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Introduction
Clinical laboratory diagnosis (CLD) – independent area of medical science is based on such fundamental disciplines, as medical biochemistry, pathological physiology, immunology, pathological anatomy, etc. Past years, this direction of medicine promptly developed in connection with new opening in biology, increase in volume of information, development of new methods of research, differentiation and specialization of medical service. Doctors of a different structure should have knowledge with CLD, be guided in modern methods of laboratory diagnosis, be able to choose correctly and meaningly methods of all-round inspection of patients, to interpret results of analysis, for necessities to pick up additional reserches for purpose (assignment) of adequate and effective therapy, monitoring of treatment.
Priority direction of medicine of XXI century becomes preventive medicine - prepared doctors –preventive maintenance, the family or domestic doctor. In this case the main object of medical attention becomes not only the patient, and the healthy person, the basic problem (task) of the doctor – an establishment, mainly, on the basis of the physical and chemical laboratory data of an individual to the certain pathology, diagnostics of hereditary defects of metabolisms risk factors, etc.
The purpose of CLD is mastering the unified and new methods of research of biological materials, consideration of pressing questions of clinical biochemistry, ordering and deepening of theoretical knowledge for competent interpretation of results of biochemical analysis, optimization of laboratory inspection, acquaintance with modern constellations and differential-diagnostic biochemical programs which are used for diagnostics and treatment of patients, improvement of practical habits and skills.
Rate of CLD (clinical biochemistry) will consist of 15 practical classes, each of which stipulates laboratory research of biological materials, reception and interpretation of results; the three-sedate control of knowledge over the base test – control, consideration of theoretical questions, decisions of situational problems (task) “with the open textbook” with further discussion, an estimation, the analysis of results, substantiation and treatment of a clinical situation; drawing up block diagrams, comparative tables of changes of laboratory parameters at different pathological conditions, selection of the most informativeсonstellation biochemical tests for an estimation of infringements of biochemical processes and physiological functions of an organism.
Within the limit rate of CLD, there is also an independent work of students which is estimated by results of obligatory home work (OHW). At the end CLD rate differential credits are
In the given methodical recommendations are the stated basic stages of preparation of students to practical classes:
1 Questions for study theme / the Questions for preparation
2 Test questions to estimate students mastering of the material by the teacher
3 The description of laboratory works, with basic practical which receptions of performance the student gets acquainted with in class
4 Clinico-diagnostic value of separate parameters, pharmacolo-gical and chemical interference
5 Situational tasks with clinical history
6 List of the basic and additional literature which is recommended for studying a theme
At the end of methodical recommendations questions to independent work on CLD, normal values of biochemical parameters of blood and urine – the most effective combinations of tests in diagnosis of diseases (on D.L. Komarov and L.P. Aksyonenko) move.
List of practical topics
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15 / Biochemical tests in clinical medicine. Monitoring of medical drugs
Normal and pathological indexes of proteins and nonprotein nitrogen metabolism
Clinical enzymology. Enzymodiagnostics
Clinical and laboratory diagnosis of breaches in carbohydrate metabolism
Laboratory investigations and biochemical diagnostics of breaches in lipids and lipoproteins metabolism
Diagnostic criteria of disturbance of water, sodium, potassium metabolism. Determination of acid-base balance
Clinical-laboratory diagnostics of disorders of hemoproteins metabolism, porphyrines and iron. Clinical-laboratory diagnosis of nucleotides metabolismdisorders
Clinical-laboratory diagnosticsof hypothalamus, hypophysis, adrenal and sexual glands disorders
Clinical-laboratory diagnostics of functional activity of thyroid and parathyroid glands. Biochemical investigations of disorders of calcium, phosphates and magnesium metabolism
A clinical estimation of biochemical indexes is at liver and biliary ways diseases
A clinical estimation of biochemical indexes in kidneys diseases
Disorders of gastrointestinal tract functions, their diagnostics and diet therapy
Laboratory diagnostics of the inherited metabolic diseases. Features of clinic-laboratory diagnostics of diseases of extreme age. Biochemical aspects of pregnancy and early child's age
Biochemical indexes under oncologic diseases. Clinical-laboratory diagnosis of the cardio-vascular system, connective tissue, breathing organs diseases
Conclusion session / 2
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Lesson 1
Topic. Biochemical tests in clinical medicine.
Monitoring of medical drugs.
Questions for preparation
1Application of biochemical analyses in diagnostics, screening, prognosis and monitoring of the diseases.
2Types of biological material. General claims for selecting samples.
3General claims for analysis of samples and arrangement of the results.
4Interpretation results of samples.
5Specificity, sensitivity and prognostic value of the biochemical analyses.
