cardiac symptoms such as tiredness, muscle weakness, nausea, vomiting, lethargy and headache and cardiac symptoms such as sinus arrhythmia, bradycardia, and various degrees of atrioventricular block.

Digitoxin

> 40 μg/L
(52 nmol/L)

Fibrinogen

< 0.6 g/L

Risk of hemorrhage.

Fibrin
monomers

Positive

Indicative of consumption coagulopathy in disseminated intravascular coagulation, sepsis, shock, multiple injury, acute pancreatitis, and obstetric complications.

Glucose

≤ 45 mg/dL
(2.5 mmol/L)

Neuroglycopenic symptoms which can range from impairment of cognitive functions to loss of consciousness.

≥ 500 mg/dL
(27.8 mmol/L)

Diabetic coma due to insulin deficiency. Development of osmotic diuresis associated with severe exsiccosis and diabetic ketoacidosis (β-hydroxybutyrate > 5 mmol/L, standard bicarbonate < 10 mmol/L).

Hematocrit

≤ 0.18 (L/L)

Corresponds to a hemoglobin concentration < 60 g/L. Supply of oxygen to the myocardium is inadequate.

≥ 0.61 (L/L)

Leads to severe hyperviscosity of the blood, causing increased circulatory resistance and ultimately cardiac failure.

Hemoglobin

≤ 66 g/L

Supply of oxygen to the myocardium inadequate.

≥ 199 g/L

Corresponds to hematocrit ≥ 0.61 and leads to hyperviscosity syndrome.

Lactate

≥ 45 mg/dL
(5.0 mmol/L)

Indicator of type A hyperlactatemia, which is caused by an adequate supply of oxygen to the tissue. Pyruvate is no longer metabolized oxidatively, but reductively.

LD

≥ 1,000 U/L

Notification depends on the patient population of the clinic or practice in question.

Lipase

> 4-fold the
upper reference
interval value

Indicative of acute pancreatitis

Magnesium

≤ 1.0 mg/dL
(0.41 mmol/L)

Characteristic symptoms include paresthesia, cramp, irritability and athetoid tetany. The patient often shows cardiac arrhythmia in conjunction with hypokalemia; arrhythmia is intensified by digitalis.

≥ 4.9 mg/dL
(2.0 mmol/L)

Reduction in neuromuscular impulse transmission, resulting in sedation, hypoventilation with respiratory acidosis, muscle weakness, and reduced tendon reflexes.

Myoglobin

≥ 110 mg/L

Myocardial infarction should be suspected in patients with angina pectoris.

Osmolality

≤ 240 mmol/kg
of H2O

Cellular edema with an increase in cell volume and development of neurological-psychiatric symptoms.

≥ 330 mmol/kg
of H2O

Cellular water loss and intracellular increase in osmotically active substances, which do not permeate the cell membrane. Result: central symptoms and coma.

Osmotic gap

≥ 10 mmol/kg
of H2O

Indicative of intoxication from non electrolytes, which increase plasma osmolality, such as ethanol, methanol, ethylene glycol, isopropanol and dichloromethane.

PCO2

≤ 19 mmHg
(2.5 kPa)

Hyperventilation

≥ 67 mmHg
(8.9 kPa)

Hypoventilation

pH /24/

≤ 6,8

Values outside this range are not compatible with life.

≥ 7,8

PO2

≤ 43 mmHg
(5.7 kPa)

Such values correspond to a hemoglobin oxygen saturation of less than 80% and are to be regarded as lifethreatening.

Na+

≤ 120 mmol/L

Severely impaired tonicity (distribution of water across the intracellular-extracellular boundary) due to dysfunction of the ADH thirst mechanism, water absorption or renal concentration and dilution capacity. Clinical symptoms of severe hyponatremia result from volume and sodium depletion.

Severe hypernatremia is primarily manifested by CNS disorders such as confusion as well as increased neuromuscular irritability including convulsions and seizures.

≥ 160 mmol/L

FT4

≥ 35 ng/L
(45 pmol/L)

Indicative of thyrotoxicosis, a condition detectable clinically and in laboratory tests; the tissues are exposed to too high a thyroid hormone concentration and react to this. Possible causes include: Graves’ disease, trophoblastic tumor, hyperfunctional adenoma, toxic nodular goiter and, in rare instances, overproduction of TSH.

T3, total

≥ 30 μg/L
(46 nmol/L)

Platelet count

≤ 20 × 109/L

Risk of hemorrhage. Exclude EDTA-induced thrombocytopenia.

≥ 1 × 1012/L

Risk of thrombosis.

Thrombo-
plastintime
(TT)

≥ 27 seconds
(INR > 2)

Decrease in the vitamin K-dependent factors II, VII and X or in factor V. Since all these factors are synthesized in the liver, a decrease in TT to values below the specified level indicates a considerable disturbance of synthesis. In patients receiving coumarin therapy, there is a risk of hemorrhage if the TT is < 15%, which corresponds roughly to an INR of > 4.

