Clinical Biochemistry

Editors:

prof. MUDr. Jaroslav Racek, DrSc., MUDr. Daniel Rajdl, Ph.D.

Authors:

Vladimír Bartoš, Milan Dastych, Milan Dastych jr., Tomáš Franěk, Milan Jirsa, Marta Kalousová, Tomáš Karlík, Petr Kocna, Viktor Kožich, Michaela Králíková, Alena Krnáčová, Pavlína Kušnierová, Richard Pikner, Věra Ploticová, Richard Průša, Jaroslav Racek, Daniel Rajdl, Václav Senft, Vladimír Soška, Drahomíra Springer, Kristian Šafarčík, Ivan Šebesta, Radka Šigutová, Zdeněk Švagera, Eva Táborská, Libor Vítek, František Všianský, Jiří Zadina, David Zeman, Tomáš Zima

Published by Charles University in Prague, Karolinum Press

www.karolinum.cz

ebooks@karolinum.cz

First edition January 2015

The publication has been pratly created within project Klinická biochemie – inovovaná, interaktivní výuka e-learningem, reg. number: CZ.1.07/2.2.00/15.0048 and is co-funded by the European Social Fund and the state budget of the Czech Republic.

License: This publication is licensed under Creative Commons license “Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0),” for further detail, please see http://creativecommons.org/licenses/by-nc-nd/4.0/

ISBN 978-80-246-3164-6 (ePub)

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CHAPTER 1

1. Pre-Analytical Effects on Laboratory Examinations

Authors: RNDr. Zdeněk Švagera, Ph.D.; Mgr. Radka Šigutová

Reviewer: RNDr. Jiří Zadina, CSc.

1.1. Introduction

The laboratory diagnostic process to obtain a result can be divided into three phases: the pre-analytical, analytical and post-analytical phases (see the diagram).

The pre-analytical phase is defined as the period from the physician’s indication of the test up to the laboratory analysis of the biological material. In other words, this phase involves an individual’s preparation for collection of the biological material, the collection itself, storage of the collected sample and its transport to the laboratory, and preparation of the sample for the assay. The importance of this phase is also supported by many publications mentioning the fact that up to 46–68 % of erroneous results are caused by failure to follow or respect the pre-analytical phase rules. That is why the primary task of the laboratory is to provide clients with all necessary instructions (on patient preparation, sample collection, biological material storage and transport, pre-analytical sample treatment) so as to minimize the risk of errors that could consequently cause harm to the patient. All this information is summarized in the manuals of testing laboratories.

The pre-analytical phase is followed by the analytical phase, involving the sample analysis itself. Each laboratory must have an established quality control system to ensure the validity of the issued results. The analytical phase ends with the post-analytical phase, defined as the period from obtaining the lab result to its hand-over to the physician.

It is necessary to keep in mind that biological samples constitute a risk of infection, and therefore personal protective equipment (rubber gloves, protective coat) should be used for work with biological material (material collection, lab work with the sample). In addition, a face mask and safety goggles must be used for highly infectious samples such as HIV or hepatitis C. If clothes or skin is contaminated by the biological material, the affected area should be washed and then disinfected. In the event of injury, the wound must be treated (let it bleed for several minutes, wash with soap, disinfect) and medical attention sought.

1.2. Pre-Analytical Phase and Its Sources of Variability

As mentioned in the introduction, the pre-analytical phase, i.e. before the analysis of the sample (specified parameter) in the laboratory, can be a source of many errors. Therefore, it is necessary to explain what factors affect the pre-analytical phase most. These factors can be divided chronologically:

1.3. Before the Biological Material Collection

Factors affecting the pre-analytical phase before the biological material collection can be further divided into controllable and uncontrollable factors. Controllable factors include, for example, adherence to some daily regimen, dietary habits, etc. Uncontrollable factors are variables such as age, gender, race, etc.