6Errors of laboratory diagnostics.
7New trends in development of clinicodiagnostic laboratories.
8Prospects of development of biochemical methods of noninvasive diagnostics.
9Monitoring of the medical drugs. Screening for presence of pharmacological drugs in the organism.
Test questions
1Mention the main functional assignments of the biochemical tests.
2Name the rules of listing up the inquiry for analysis.
3Name the factors which influence the results of biochemical study. Give some examples.
4Enumerate the main rules of taking samples to perform biochemical analysis of blood.
5Name the main characteristic of the “ideal” analytic method.
6Explain the notion “analytical variability” in the biochemical analysis.
7Explain the notion “biological variability” in the biochemical analysis.
8Explain the notion “specificity of diagnostic test”.
9Explain the notion “sensitivity of diagnostic test”.
10 Explain how the effectiveness of analysis can be estimated.
11 Name the main claims of the screening tests.
12 Mention the cases in which estimation of medical drugs in liquid media of the organism is essential.
13 Name the claims for the methods of estimation of concentration of medical drugs in plasma.
14 What concentration of pharmacological drugs in plasma is defined as “therapeutic” value?
15 Justify the importance of performing the monitoring of phenytoin.
16 Name the anticonvulsant drugs, concentration of which must be estimated in plasma. Justify your answer.
17 Name the pathological and physiological factors which influence on sensitivity to digioxin.
18 Justify the importance of performing monitoring of digioxin.
19 Justify the importance of performing monitoring of lithium.
20 Justify the importance of performing monitoring of theophylline.
21 Justify the importance of performing monitoring of cyclosporine.
22 Justify the importance of performing monitoring of aminoglycoside antibiotics.
23 Justify the importance of performing monitoring of methotrexate.
24 Describe the opportunities of noninvasive diagnostics during monitoring of medical drugs.
25 Explain the biochemical mechanisms of toxicity of paracetamol when it’s overdosed.
26 Explain the biochemical mechanisms of influence of the salicylates on metabolic processes when they are overdosed.
27 Explain the biochemical mechanisms of influence of the salicylates on the processes of oxidative phosphorylation when they are overdosed.
28 Explain in what cases monitoring of alcohol is expedient.
29 Explain in what cases screening for medical drugs in blood is necessary.
30 Describe the general principles of performing electrophoresis. Name the drawbacks of performing paper electrophoresis and benefits of gel electrophoresis.
31 Give the examples for application of method of electrophoresis in diagnostics.
32 Describe the general principles of performing adsorption chromatography.
33 Describe the general principles of performing partition chromatography.
34 Describe the opportunities of application of chromatographic methods in clinical laboratory examinations.
35Mention the principle of radioimmunoassay. Give the examples of its application in clinical laboratory examinations.
36Mention the principle method of atomic adsorption analysis, give the examples of its application for studying the mineral composition of blood.
Literature
I - p. 1-12, 301-304
Lesson 2
Topic. Normal and pathological indexes of proteins and nonprotein nitrogen metabolism
Questions for preparation
1Biological role of plasma proteins in an organism.
2Characteristics of methods of proteins research (electrophoresis, immunoelectrophoresis, chromatography).
3Technique and conditions for performing electrophoresis of blood serum proteins. Graphical presentation of the results obtained.
4Clinical significance of the determination of the total blood serum proteins. Main causes of hyper- and hypoproteinaemia.
5Characteristics of the main blood fractions of proteins.
6Explain what are disproteinaemias, paraproteinaemias, M-gra-dient,and Bence Jones proteins.
7Electrophoregrams of blood serum proteins in normal and in pathological state.
8Characteristics of the most investigated separated proteins of blood plasma and their applications for diagnostics.
9Explain what is proteinuria. Uroproteinograms.
10Proteins of other liquid mediums in an organism.
11Nonprotein nitrogen of blood. Classification of azotemias.
12Clinical significance for the definition of the rest nitrogen components in blood (urine, free amino acids, creatine, creatinine, indican, ammonia, uric acid).
Perform and interpret the results of these experiments:
1. Determination of the total proteins in blood serum by the biuretic method.
Method principle: the method is based on formation of a violet-coloured complex of peptide bonds of protein with copper sulphate in an alkaline medium. The intensity of the colour is directly proportional to the protein concentration in blood serum and is estimated by the photometric method.
Process of work: 1 ml of blood serum is placed into the first tube (test sample), and into the second one – 0,1 ml of standard protein solution (standard sample), into the third one – 0,1 ml of the 0.9% solution of sodium chloride (control sample). In each of these tubes, 5 ml of a biuretic reagent is added, then the contents of the tubes is carefully mixed and after 30 minutes extinction is estimated with the help of photoelectrocalorimeter in the cuvettes in 10 ml through a green colour filter (540 nm) against the control solution.