Troponin

Elevated

Indicative of myocardial infarction.

Uric acid

≥ 13 mg/dL
(773 mmol/L)

Acute urate nephropathy with tubular blockade and renal failure. The uric acid/creatinine ratio in spontaneous urine in such cases is > 1.0 mg/mg.

Urea

≥ 214 mg/dL
(35.6 mmol/L)

Indicative of acute renal failure; unlike pre-renal and post-renal kidney failure, no disproportionate increase in urea compared to creatinine in serum.

BUN

≥ 100 mg/dL
(35.6 mmol/L)

Table 53.3-3 Neonatal quantitative limits of laboratory results that need urgent notification by the physician /5/

Parameter

Value

Note

Bilirubin

≥ 14 mg/dL
(239 μmol/L)

On the first day of life, e.g. in hemolytic disease of the newborn, risk of kernicterus

C-reactive
protein

≥ 50 mg/L

Indicative of neonatal sepsis

Glucose

≤ 30 mg/dL
(1.7 mmol/L)

Hypoglycemia, caused for example, by congenital metabolic disorder or hyperinsulinism due to maternal diabetes mellitus. Glucose concentrations < 25 mg/dL (1.3 mmol/L) should be treated with parenteral administration of glucose.

≥ 325 mg/dL
(18 mmol/L)

Urgent clarification of pathogenicity required

Hematocrit

≤ 0.33 (L/L)

Indicative of marked anemia with inadequate supply of oxygen to tissue.

≥ 0.71 (L/L)

Hyperviscosity of the blood with increased circulatory resistance.

Hemoglobin

≤ 85 g/L

Risk of multiorgan failure, especially if the patient has combination of ischemia and hypoxia

≥ 230 g/L

Abnormal flow kinetics (hyperviscosity) with increased circulatory resistance and increased load on the heart

IgM

≥ 20 mg/dL

Umbilical cord blood IgM concentration above the limit can be linked to intrauterine infection

Potassium

≤ 2.6 mmol/L

Occurrence of neuromuscular symptoms with hyporeflexia and paralysis of the respiratory muscles

≥ 7.7 mmol/L

Clinical consequences are heart rhythm disturbances, weakness of the skeletal muscles, and respiratory paralysis.

Leukocyte
count

≤ 5 × 109/L

Values below and above these limits can be indicative of neonatal sepsis

≥ 25 × 109/L

PO2

≤ 37 mmHg
(4.9 kPa)

Decrease in hemoglobin oxygen saturation to below 85%.

Platelet
count

≤ 80 × 109/L

In normal weight neonates, such platelet count should give rise to further clinical and laboratory investigations. In a birth weight below 2500 g, the cutoff value is ≤ 50 × 109/L

TSH

≥ 40 mIU/L

Suggestive of congenital hypothyoidism

Table 53.3-4 Adult and pediatric quantitative and qualitative limits which, need urgent notification of the physician /5/

Cerebrospinal fluid

  • Increased cell count
  • Leukocytosis, tumor cells
  • Glucose lower than in serum
  • Lactate ≥ 20 mg/dL (2.2 mmol/L)
  • Detection of pathogens by Gram staining or agglutination test

Urine

  • Strongly positive test strip reaction for glucose and acetone
  • Red cell casts or > 50% dysmorphic erythrocytes
  • Severe hemoglobinuria (no erythrocytes on microscopic examination
  • Detection of drugs

Differential blood count

  • Leukemoid reaction
  • Suspected leukemia, especially promyelocytic leukemia
  • Suspected aplastic crisis
  • Sickle cells
  • Malaria parasites

Microbiology

  • Detection of pathogens by Gram staining of blood culture or of exudates and transudates of body cavities
  • Antigenic detection of pathogens with rapid tests such as latex agglutination, immunofluorescence or immunoassay, e.g. group B Streptococci sp., Legionella sp., Pneumocystis carinii, Cryptococcus sp., Hepatitis virus
  • Detection of acid-fast bacilli or detection of M. tuberculosis after amplification (PCR)
  • Cultural detection of Salmonella sp., Shigella sp., Campylobacter sp., C. difficile, C. perfringens, N. gonorrhoeae, B. pertussis, N. meningitidis, C. diphtheriae as well as pathogenic fungi such as Aspergillus sp., Blastomyces sp., Coccidioides sp., Histoplasma sp. and Cryptococcus sp.
  • Detection of HIV antibodies
  • Detection of methicillin-resistant S. aureus
  • Detection of Norovirus
  • Detection of enterohemorrhagic E. coli
  • Detection of multiresistant Gram negative bacterium
  • Detection of COVID-19

Table 53.3-5 Influence factors to be considered in the medical assessment of laboratory results

Influence factors

Gender

Gender specific reference ranges are specified for numerous laboratory tests. The differences between men and women in some analytes are presumably due to the differences in body weight, body surface and muscle mass. Values of men are higher than those of women in the following biochemical assays: GGT, triglycerides, uric acid, creatinine, ammonia, CK, AST, ALP, iron, urea, cholesterol /7/.

