Chapter 1 The Clinical Biochemistry Laboratory the use and the requirements of laboratory Objective of the session 1, To make the students aware of the basic setup of laboratory, the procedure for sample collection, separation to the analysis. Introduction : Clinical Biochemistry tests comprise over one third of all hospital laboratory investigation. clinical biochemistry is that branch of laboratory medicine in which chemical and biochemical methods are applied to the study of disease while in theory this embraces all non-morphological studies, in practice it is usually, though not exclusively, confined to studies on blood and urine because of the relative ease in obtaining such specimens although analysis are made on other body fluids such as gastric aspirate and cerebrospinal fluid, The use of Biochemical tests :- Biochemical investigations are involved, to varying degrees, in every branch of clinical medicine. © The results of biochemical tests may be of use in diagnosis and in the monitoring of treatment. * Biochemical tests may also be of value in screening for disease or in assessing the prognosis once a diagnosis has been made (fig. 1)¢ The biochemistry laboratory is often involved in research into the biochemical basis of disease and in clinical trials of new drugs. Treatment Diagnosis Biochemistry Report Sugar —110mg/dl Urea — 62mg/dl Creatinine-1.8my/dl Screening Progn Fig. | How Biochemical Tests are used The use of the laboratory :- Every biochemistry analysis should attempts to answer a question which the clinician has posed about the patient obtaining the correct answer can often seem to be fraught with difficulty. Basic steps for drawing a blood specimen 1 Preparation for blood collection. (A) The information given on the blood request form should be recorded on the specimen labels essential items include the following. a Patients complete name and age. b. Identification number.(B) The specimen containers should be labeled appropriated before the specimen collection. Ascertaining whether the patient to fast such care is needed to ensure accurate results. The technician must gain the patients confidence and assure him that, although the venipuncture will be slightly painful, it will be short duration. Positioning the patient (a) The patient should be made to sit comfortably in a chair and should position his arm straight from the shoulder and it should not bent at the elbow. (b) If the patient wants to lie down, let the patient to lie comfortably on the back, the patient should extent the arm straight from the shoulder. Requirement of Blood Collection i ew Blood collection : Collection tubes. Sterilized syringes and needles. Sprit or 70% ethanol. Cotton. Compare the requisition form and labeling the tubes. Selecting Vein site. Applying the tourniquet. Cleaning the area. Inspecting the needles and syringes. Performing the venipuncture. Separation of serum :- 1. 2. 3, Allow the blood to clot. Loosen the clot slowly and centrifuge the supernatant fluid, By using a pipette, separate the serum from blood cells and store it in aclean & day test tube. Sampling errors There are a number of potential errors which may contribute to the success or failure of the laboratory to provide the correct answers to the clinician's question. Some of these problems arise when a clinician first obtains specimens from the patient. Blood sampling technique. Difficulty in obtaining a blood specimen may lead to haemolysis with consequent release of potassium and other red cells constituents. results for these will be falsely elevated. Prolonged stasis during venepuncture. Plasma water diffuses into the interstitial space and the serum or plasma sample obtained will be concentrated. Proteins and protein-bound components of plasma such as calcium or thyroxine will be falsely elevated. Insufficient specimen. Fach biochemical analysis requires a certain volume of specimen to enable the test to be carried out it may prove to the impossible for the laboratory to measure everything requested on a small volume specimen.© Errors in timing. The biggest source of error in the measurement of any analyte in a 24-hour urine specimen is in the collection of an accurately timed volume of urine. © Incorrect specimen container, For may analyses the blood must be collected into a container with anticoagulant and preservative. For example, samples for glucose should be collected into a special container containing fluoride which inhibites glycolysis; otherwise the time taken to deliver the sample to the laboratory can affect the result. Ifa sample is collected into the wrong container, it should never be decanted into another type of tube. for example, blood which has been exposed even briefly to EDTA (an anticoagulant used in sample containers for lipids) will have a markedly reduced caleium concentration, approaching zero. © Inappropriate sampling site. Blood samples should not be taken ‘down-stream! from an intravenous drip. It is not unheard of for the laboratory to receive a blood glucose request on a specimen taken from an intravenous drip. It is not unheard of for the laboratory to receive a blood glucose request on a specimen taken from the same arm into which 5% glucose is being infused. Usually the results are biochemically incredible but it is just possible that they may be acted upon, with disastrous consequences for the patient. * Incorrect specimen storage. A blood sample stored ovemight before being sent to the laboratory will show falsely.Clinical Question