Calculations are made according to the formula:
,
where Cex is concentration of protein in the examined blood serum, g/l;
Eex is extinction of the examined blood serum;
Cst is standard concentration of protein in the standard solution, g/l;
Est is extinction of standard protein solution.
Clinical-diagnostic significance. Concentration of proteins in the blood serum of adults are 65-85 g/l. Increase of the total protein concentration in blood serum (hyperproteinemia) can be relative (water loss in case of burns, diarrhea) or absolute (myelomatosis, infectious diseases and rheumatism). Hypoproteinemia is decrease of the protein concentration in blood serum, it occurs mainly due to decrease of albumins and together with nephrotic syndrome, liver pathologies, increased permeability of vascular walls, protein starvation.
Pharmacological interference. The results of investigation are overestimated by: amino acids (when introduced intravenously), anabolic steroids, androgens, ACTH (adrenocorticotropic hormone), acetylsalicylic acid, butamide, imipramine, insulin, corticotropin, corticosteroids, progesterone, roentgenopaque drugs, chloramphenicol, streptomycin sulphate, sulfanilamides, tetracycline, phenothiazines, miscleron, bromsulfalein.
The results of research are underestimated by: trasinamide, ammonium ions, purgatives.
2. Distribution of the proteins fractions using the method of electrophoresis in a polyacrylamide gel
Normal index: albumins – 35-50 g/l (52-65%);
globulins – 23-35 g/l (35-48%)
Clinical-diagnostic significance. With the help of electrophoresis proteins of blood serum are separated into fractions which give characteristic for the state of an organism and are used for diagnostics. Decrease in number of albumins points to liver diseases or their loss with urine in renal pathologies. Increase in the number of γ-globulins is observed in infectious processes and decrease of this fraction can indicate AIDS. Significant decrease of albumins and increase of all globulin fractions occur with malignant tumors.
Pharmacological interference. Increase of α1-globulins is detected when peroral contraceptives are ingested. Decrease of γ-globulins content is observed with corticoid or ACTH treatment.
3. Sedimentation tests: thymol, sulemic, Weltman’s tests.
3.1. Thymol test
Method principle: pathologically increased β-globulins, γ-globu-lins and lipoprotein are precipitated from blood serum inpH=7.55 buffer solution that saturated by thymol. The intensity of opacification is measured which depends on the content of proteins fractions and their correlation.
Process of work: 4,8 ml of thymol reagent are put into a tube, then 0,08 ml of blood serum is added and then mixed. They are left for 30 minutes at room temperature. Then the solution is processed by photocalorimeter against a thymol reagent at 630-690 nm. The intensity of opacification is determined according to the gauging graph.
Clinical-diagnostic significance. Normal index – 0-4 units S-H (after Shank and Hoagland). Considerable increase of the index is observed when the liver parenchyma is affected, especially in cases of acute hepatitis. The level of this increase correlates to the extent of the organs affection. The test is positive in 90-100% cases in Botkin’s disease (on the preicteric stage and anicteric form).
It is used for the differential diagnosis between obstructive jaundice and hepatocellular jaundice. In case of obstructive jaundice the index is within the bounds of norm and it becomes positive if the process is complicated with hepatocellular jaundice. In the case of obstructive jaundice, the thymol test is negative, Burstein’s test (for β- and pre-β-lipoproteins) is sharply positive, in case of hepatocellular jaundice both tests are positive. The thymol test is not specific for liver diseases and is of importance for all diseases which are accompanied by disproteinemia.
The index is increased in case of chronic hepatitis, liver cirrhosis, a less considerable increase is observed in cases of collagenoses, malaria, viral infections.
Pharmacological interference on the received results. The results of investigation are overestimated by: anabolic steroids, androgens, phenylbutazone, heparin, phenytoin, indometacin, insulin, MAO inhibitor, clofibrate, corticotrophin, corticosteroids, lincomycin hydrochloride, chloramphenicol, mercaptopurine, methyluracyl, methyldopa, procainamide hydrochloride, oleandomycin phosphate, progesterone, sulfonamides, tetracycline, phenothiazines, chlorpro-pamide, erythromycin, oestrogens.
3.2. Sulemic test
Clinicodiagnostic significance. Normal indices: 1,6-2,2 ml HgCl2. In cases of Botkin’s disease – 1,0-1,65. With recovery it goes back to norm. Sulemic test is positive in cases of liver cirrhosis, silicosis.
3.3. Weltman’s test
Method principle: adding of CaCl2 to the blood serum and their heating results in the disturbance of the colloid constance of proteins in blood serum.