Race

Race is insignificant for the reference intervals of enzymes, except for CK. Analytes such as cholesterol, triglycerides and uric acid are more influenced by dietary habits within social classes than by racial differences. Racial differences play an important role in the frequency distribution of blood group types and in the correlation between the phenotype and the concentration of certain plasma proteins, for example, in haptoglobin and α1-antitrypsin.

Age

The following changes occur:

  • Increase: glucose, creatinine, urea, cholesterol, ALP, α-amylase, LD, fibrinogen, ESR, CRP. An increase in monoclonal gammopathies has also been confirmed.
  • Decrease: calcium, phosphorus, total protein, albumin, immunoglobulins, glucose tolerance, estimated GFR, vitamin B12, folic acid, white blood cell count and platelet count. Albumin decreases by 0.53 g/L per decade of life.

Enzyme activities are higher in childhood than in adulthood, but iron, copper and immunoglobulin concentrations are lower. All blood cell count parameters of newborns are higher than those of infants and adults /9/.

Body mass index

Body mass index is the measure for determination of nutritional status during pediatric years and in adults. It is determined from the ratio of body weight (kg)/(body height in m)2. As the body mass rises, so do first order risk factors such as blood pressure and lipoprotein concentration; glucose tolerance decreases. This also applies to second-order risk factors such as uric acid, triglycerides and CRP /10/.

The concentrations of uric acid, cholesterol, LD, insulin and postprandial glucose are higher in obese men and women than in individuals with a normal body weight. Obese men have higher AST, creatinine, total protein and uric acid levels than normal men. Phosphate is lower in obese than in non obese individuals of both genders and calcium is lower in obese than in normal women.

Fasting duration

Even if blood samples are collected after fasting, the concentration of metabolic markers may still differ in relation to fasting duration. Non fasting men have lower mean LDL cholesterol and higher glucose, insulin and triglyceride levels than fasting men. These differences are more marked among diabetics and larger with fasting duration above 6 h /11/.

Time of blood specimen collection

Marked differences are found if blood samples are collected from fasting individuals at 8:00, after breakfast at 9:30, and again at 11:00. In a study /12/ the coefficient of variation (CV,%) of the patient results was compared with the analytical CV. The observed results of the individuals’ test results far exceeded the analytical CV, the average was 3.5 for enzymes, metabolic parameters and electrolytes, 5 for cellular components of the complete blood count, 5 for TSH, and 1.5 for FT4.

Time of day

A distinction was made between individual, non-regular variance in blood components during the course of the day and the circadian variance (endogenous rhythm of the 24-h periodicity) /13/.

Complete blood count: the time component of variance does not require blood specimen collection at constant times of day.

Clinical chemistry: Na, K, Ca, Mg have peak levels between 21:00 and 6:00, while the phase of the phosphate rhythm is shifted by 180°.

Hormones: cortisol and ACTH reach peak levels in the morning and trough levels at night. Testosterone reaches its minimum between 16:00 and 20:00 and increases at night to reach peak values at 6:00. The concentration of prolactin elevates with increasing duration of sleep; growth hormone increases during the early period (first 2 h) of sleep.

Acid-base parameters: only slight changes; nocturnal pH decrease.

A change in circadian rhythm is observed if times of day are shifted, for example, in journeys along the degrees of latitude. The body needs 6–8 h to adapt to the new time of day. However, diurnal variance and circadian rhythms do not change in all individuals. The changes may be masked by short term oscillations of the blood components, e.g. cortisol levels varying within minutes in the morning, or by longer-term oscillations. Individual variances, circadian rhythms and long term oscillations only play a role in longitudinal assessment of the biochemical marker concentrations and are less significant for transverse assessment because they stay within the reference intervals.

Stress

The excretion of catecholamines and 17-hydroxycorticoids is elevated and serum concentrations of cortisol, renin, aldosterone, growth hormone, TSH and prolactin increase.

Bed rest

The excretion of sodium, calcium, chloride, phosphorus and ammonia increases, as do serum ALP concentrations.

Pregnancy

Increase in ALP, cholesterol, triglyceride, copper, ceruloplasmin, transferrin, WBC count, progesterone, estradiol, estriol, prolactin; new occurrence of hCG and α-fetoprotein /14/.

Decrease in iron, magnesium, calcium, total protein, albumin, cholinesterase, hemoglobin, hematocrit, RBC count, glucose, HbA1c (see also Section 38.1 – Laboratory findings in normal pregnancy).

Alcohol intake

A few minutes after alcohol consumption, AST increases mildly and, after 3 h, reaches a peak which in nondrinkers is usually within the reference interval. A mild increase in GGT occurs slightly delayed. The influence of alcohol intake on the activities of other enzymes is not measurable. A pronounced increase in triglyceride level for hours or days is measured in some individuals after the consumption even of small amounts of alcohol. Chronic alcoholics can have permanently elevated GGT.