1.3.1. Controllable Factors before the Biological Material Collection

Food is an important controllable factor before the biological material collection. Blood should ALWAYS be collected after a fasting period. Where this is not the case, increased levels of some metabolites can be observed due to ingested nutrient metabolism. Glucose, triacylglycerol, free fatty acid and lipoprotein levels are elevated. People whose diet is rich in fats will primarily have an elevated serum triacylglycerol concentration on the one hand, and a decreased serum nitrogen substance concentration on the other. Protein-rich food leads to increased ammonia and urea levels. At the same time, postprandial hormones (e.g. insulin, which reduces potassium and phosphate levels) are released. Food composition may also affect the pH of urine. For example, vegetable and fruit consumption makes urine more alkaline, while meat, fat and protein-rich food makes it more acidic. Some metabolite levels may also be influenced by the consumption of certain beverages (caffeine increases the glucose level in blood). Alcohol also significantly affects biochemical assays. After alcohol ingestion, the blood lactate concentration increases almost immediately, while hydrogencarbonate and glucose levels go down. Long-term alcohol burden in the body leads to liver damage, which is manifested by increased alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) levels. Triacylglycerol and cholesterol concentrations are also elevated.

Another factor that may affect the final result is physical strain before the collection. The impact on the result will depend on the type of physical activity: either a short-term activity, with high-intensity anaerobic metabolism of the body, or a long-term (endurance) activity where the body predominantly employs aerobic metabolism. Medium physical exertion increases the glucose level and insulin secretion is stimulated. Muscular activity also increases levels of AST, lactate dehydrogenase (LD) and creatine kinase (CK) enzymes as well lactate and fatty acid levels. Long-term strenuous activity results in a decrease in blood sugar, an increase in creatinine, and multiple-fold increase in lactate levels. Cholesterol and triacylglycerol levels are also reduced.

Another controllable factor before the biological material collection is mechanical trauma; for example, muscle trauma, including intramuscular injections, causes the release of enzymes (CK, ALT, AST) and muscle tissue proteins (e.g. myoglobin). Cycling may cause mechanical trauma to the prostate, which may manifest itself by the release of prostatic serum antigen leading to a false positive result for this test. Marathon running and heart valve defects lead to the mechanical haemolysis of erythrocytes.

A very common problem, which is very difficult to control, is the effect of drugs. Drugs may affect the level of some monitored analytes; for example, acetylsalicylic acid (aspirin) increases serum AST and ALT and urine protein levels, furosemide increases serum glucose, amylase (AMS) and alkaline phosphatase (ALP), and decreases sodium cation levels. Drugs may also interfere with the analytical assay procedure. For example, since vitamin C has strong reduction properties, it causes a false decrease in the level of analytes detected using peroxide. Drugs may also affect the rate of metabolism or monitored analyte elimination, or damage certain organs – the hepatotoxicity of narcotic agents being an example.

Stress is also a major factor. Stress situations cause the release of stress hormones such as renin, aldosterone, somatotropin, catecholamines, cortisol, glucagon and prolactin. This is why blood collection for prolactin assays should be performed within three hours after waking up. Another example might be the 60% drop in cholesterol compared with the initial level within 24 hours after acute myocardial infarction. It takes many weeks before its concentration reverts to normal. For this reason, blood collection for cholesterol, HDL and LDL cholesterol assays is not recommended when patients with suspected acute myocardial infarction are being hospitalized. In contrast, slight stress may increase cholesterol concentration. Post-operative stress decreases the concentration of thyroidal hormones and transferrin, and secondarily increases the concentration of ferritin.

1.3.2. Uncontrollable Factors before the Biological Material Collection

Uncontrollable factors before biological material collection include age, gender, race and biological rhythms. A further uncontrollable factor which might be included here is pregnancy. However, since this example of influence on the pre-analytical phase is too specific, it will not be described in this communication. Except for biological rhythms and pregnancy, these effects do not require any special attention as they are beyond our control and are considered through reference limits for the relevant analyte.