Process of work: 0,1 ml of blood serum and 4,9 ml H2O are put into a tube; thoroughly shaken off and 0,1 ml 0,5 % CaCl2 are added. It’s all heated till the first boiling of the mixture. Then it’s cooled, if there is no opacification, 0,1 ml CaCl2 is added and boiled again. The process of adding CaCl2 and boiling is repeated until white flakes appear. The result is expressed through amount of added CaCl2 (ml).
Clinical-diagnostic significance
Normal indices are 0,4-0,5 ml CaCl2. The increased indices are observed in cases of rheumatism, pulmonary tuberculosis.
The decreas is observed in cases of malaria, parenchymatous liver damage.
4. Determination of the rest nitrogen (residual nitrogen) in blood
Clinical-diagnostic significance. The increase of the rest nitrogen of blood – azotemia – can be of two types: absolute (accumulation of rest nitrogen components in blood) or relative (dehydration of the organism, for example in case of emesis, diarrhea). Absolute azotemia is subdivided into retentive and productive. Retentive azotemia occurs as a result of insufficient excretion of nitrous compounds with urine in case of their normal falling into blood. Retentive azotemia can be renal and nonrenal.
Productive azotemia is conditioned by rebundant falling of decay products of tissue proteins into blood. The function of kidneys is not affected at the same time. Azotemia of such type is observed in cases of cachexia, leucosis, malignant tumors, treatment with glucocorticoids.
The decrease of rest nitrogen is observed in cases of nutrition deficiency and sometimes in case of pregnancy.
Clinical interference:increase of rest nitrogen is caused by nephrotoxical medical drugs (heavy metals, analgesics, antibacterial agents, contrast agents, organic solvents, etc.)
Chemical interference: ethylene diamine tetraacetate (EDTA),which leads to the overestimating the rest nitrogen index.
Norm. The content of rest nitrogen in blood is 14,2-28,5 mmol/l, or 20-40 mg/l, or 0,2-04 g/l.
5 Definition of the C-reactive protein
Clinical-diagnosticvalue. Increased level of C-reactive protein is observed in cases of intrauterine infections (its level in blood from umbilical cord can reach 25600 mg/100 ml); in cases of acute inflammatory diseases (rheumatism, rheumatoid arthritis), tumors, acute myocardial infarction (≈15000 μg/100 ml), some viral infecti-ons and immunodeficiency states (insignificant increase of its level).
Test questions
1What proteins detected in the composition of the α1-, α2-, β- and γ-globulins fractions can be diagnostically useful?
2What does the notion “disproteinaemia” mean?
3What sequences does hypogammaglobulinaemias have?
4Name the causes of secondary hypogammaglobulinaemia.
5In case of what diseases are there increased contents of gammaglobulins observed?
6What does the notion “paraproteins” mean? How are they classified?
7How can the M-gradient located in the area of β-globulins be detected?
8How can the Bence Jones protein be detected?
9What is “selective” and “nonselective” proteinuria?
10How can a multiple myeloma be detected using laboratory methods?
11What are the main causes of hypoproteinaemia?
12What are the main causes of hyperproteinaemia?
13Depict the electrophoregram of blood serum in case of nephrotic syndrome. Explain it.
14Depict the electrophoregram of blood serum in case of an acute inflammatory process. Explain it.
15Depict the electrophoregram of blood serum in case of a chronic inflammatory process. Explain it.
16Depict the electrophoregram of blood serum in case of liver cirrhosis. Explain it.
17Depict the electrophoregram of blood serum in case of a malignant tumor. Explain it.
18Depict the electrophoregram of blood serum in case of a myeloma. Explain it.
19Depict the electrophoregram of blood serum in case of an myocardial infarction. Explain it.
20Depict the electrophoregram of blood serum in the case of a coronary heart disease (CHD). Explain it.
21Name the main causes and consequences of hypoalbuminaemia.
22Concentration of albumins in blood serum is used as a test of functional state of liver. In what case is it expedient to use such index – for diagnosing acute or chronic affections of liver? Justify your answer.
23Explain the biological role of “proteins of acute phase”, give examples of their application in diagnostics.
24Explain in what cases are the increase and decrease in number of 1-antitrypsin are used as a diagnostic test.
25Give the examples of use of-fetoprotein in dagnostics.
26Explain the biological role of 2-macroglobulin, its use in diagnostics.
27Explain the biological role of haptoglobin, its use in diagnostics.
28State the diagnostic importance of detection of C-reactive protein in blood serum.
29Name the causes of hypo--globulinaemia. Explain why the method of electrophoresis cannot be used for diagnosing hypo--globulinaemia?