Contraceptives

Second-generation hormonal contraceptives cause an increase in cholesterol. This effect does not occur in third generation contraceptives (quantity of ethinylestradiol below 50 μg). However, the CRP is elevated to above 3 mg/L in a third of women on third generation contraceptives. Thyroxin binding globulin and ceruloplasmin are elevated in 44% and 70%, respectively, of women on contraceptives /1516/.

Surgery

The effects on the complete blood count, enzyme activities and serum analyte concentrations are dependent on the underlying disease and the type of surgical intervention. The following changes generally occur in the first 24 h after surgery: decrease in hemoglobin, leukocytosis, elevated ESR, increase in acute phase proteins (e.g., CRP) mild transient hyperbilirubinemia and increase in urea. Increases in CK are observed, in particular, in abdominal and thoracic surgery and can amount to 3–10-fold the upper reference interval value. Mild increases in aminotransferases are frequently encountered in cholecystectomy and less frequently in other surgical interventions /17/.

Ionizing radiation

Ionizing radiation therapy causes a decrease in platelets and white blood cells and an increase in uric acid up to 30 mg/dL (1785 μmol/L) by damaging tumor tissue.

Gammopathy interference

Monoclonal immunoglobulins (mIg) cause interference in clinical chemistry assays because of the formation of turbidity. The interference may be prevented by optimizing the buffering conditions of the reagents to avoid the formation of turbidity or by removal of the mIg prior to analysis of the sample. Examples of mIg interference for the analytes glucose, bilirubin, γ-glutamyltransferase, urea and ferritin are presented in Ref. /18/.

Patient hydration

Variations in patient hydration can overwhelm the improvement in analytical precision and accuracy /19/. Large numbers of tests are performed without recognition that the major daily shifts are common place. Variations up to 15–23% over a period are possible solely on the fluctuation in plasma volume /20/.

Within subject biological variation in health and disease

In a study /21/ the within-subject variation (CV) for 66 laboratory tests in 34 disease states was investigated and the results compared with the CV determined in healthy individuals. For the majority of quantitative tests studied, CV values were in the same order in disease and health. However, the CV was higher than that in healthy individuals in organ specific diseases, namely:

  • Alpha fetoprotein in hepatic diseases
  • ALP in chronic renal failure, in chronic liver disease, and in Paget’s disease
  • CA 125 in ovarian cancer
  • CA 15-3 in breast cancer
  • CEA in colorectal cancer
  • Creatinine in kidney disease and post-transplantation patients
  • HbA1c, Lp(a) and first morning urine albumin in patients with diabetes mellitus.

Table 53.3-6 Drug-induced effects on serum parameters /222324/

Test

Effect

Glucose

I

Nicotinic acid ester, phenytoin, prednisolone, propranolol, thiazide, chlorpromazine, indomethacin, levodopa oral antidiabetic drugs

D

Cimetidine, clofibrate, disopyramide, paracetamol, pentamidine

Cholesterol

I

Chlorthalidone, hydrochlorothiazide, oral contraceptives (not the micropill)

D

Vitamin C (prolonged intake)

Uric acid

I

Acetazolamide, bumetanide, hydrochlorothiazide, cyclosporine, ethambutol, furosemide, methoxyflurane, nicotinic acid ester, pyrazinamide. See also Section 5.4 – Uric acid.

D

Allopurinol, alprenolol, salicylic acid, clofibrate, phenylbutazone, tienilinic acid, azlocillin

Creatinine

I

Amoxapine, salicylic acid, cimetidine, cotrimoxazole, cyclosporine, mefenamic acid, methoxyflurane, tienilinic acid, trimethoprim-sulfamethoxazole

Calcium

I

Tamoxifen, thiazide

D

Antiepileptic drugs, lithium, propranolol

Phosphate

I

Propranolol, intralipid

D

Cimetidine

Bilirubin

I

Acetaminophen, amsacrine, androgens, aspirin, azathioprine, captopril, carbamazepine, carbimazole, chlorpromazine, cotrimoxazole, erythromycin, gold salts, halothane, heroin, hydralazine, intralipid, isoniazid, ketoconazole, mercaptopurine, methotrexate, α-methyldopa, methyltestosterone, naproxen, nitrofurantoin, oxyphenasitin, paracetamol, perhexiline, penicillamine, phenylbutazone, phenytoin, propylthiouracil, ranitidine, rifampicin, sulfamethoxazole/ trimethoprim, sulfasalazine

ALP

I

Allopurinol, amsacrine, carbamazepine, cotrimoxazole, cyclophosphamide, disopyramide, erythromycin, gold salts, isoniazid, ketoconazole, mercaptopurine, methotrexate, methoxyflurane, α-methyldopa, methyltestosterone, oxacillin, oxyphenisatin, papaverine, penicillamine, perhexiline, phenobarbital, phenylbutazone, phenytoin, primidone, propylthiouracil, ranitidine, trimethoprim/sulfamethoxazole, sulfasalazine, valproic acid. See also Section 1.3 – Alkaline phosphatase (ALP).