Age is a very important uncontrollable factor, since most monitored analytes are age related. An older person will have higher cholesterol levels than a younger person. Children and adolescents exhibit higher total alkaline phosphatase activity than adults due to a higher production of the bone isoform of this enzyme as the body grows. The reason is that the assay includes total alkaline phosphatase activity, including the bone isoform. Attention must also be paid to the higher total ALP level in pregnant women due to the higher production of the placental isoform of this enzyme.

Gender also has a major influence on the result of the assay. It is commonly known that many parameters depend on the hormone set and physical constitution. For example, men have higher levels of creatine kinase (CK), ALT, AST, ALP, uric acid, urea, haemoglobin, ferritin, iron and cholesterol than women.

Furthermore, the non-Caucasian population is increasing in the Czech Republic. For example, the CK and AMS activity or the granulocyte count rise in ascending order from Caucasian through Asian to African-American populations (African-Americans have up to twice as much CK activity and Asians have a higher salivary amylase activity and a higher total bilirubin concentration).

Other effects that should be considered are biological rhythms with their different time periods, either occurring within a single day (circadian) or cycles taking roughly a year to complete (circannual). Circadian changes vary for different parameters; for example, there is up to 50 % change in iron levels during the day. Other parameters such as AST, ALT, LD and ALP show changes in the range of tens of percent. Maybe the most notable circadian change occurs in cortisol – about 250 % with minimal levels in the evening. An example of circannual rhythm is the change in vitamin D concentration, with maximum levels in summer months due to skin exposure to intense sunlight.

1.4. During the Biological Material Collection

Factors influencing the pre-analytical phase during the biological material collection are primarily related to the work of the sample-collecting nurse, who has to keep in mind the basic sampling principles that may affect the result of the test. In particular, such principles include collection timing, selecting the appropriate collection set, the patient position during the collection, venostasis and local metabolism effects, as well as the effect of infusion and transfusion in the hospital environment.

1.4.1. Collection Timing

Collection timing plays a very important part in the strategy to obtain valid results. Most often, collections take place in the morning when we can be sure that the patient has fasted (provided the patient respects general pre-collection recommendations) and the circadian rhythm effect mentioned in the chapter above is limited. A different example is blood sugar monitoring (blood sugar profile) or pharmacotherapy monitoring, where samples are taken based on drug elimination half-life.

1.4.2. Patient Position during the Collection

Patient position during the collection is also important. It must be kept in mind that the difference in protein concentration when comparing a standing vs. sitting position for 15 minutes is 5–8 %, and about 10–20 % for a standing vs. recumbent position. In the standing position, water transfers from the intravasal to the interstitial space, which subsequently leads to a rise in high-molecular substances, primarily proteins, lipoproteins and protein-bound substances such as calcium cation and hormones (cortisol, thyroxin), or some drugs. In general, biological material should always be collected in the same position, preferably the standard sitting position, which is not always possible in hospitalized patients, though.

1.4.3. Use of Tourniquet and Local Metabolism Effect

The effect of local metabolism when a tourniquet is used for collection is also interesting. The evidence shows that one minute after constricting the arm with a tourniquet there is already a significant transfer of water and ions from the vessel to the interstitium, with a subsequent rise in protein and blood protein-bound substance concentration. Long-term constriction or overcooling of the arm leads to a change in local metabolism due to hypoxia, which results in a rise in partial carbon dioxide pressure and potassium and lactate concentration, which in turn results in a drop in pH. In addition, there are homeostasis changes connected with the release of the tissue factor. Exercising the arm is not recommended, or it is even forbidden, as it primarily causes an increase in potassium concentration. For these reasons, the period for which the arm is constricted should not exceed one minute, and the tourniquet should be released immediately after the venipuncture.

1.4.4. Choosing the Collection System and the Effect of Anticoagulants

The choice of the collection system is also very important. Options include a closed or an open sample collection system. The open collection system consists of a classical needle and a Luer-taper syringe. Following venipuncture, freely flowing blood is taken directly into the test tube or by gently pulling the plunger. Collection into a closed system is the preferred option today as it minimizes the risk of contaminating the collecting person through the blood, and collection tubes are colour coded depending on the added preservative or anticoagulant. Another advantage of the closed system is that the ratio of anticoagulant (preservative) to collected blood is maintained.

As mentioned above, anticoagulant (heparinate, citrate, oxalate, etc.) can be chosen depending on the required test. Nevertheless, attention needs to be paid when choosing the anticoagulant for cation tests, since the anticoagulant must not contain the cation being determined. For example, the use of EDTA with potassium will lead to highly pathological potassium concentrations in the sample! EDTA is not suitable for determining bivalent cation concentration as it acts as a chelating agent, binding these cations to form a complex, and it results in finding a falsely low concentration of these ions. In some cases, another substance (preservative) such as sodium fluoride is added to the anticoagulant in order to determine glucose concentration. The addition of sodium fluoride will cause glycolysis inhibition in red blood cells, thus preventing a drop in glucose concentration over time.

In addition, we must keep in mind that if a collection set containing an anticoagulant is used, we should gently mix the collected blood immediately after the collection. Without mixing, the anticoagulant effect is limited and undesired blood clotting will occur. A suitable needle lumen should be selected for blood collection to avoid red blood cell haemolysis.

1.4.5. Effect of Infusion and Transfusion

Patients in a critical condition have to receive transfusion and infusion products containing high concentrations of selected substances and low concentrations of others. Infusion may therefore affect the determination of some substances, usually by direct contamination during collection or just due to their properties. For example, the infusion of glucose with potassium results in a false increase in glucose and potassium levels. The infusion of lipid emulsion causes serum chylosis and Hartmann infusions containing high lactate concentration (>15 mmol/l) cause a false increase in lactate concentration. On the other hand, Plasmalyte infusion causes a false normalisation of ion concentration in the collected sample. This is why certain rules should be followed during the sample collection following an infusion. Ideally, collect blood from the other arm, i.e. where the infusion was not applied, or stop the infusion for 15 minutes and then take the sample.

With respect to the pre-analytical phase, the age of transfusion must be taken into account. With the growing age of the erythrocyte concentrate, sodium and glucose concentrations decrease due to red erythrocyte metabolism, whereas, in contrast, potassium and lactate concentrations increase.

1.5. Between Biological Material Collection and Analysis

This period includes the time from the collection of biological material until its analysis in the laboratory, and involves handling the sample following the collection, its subsequent transport to the laboratory, and centrifuging or pre-treatment before the analysis.

In general, if anticoagulated blood is taken (collection container with anticoagulant), the test tube should gently be shaken immediately after the collection. If non-anticoagulated blood is taken, wait about 30 minutes before transporting the sample to allow sample clotting (exact time required for clotting is indicated by the manufacturer of the collection set). Immediate transport of the biological material after the collection may cause haemolysis and sample deterioration. The problem of haemolysis interfering with the assay is not only related to the release of erythrocyte content into the serum or plasma with a subsequent increase in the concentration of these substances in the tested material, but also to the release of haemoglobin, whose colour interferes directly with a photometric assay or with the agent used for the assay. Take care – haemolysis may also occur due to sample overcooling, high centrifuge speed or a narrow sampling needle. The following table describes the effect of haemolysis on selected biochemical assays.