D

Clofibrate, oral contraceptives

AST and ALT

I

Acetaminophen, amiodarone, salicylic acid, carbamazepine, disopyramide, heparin, megalatran, mevinacor, oxacillin, oxyphenisatin, papaverine, paracetamol, penicillamine, perhexiline, phenylbutazone, phenytoin, ranitidine, rifampicin, streptokinase, trimethoprim/sulfamethoxazole, valproic acid. See also Section 1.6 – Alanine aminotransferase (ALT), Aspartate aminotransferase (AST).

CK

I

Statins, olanzapine

Intox

Amphetamines, barbiturates, ethanol, heroin, theophylline. Refer to Tab. 1.8-4 – Creatinkinase.

D

Prednison

GGT

I

Carbamazepine, erythromycin, oral contraceptives (other than the micropill), oxacillin, phenytoin. See also Section 1.9 – Gamma-glutamyl transferase (GGT).

D

Clofibrate

LD

I

Allopurinol, amiodarone, androgens/anabolic steroids, aspirin/salicylates, captopril, carbamazepine, chlorpromazine, cisplatin, clozapine, coumarin, dacarbazine, diltiazem, erythromycin, fluphenazine, gold salts, α-methyldopa, naproxen, paracetamol, papaverine, penicillamine, perhexilline, phenytoin, phenylbutazone, propylthiouracil, ranitidine, sulfasalazine, tienilinic acid, valproic acid, verapamil.

D

Clofibrate, oral contraceptives.

I, increase; D, decrease; Intox, intoxication; LD, lactate dehydrogenase

Table 53.3-7 Terms characterizing the conclusiveness of laboratory findings /26/

Criterion

Note

Diagnostic (clinical)
sensitivity

Reflects the fraction of disease cases that the assay correctly predicts (Tab. 53.3-9 – Clinical sensitivity and specificity based on the number of positive and negative results).

Diagnostic (clinical)
specificity

Reflects the fraction of individuals with absence of disease that the assay correctly predicts (Tab. 53.3-9).

Hazard ratio

Describes the cumulative risk ratio over time for two analyzed cohorts. If, in a pharmacological study, for instance, twice as many tested individuals in the untreated cohort die as in the treated cohort, the hazard ratio of non treatment is 2. The hazard ratio is 1 if there is no difference between the two cohorts.

Prevalence

Prevalence (pretest probability): the frequency of patients with a certain disease in the group being tested with the measurement (Tab. 53.3-8 – Dependence of positive predictivity on prevalence of disease and clinical sensitivity and specificity of the test).

Positive predictive
value

Predictive value (post test probability) of a positive test: probability of a disease being present if the test is positive (Tab. 53.3-10 – Positive and the negative predictive values based on the number of positive and negative results).

Negative predictive
value

Predictive value (post test probability) of a negative test: probability of a disease being absent if the test is negative (Tab. 53.3-10).

Diagnostic efficiency

Describes the ratio between the true results and the total of all results for a cohort. Depends on the diagnostic sensitivity and spe­cificity of the test and the prevalence of the disease (Tab. 53.3-12 – Calculation of the diagnostic efficiency of a laboratory test).

Likelihood ratio (LR)

Identifies the fraction of true-positive and false-positive results in a cohort of diseased individuals. LR = sensitivity/non-specificity. 1 – specificity = non-specificity.

Measurand

Measurable quantity subject to measurement.

Measurement

Set of operations that have the object of obtaining a value of a measurable quantity.

Odds ratio

The ratio between the probability P of the occurrence of an event E and the probability of the non-occurrence of this event (1–P). ORE = PE/1–PE. If OR = 1, then the probability is 50 : 50. The difference between two correlations at OR 10 is five times as high as at OR 2.

Reference
measurement

Thoroughly investigated measurement procedure shown to have an uncertainty of measurement commensurate with its intended use, especially in assessing the trueness of other measurement procedures for the same quantity and in characterizing reference materials (prEN 12286, 3.7: 1997).

Reference standard

Standard, generally having the highest metrological quality available at a given location or in a given organization, from which measurements made there are derived (VIM, 6.6: 1993)

Reproducibility

Closeness of the agreement between the results of measurements of the same measurand carried out under changed conditions of measurement.

Result

Value attributed to a measurand, obtained by measurement (VIM, 3.1: 1993)

Sample

An aliquot of a specimen that has been appropriately collected and transported and processed in the laboratory to provide material for a specific laboratory test.

Sampling

Process of drawing or constituting samples.

Specimen

A volume of blood appropriately collected to perform one or more laboratory tests.

Test

A technical operation that consists of the determination of one or more characteristics or performance of a given product, material, equipment, organism, physical phenomenon, process or service according to a specified procedure (ISO guide 25, 3.5: 1990)

Traceability

Property of the result of a measurement or the value of a standard whereby it can be related reference, usually national or international standards, through an unbroken chain of comparison all having stated uncertainties. Note 1: the concept is often expressed by the adjective traceable. Note 2: the unbroken chain of comparisons is called traceability chain.