Increase in concentration or activity

Potassium, magnesium cation, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatine kinase, acid phosphatase

Decrease in concentration or activity

Gamma-glutamyltransferase, alkaline phosphatase, amylase

Storage of the sample before the transport and the very transport of the biological material are very important and must be given adequate attention, especially if samples are transported from practitioners in the periphery and brought to a specialized laboratory. The transport time will vary; however, always avoid exposing the sample to extreme conditions (heat/freezing) during the transport, minimize shaking the sample and avoid complete deterioration which will occur if the sample is spilled. This is why samples have to be transported in temperature-controlled transport boxes protected against spillage. Some samples (tissues) must be transported frozen even at very low temperatures (−80°C) on dry ice. If the maximum time before sample processing is exceeded or transport conditions are not adhered to, some substance concentrations in the material for testing will change. One example is a decrease in glucose concentration or an increase in lactate concentration due to the anaerobic glycolysis of blood elements. Some analytes in biological material are thermolabile at room temperature (most parameters) and some, paradoxically, at 4°C (e.g. ALT activity decreases or potassium concentration increases due to the ATPase inhibition in the erythrocyte). Some analytes are photo-sensitive (e.g. bilirubin and porphyrins), and their amount drops unless transported and stored in the dark. For these reasons, some analytes have specific recommendations for storage and transport. For example, the recommendations for a plasma ammonia assay are as follows: carry out the anaerobic collection, prevent haemolysis, maintain the anticoagulant to blood ratio and transport in a transport container or on melting ice; analyse within 20 minutes after the collection.

As soon as the samples are delivered to and received by the laboratory, they are either analysed directly (when whole blood is used), or must be centrifuged to obtain serum or plasma. The required conditions must be adhered to during centrifuging to achieve perfect serum (plasma) separation from erythrocytes and perfect leukocyte sedimentation in the plasma. If the speed (relative centrifugal force) is too high during centrifuging, the cells may break and their content may get released. Many analytes require centrifuging at lower ambient temperatures (cooled centrifuges), for hormone assays, for example.

Urine analysis requires a chapter to itself, since it requires the use of collected, first morning or single random specimens. Very often, patients are not instructed about the collection rules; they typically collect urine for a longer or a shorter time than required; moreover, obtaining an exact reading of the quantity of urine collected over the collection period, usually 24 hours, is always problematic. Nor it is possible to ensure the required storage of the collected urine in the fridge or the urine pre-treatment needed to stabilize the tested parameter. First morning urine collection poses a similar problem, since it has to be delivered for sediment analysis within one hour of collection. There is often a delay in delivering the collected urine to the laboratory, which leads to false negative or false positive results (increase in the bacteria count, increase in the pH value due to the urease of bacteria and cell element degradation).

In general, the transport and storage conditions required for transported samples/material must be followed. Material transport in extreme (very hot, very cold) conditions requires special care.

Ten pieces of advice for obtaining correct results

- Instruct the patient (why they are being tested, diet, physical strain)

- Time the collection correctly

- Fill in the order slip correctly

- Choose the right collection procedure

- Choose the right test tube

- Take the recommended amount of material

- Do not spill any biological material

- Label the test tubes correctly

- Ensure appropriate storage for biological material before transport

- Ensure appropriate transport to the lab

CHAPTER 2

2. Reference Values and Methods for Their Determination

Authors: Ing. Vladimír Bartoš, Ph.D.; RNDr. Pavlína Kušnierová, Ph.D.; Ing. František Všianský

Reviewer: prof. MUDr. Richard Průša, CSc.

2.1. Introduction

Laboratory test results are indisputably a very important source of information when choosing the right treatment for the patient. Among other things, they help determine or specify the diagnosis, select, optimize and monitor the therapeutic procedure, or determine the prognosis of the patient’s condition.

When the results are interpreted, in most cases, the extent to which a result is consistent with values that might be reasonably expected in the selected reference population is considered. In other words, a specific result is compared with the limits defining the interval of result values obtained in the same laboratory test of a sample of the reference population, i.e. with the reference interval.

2.2. Basic Terms and Definitions

Reference interval (reference range): A generally accepted definition of this term is: the determination of limits between which 95 % of reference values fall (an interval encompassing up to 99 % is used for certain parameters).

This is usually the interval between the lower and the upper reference limits, which are typically the 2.5th and 97.5th percentile of a set of values obtained through the analysis of a sufficiently homogeneous and large sample of the defined reference population.

Sometimes, from a clinical point of view, only the upper or the lower reference limit may be important, which corresponds to the 95th percentile, or the 5th percentile, respectively.

Examples of some specific variables and their assignment to the reference interval type are shown below (Table 1).

Reference values: These are the values (results of measuring the relevant variable) obtained from a selected group of individuals with a defined health condition.

Reference value distribution: Distribution of individual measurement results corresponding to some of the statistical distributions. The relevant type of distribution (normal, also called Gaussian, log-normal, Laplace distribution, etc.) is tested using suitable statistical methods.

Reference population: The set of all individuals meeting certain criteria concerning their health status or other defined requirements (age, gender, race). Absence of a certain disease is usually required; it is clear, however, that this defined notion of “health” is very relative and in its way imperfect.

Reference individual: An individual selected from the set of reference population.

Reference population selection (reference sampling group): A randomly selected part of the reference population from whom reference sampling values are obtained by measurement in order to estimate the reference limits. It is unrealistic to make measurements on the entire reference population, and therefore only a randomly selected sample is measured. Sampling characteristics obtained by measurement of this sample are thereby a more or less plausible estimate of actual values.

Likelihood estimation: Agreement between the estimate and the actual value typical of the entire population. This depends on many factors, the primary one being the size of the reference population sample. At least 120 reference individuals are typically required as a minimum; the confidence interval of estimates contracts considerably with the size of the reference sampling group. The likelihood estimation is further affected by pre-analytical aspects (method of collection, transport and storage of samples), the measurement method and the statistical evaluation method used.

Reference Interval Type

Measured Variable (Parameter)

LRL – URL

Na+, K+, Ca2+, glucose, transferrin, thyroid hormones

below URL

bilirubin, AST, ALT, CK, troponin I, tumour markers

above LRL

cholinesterase, prealbumin

Table 2.1. Examples of reference interval types relative to the clinical importance of reference limits (LRL – Lower Reference Limit, URL – Upper Reference Limit)

Figure 2.1. Relationships between terms in connection with determining the reference interval

2.3. Options for Determining Reference Intervals

One of two methods is usually chosen to determine reference intervals from actual measurement results.

The first method, referred to as the direct (inductive) method of estimating reference limits, is applied when the possibility exists of obtaining a sufficiently large reference sampling population on which the studied variable is measured, providing the relevant pre-analytical conditions are adhered to. Selecting the requirements for reference individuals is quite a complicated task which may substantially affect the resulting reference limit values. In the NORIP study (Malmø, 27/4-2004), some of these requirements were proposed together with the pre-analytical conditions that should be followed when determining reference intervals. The conditions that reference individuals should meet include:

- Be feeling subjectively well;

- Have reached the age of 18;

- Not be pregnant or breast-feeding;

- Not have been hospitalized or seriously ill during the last few months;

- Not have had more than 24 g of alcohol in the last 24 hours;

- Not have given blood as a donor in the last 5 months;

- Not have taken prescribed drugs during the last 2 weeks;

- Not have smoked during the last few hours prior to sampling.

The second method, also referred to as the indirect (deductive) method, is applied if no suitable reference sampling population is available, and only the results of the variable obtained by measurement of a “mixed” population comprising healthy and ill individuals can be used.

2.3.1. Direct Method of Reference Interval Estimation

This method is based on the statistical processing of measured reference values. Data obtained by measurement can be evaluated either parametrically or non-parametrically, depending on whether parameters characterizing the reference value distribution are used to establish the reference limits, which are then used to derive the numerical values of corresponding quantiles determining the reference limits. Although the non-parametric procedure is more universal, in this case the quantile likelihood estimates are lower.

2.3.1.1. Parametric Procedure

Use of the parametric procedure is only legitimate in selections coming from a normal (Gaussian) distribution, or from distributions that can be transformed into a normal distribution. Only in these cases is it possible to use the parameters of this distribution, i.e. the sample mean and sample standard deviation, as the best estimates of position and variance characteristics of this distribution, and to derive from them the relevant percentile values.