Trueness

The closeness of agreement between the average value obtained from a large series of test results and an accepted reference value (ISO 3435-1, 3.2: 1993)

Uncertainty

Measurand, associated with the result of a measurement, that characterizes the dispersion of the values that could be reasonable attributed to the measurand (VIM, 3.9: 1993)

Validation

Confirmation by examination and provision of objective evidence that the particular requirements for a specific intended use are fulfilled (ISO 8402, 2.18: 1994).

Verification

Confirmation by examination and provision of objective evidence that the particular requirements for a specific requirement are fulfilled (ISO/IEC 25; 3.2: 1997)

Z-score

The z-score, also referred to as standard score, describes the number of standard deviations a measured value is above the mean, based on the prerequisite that the correlated mean stems from a normal distribution. Z = x – m/s; Z, score; x, measured value; m, mean; s, standard deviation of the normally distributed population.

Table 53.3-8 Dependence of positive predictivity on prevalence of disease and clinical sensitivity and specificity of the test

Prevalence
(%)

Predictive
value (%)

Sensitivity
(%)

Specificity
(%)

0.1

1.9

95 

95

9.0

99

99

1.0

16.1

95 

95

50

99

99

2.0

27.9

95 

95

66.9

99

99

5.0

50

95 

95

83.9

99

99

50

95

95 

95 

99

99

99

Table 53.3-9 Clinical sensitivity and specificity based on the number of positive and negative results

Clinical sensitivity (%) = Number of true-positive results × 100 Number of true-positive results + false-negative results Clinical specificity (%) = Number of true-negative results × 100 Number of true-negative results + false-positive results

Table 53.3-10 Positive (PVpos) and negative (PVneg) predictive values based on the number of positive and negative results

PV pos (%) = Number of true-positive results × 100 Total number of positive results (true-positive + false-positive) PV neg (%) = Number of true-negative results × 100 Total number of negative results (true-negative + false-negative)

Table 53.3-11 Positive and negative predictive values (PV) if the clinical sensitivity, specificity and prevalence are known

PV pos (%) = Prevalence × Sensitivity × 100 Prevalence × Sensitivity + (100 – Prevalence) × (100 – Specificity) PV neg (%) = (100 – Prevalence) × Specificity × 100 (100 – Prevalence) × Specificity + Prevalence × (100 – Sensitivity)

The prevalence, clinical sensitivity and clinical specificity must be entered into the equations in %.

Table 53.3-12 Calculation of the diagnostic efficiency of a laboratory test

Efficiency = Number of true-positive + true-negative results Number of all results Efficiency = Prevalence × Sensitivity + (1 – Prevalence) × Specificity

Table 53.4-1 Important business management terms

Designation

Description

Example

Cost object

Product or service generating proceeds

Car, project, laboratory parameter

Direct cost (individual cost)

Costs directly assignable to the cost object

Employment costs incurred during cost object processing.

Direct material costs; material required by, and directly assignable to, the cost object itself.

Indirect cost (overhead cost)

Costs allocated using an apportionment key

Administration costs

Infrastructure costs

Marketing costs

Fixed cost

Associated with fixed factors; independent of the output level /2/

Building costs, IT, insurances, indirect employment costs not related to the provision of an activity.

Variable cost

Output and quantity-based costs

Direct material costs (e.g., reagent for analysis). Material costs are variable (i.e., they increase continuously) or as a function of synergy effects with the output quantity.

Total costs

Total of fixed costs and variable costs

A distinction is made between total and partial absorption costs.

Full cost accounting

Total absorption costs per cost object

All costs incurred are allocated to the cost objects.

Partial cost accounting

Total costs per cost object and (infrastructure costs + overhead costs per x)

Only part of the costs are directly allocated to the cost objects; the remaining part enters the operating result in a different way (e.g. by contribution costing).

Profit contribution

Profit contribution = Proceeds – variable costs (total profit contribution) /3/

Provides information on the controllability of the costs and the cost structure of goods and services. The profit contribution is the amount available for covering the fixed costs. It can be related to both the total quantity of a product and the quantity unit (unit size).

Break even analysis, contributing costing

The profit contribution is the operand of contribution costing

Within multi-level contribution costing (fixed charge costing), a distinction is made between various levels depending on the business level analyzed (i.e., which fixed business costs are taken into consideration). Some companies have established 5-level contribution costing or even 13-level contribution costing or even more /4/.

Internal transfer pricing (ITP), internal cost allocation

Internal cost apportionment due to cross-departmental provision of activities

Internal transfer pricing or fixed apportionment accounting can be performed depending on the type of activity/service /2/.