An important step in selecting the reference limit method is to confirm the assumption of normality for the distribution of obtained reference values, with the primary requirement being the symmetry of distribution. Various statistical tests such as Kolgomorov-Smirnov, Anderson-Darling or D’Agostino-Pearson tests are used to evaluate this normality. However, it should be kept in mind that different statistical tests can have different predicative abilities, and so are more likely to inform us that there is something wrong with the anticipated normality of the distribution. These tests are often used in combination with graphical methods; this has the advantage of enabling a comparison between the distribution of measured reference values (usually shown in a histogram) and a normal distribution with the same parameters using a frequency function diagram (probability density). Common statistical programs also enable the plotting of diagrams called rankit-plots, which are used to compare sampling distribution quantiles with normal distribution quantiles. If the sampling distribution matches the normal distribution, the dependence is linear.

Figure 2.2. The histogram of distribution of the reference values obtained (x-axis: measurand value, y-axis: frequency of occurrence) and its approximation using the frequency function with the same parameters, and the rankit plot comparing empirical distribution quantiles (y-axis) and normal distribution quantiles (x-axis)

So that the parameter estimate is unbiased, a test must be conducted to ensure that the reference values obtained by measurement do not contain outlying results or gross errors. Some common tests for outliers (Grubb’s test, Dean-Dixon test, etc.) or some graphical techniques can be used for this purpose. If the data contain outliers or gross errors, the next step must be their exclusion.

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Table of Contents

Copyright

1. Pre-Analytical Effects on Laboratory Examinations

1.1. Introduction

1.2. Pre-Analytical Phase and Its Sources of Variability

1.3. Before the Biological Material Collection

1.4. During the Biological Material Collection

1.5. Between Biological Material Collection and Analysis

2. Reference Values and Methods for Their Determination

2.1. Introduction

2.2. Basic Terms and Definitions

2.3. Options for Determining Reference Intervals

3. Analytical Properties of the Laboratory Method, Quality Control

4. Diagnostic Sensitivity, Specificity of the Method, Methods of Determination, Interrelations, Laboratory Screening

5. Basic Urine Tests

6. Kidney Function Tests

7. The Importance of Plasma Protein Assays

8. The Importance of Na, K, Cl Assays in Clinical Practice

9. Metabolism of Calcium, Phosphorus and Magnesium

10. Trace Elements

11. Vitamins

12. Thyroid Gland

13. Hormones of hypothalamus and hypophysis

14. Adrenal Cortex Hormones

15. Disorders of Acid-Base Balance

16. Importance of Oxygen Assays

17. Importance of Osmolality Tests, Water Metabolism

18. Serum Lipids and Lipoproteins, Relation to Atherogenesis

19. Risk Factors for Atherosclerosis (Except Lipids)

20. Free Radicals, Relation to Diseases and Protection against Them

21. Biochemical Tests for Liver Diseases

22. Laboratory Diagnosis of Jaundice

23. Bone Metabolism

24. Laboratory Diagnostics in Gastroenterology

25. Diabetes Mellitus

26. Cardiac Markers

27. Laboratory Signs of Malignant Tumours

28. Cytochemical Examination of Cerebrospinal Fluid

29. Inherited Metabolic Diseases – Laboratory Diagnostics

30. Laboratory Test for Urolithiasis

31. Laboratory Examinations during Pregnancy

32. Specificities of Laboratory Examination during Childhood

33. Basics of Toxicology in Clinical Laboratory

34. Laboratory investigation of Ovarian and Testicular Disorders

35. Therapeutic Drug Monitoring

36. Trends in Laboratory Medicine (POCT, Automation, Consolidation, EBM, Omics)

37. Anticoagulant Therapy Monitoring

38. Clinical Nutrition and Metabolic Balance