Key performance indicator

Figure with or without unit of measurement, allowing a relevant statement (e.g., error rate)

Manufacturing costs per unit

Full time equivalent for unit

Units per stuff member

Productivity

Quantitative output divided by quantitative input

Correlates the quantitative production result with the production factors used (e.g., reported results per hour).

Labor productivity

Number of identical activities per working hour

Partial productivity indicator indicating the performance per personnel (e.g. reported results per full time equivalent).

Average cost/unit cost

Costs per output unit

Refers to the fixed costs, variable costs or total costs. The average costs change as a function of quantity; as quantities increase, fixed costs are apportioned to a higher number; thus, average fixed costs decrease with increasing quantity. Average variable costs are constant; in special cases, however, they can also increase as a function of quantity. Both effects combined yield a U-shaped average curve for the total cost.

Marginal cost

Change in cost/change in output

Increase in total cost caused by the production of the last output unit /1/.

Proceeds

Price of a good multiplied by the amount sold

The change in proceeds correlates with the elasticity of demand. For example, the price of a product increases, the quantity of sold products decreases; the proceeds may increase or decrease. Therefore, the direction of the change in proceeds depends on the response of demand to the change in price (= elasticity) /2/.

Return

Value of all activities/services rendered within a period of time

Revenue leads to an increase in net worth (= capital); capital gain of a business within a defined period of time.

Expenditure

Value of all activities/services consumed within a period of time

Expenditure leads to a decrease in net worth (= capital); consumption of goods (commodities and services) within a defined period of time.

Profit

Proceeds – expenditure

The relation between amount of profit and capital investment allows the assessment of success /1/.

Table 53.4-2 Calculation of the internal point value (IPV)

 

2006 €

2007 €

2008 €

2009 €

2010 €

Deviation*

Σ total costs

110,000

120,000

125,000

130,000

140,000

7.7%

Σ MFS points

12,000,000

13,500,000

14,000,000

14,500,000

15,000,000

3.5%

IPV

0.00917

0.00889

0.00893

0.00897

0.00933

4.1%

* Deviation from previous year

Table 53.4-3 Allocation of costs to different locations

Total costs

2008 €

2009 €

2010 €

Deviation*

Total

125,000

130,000

140,000

7.7%

Central laboratory A

101,875

111,800

121,100

8.32%

Laboratory B

18,750

12,350

12,600

2.02%

(POCT)

4375

5850

6300

7.69%

* Deviation from previous year

Table 53.4-4 Location based calculation of the internal point value (IPV)

IPV

2008

2009

2010

Dev.*

Laboratories A+B

  • Total

0.00893 €

0.00897 €

0.00933 €

4.1%

  • MFS fraction

15.32%

15.38%

16.01%

Central lab A

  • Total

0.00758 €

0.00798 €

0.00829 €

4.0%

  • MFS fraction

13.00%

13.69%

14.22%

Laboratory B

  • Total

0.03348

0.02505

0.02800

11.8%

  • MFS fraction

57.44%

42.98%

48.04%

* Deviation from previous year

Table 53.4-5 Example of single-level contribution costing

 

Product A

Product B

Total

%

%

%

Turnover

300,000

100

500,000

100

800,000

100

Variable cost

140,000

47

250,000

50

390,000

49

Profit
contribution

160,000

53

250,000

50

410,000

51

Fixed cost

 

260,000

33

Result

 

150,000

19

Table 53.4-6 Example of single level contribution costing

 

Product

Product

Product

Product

Total

A-1 €

A-2 €

B-1 €

B-2 €

Turnover

300,000

500,000

160,00

200,000

1,160,000

Variable cost

–140,000

–250,000

–60,000

–120,000

–570,000

Profit
contribution I

160,000

250,000

100,000

80,000

590,000

Product fixed
cost

–60,000

–130,000

120,000

–30,000

–340,000

Profit
contribution II

100,000

120,000

–20,000

50,000

250,000

Task/location
fixed cost

 

–110,000

 

–20,000

–130,000

Profit
contribution III

100,000

10,000

–20,000

30,000

120,000

Business fixed
cost

 

–15,000

Operating result

 

105,000

Table 53.4-7 Calculation of profit contribution I

Profit contribution I

2008

2009

2010

Deviation*

Σ direct profits

+3750 €

+9880 €

+13,440 €

36%

Total costs
(annual accounts)

–125,000 €

–130,000 €

–140,000 €

7.7%

Σ profit contrib. I
(annual accounts)

–121,250 €

–120,120 €

–126,560 €

5.4%

MFS points

14,000,000

14,500,000

15,000,000

3.4%

Ø IPV, profit contrib. I

0.08661 €

0.00828 €

0.00844 €

1.9%

Fraction
of MFS points*

14.86%

14.21%

14.48%

1.9%

* 1 MFS point = 0.0582873 €; *Deviation from previous year

Table 53.4-8 Key indicator for profit contribution I per point

Key indicator**

2008

2009

2010

Deviation*

Σ direct profits

3750 €

+ 9880 €

+ 13,440 €

36%

MFS points

14,000,000

14,500,000

15,000,000

3.4%

Profit
contribution*

0.00027 €

0.00068 €

0.00090 €

31.5%

** Profit per MFS point; *Deviation from previous year

Table 53.4-9 Calculation of profit contribution II

Profit contribution II

2009

2010

Deviation*

Σ profit contrib. I
(annual accounts)

–120,120 €

–126,560 €

5.4%

Σ credits, medical SP

+140,000 €

+150,000 €

7.1%

Profit contrib.
+ ITP credit

=19,880 €

=23,440 €

17.9%

Σ charges, medical SP

–15,000 €

–16,000 €

6.7%

Profit contrib. II

= 4880 €

= 7440 €

52.5%

* Deviation from previous year

Table 53.4-10 Calculation of profit contribution III

Profit contribution III

2009

2010

Deviation*

Σ profit contribution II
(annual accounts)

–4880 €

+7440 €

52.5%

Σ apportionment

–1200 €

–1400 €

16.7%

Profit contribution III

= 3680 €

= 6040 €

64.1%

* Deviation from previous year

Table 53.4-11 Profit contribution III – Fractions of Central Laboratory A, Laboratory B and POCT

 

Fraction of infrastructure costs

 

2008

2009

2010

2008

2009

2010

Manpower costs (MC), total

47,500 €

49,400 €

53,200 €

1200 €

1250 €

1400 €

Profit contrib. III/€ MC

 

0.02526 €

0.02530 €

0.02632 €

Central laboratory A

40,138 €

41,743 €

44,954 €

1014 €

1056 €

1183 €

Laboratory B

5938 €

6175 €

6650 €

150 €

156 €

175 €

Point of care test (POCT)

1425 €

1482 €

1596 €

36 €

38 €

42 €

FTE, total

2.7

3.1

3.20

1200 €

1250 €

1400 €

Profit contrib. III/FTE

 

444 €

403 €

437 €

Central laboratory A

2.28 €

2.62 €

2.70 €

1014 €

1056 €

1183 €

Laboratory B

0.34 €

0.39 €

0.40 €

150 €

156 €

175 €

Point of care test (POCT)

0.08 €

0.09 €

0.10 €

36 €

38 €

42 €

MFS points, total

14,000,000

14,500,000

15,000,000

1200 €

1250 €

1400 €

Profit contrib. III/MFS points

 

0.00009 €

0.00009 €

0.00009 €

MFS points/ Labo­ratory A

13,720,000

13,920,000

14,550,000

0.00007 €

0.00008 €

0.00008 €

MFS points/ Labo­ratory B

280,000

580,000

450,000

0.00054 €

0.00027 €

0.00039 €

Table 53.4-12 Location-based IPV calculation in consideration of profit contribution III

Internal point value – profit contribution III

2008

2009

2010

IPV, Central laboratory A only

0.00758

0.00798

0.00829

  • MFS fraction,
    Central laboratory A

13.00%

13.69%

14.22%

Profit contrib. III per MFS point,
Central laboratory A

0.00007 €

0.00008 €

0.00008 €

  • Fraction of profit contrib. III,
    Central laboratory A
    per MFS point

0.13%

0.13%

0.14%

IPV, Central laboratory A,
incl. profit contrib. III

0.00765 €

0.00806 €

0.00837 €

MFS, Central laboratory A,
incl. profit contrib. III

13.13%

13.82%

14.36%

IPV, Laboratory B only

0.03348 €

0.02505 €

0.02800 €

  • MFS fraction, Laboratory B

57.44%

42.98%

48.04%

Profit contrib. III per MFS point,
Laboratory B

0.00054 €

0.00027 €

0.00039 €

  • Fraction of profit contrib. III,
    Laboratory B
    per MFS point

0.92%

0.46%

0.67%

IPV, Laboratory B,
incl. profit contrib. III

0.03402 €

0.02532 €

0.02839 €

MFS, Laboratory B,
incl. profit contrib. III

58.36%

43.44%

48.71%

Figure 53.3-1 Framework for laboratory testing in medical decision making /8/.

AnamnesisPhysical examinationSuspected diagnosisDifferential diagnosisFinal diagnosisMonitoring of the disease progressionLaboratory results of:– Severity of the disease– Evaluation of the progress– Evaluation of the prognosis– Evaluation of the therapy Imaging techniquesLaboratory results for confirmation or for an exclusion of a suspected diagnosis Laboratory results of a limited number of tests for an examination of frequent diseases

Figure 53.3-2 Frequency distribution curves of healthy and diseased populations. A shift of the decision threshold towards lower test values increases the diagnostic (clinical) sensitivity of the test, but the number of false-positive results in the healthy population increases (decrease in clinical specificity). A shift of the decision threshold towards higher test values reduces the number of false-positive results (increase in clinical specificity) but the clinical sensitivity decreases.

Probability density Diagnosticsensitivityincrease Diagnosticspecificityincrease Threshold False positive False negative Test value Truepositiveresults Truenegativeresults
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