Bochemical Anwar roe Interpretation Patient sampled nterpretat Collation ‘Transit to ab C J ‘Reception and Quality control ee” Fig. 2 Circuit diagram of clinical biochemistry process. Analysing the specimen Once the form and specimen arrive at the laboratory reception, they are matched with a unique identifying number or bar code, The average lab receives many thousands of requests and samples each day and it is important that all are clearly identified and never mixed up. Samples proceed through the laboratory as shown in figure 2. All analytical procedures are quality controlled and the laboratory strives for reliability. Once the results are available they are collated and a report is issued. Cumulative reports allow the clinician to see at a glance how the most recent result (s) compare with those tests performed previously, providing an aid to the monitoring of treatment.Methodology of its teaching :- lecture with few C.D. demonstration, Evaluation of the session :- Asking queries regarding the lecture.Chapter 2 The Interpretation Of Results Objective of the session :- 1. To teach the interpretation of results. The laboratory report It can take considerable effort, and expense, to produce what may seen to be just numbers on pieces of paper, understanding what these numbers mean is of crucial importance if the correct diagnosis is to be made, or if the patients treatment is to be changed. Usually results of tests carried out in clinical chemistry are reported in units of concentration or of activity. CONCENTRATION Units of concentration contain both units of quantity and units of volume. ‘The amount of substance present can be expressed in grams, equivalents, or moles or divisions of these ie. milligrams, milliequivalents millimoles etc similarly the volume can be expressed in liters milliliters or deciliters (100 mb). For many substances the usual units of concentration has been in mg per 100 ml. This form had replaced the earlier less defined form of mg% which should no longer be used as the use of % to mean per 100 ml here differs from the general use of (°%) to more recently deciliter has been proposed as a term for 100 ml. Just milliliter is one thousand of a liter so a 100 ml. is one tenth of a liter (a deci-litre) so units have changed from mg % to mg/100 mal to mg (dl) without any numerical change.Serum electrolytes are usually expressed in mill equivalent per liter and protein and albumin in grams per 100 ml. "S. 1." UNITS For many purpose e.g., calculation of osmolarity or electrolyte balance it would be convenient to have all results expressed in the same units, this would also help avoid confusion when results are compared from one laboratory to another or in the evaluation of results by medical staff are used to different units, for these reasons standard international units for reporting results have been recommended. As with many changes there has been resistance to the change over to the new units especially among the medical profession, But as the newer text books and reviews use these units. they are gaining international acceptance. The S.1. (Standard International) units apply to clinical chemistry as follows. 1. Where the molecular weight of the substance being measured in known, the units of quantity should be the mole_submultiple of a mole. ¢.g., milimoles and Micromoles. 2. The units of volume should be the liter. Units of concentration. will there fore be millimoles per liter ete. e.g, sodium of .140 m eg/l in S.1. units is 140 m mol/l. glucose of 180 mg/100 ml in S.L. units is 10 m mol/L 3, When the molecular weight is not known, the for example for serum protein or albumin determinations the concentration should be expressed in grams per liter i.e. 7.0 g/100 ml becomes 70 g/l.Units of activity Usually activities are measured in clinical chemistry as an index of the concentration of certain enzymes, or enzymatic processes ¢.g, prothrombin activity. Activity is a measure of the rate at which a process takes place. Enzymatic activity is usually estimated by measuring the rate at which a substrate is converted to a product. This activity is affected by many things e.g. temperature, time over which the activity is measured, incubation conditions etc. All of these must be defined when reporting the activity and so units or activity include fixed values for each variant. As the combination of the variations as large so many different such units are in use. Intemational standardization of all these variables is almost impossible and as such the international unit of enzyme activity has not been widely accepted the units for each such test in the laboratory should be clear to all concemed. Reporting results There is a certain error in all results, Usually laboratory will claim 95 % confidence in its result i.c. plus or minus two standard deviation, Thus a blood sugar report of 100 mg/100 ml with a standard deviation of 1 mg per 100 ml would really be 100 + 2 mg/100 ml. For simplicity the variation is not usually reported with the individual result. ‘The standard deviation for the method should be indicated and the result given with the understanding that the variation is understood. 10Significant figures From mathematical calculations results may be obtained to many decimal places but these will not usually be significant. The final result cannot be accurate to a greater degree than the sum of the errors through the test allow e.g. error in pipetting sample. or standard error in reading spectrophotometer etc. When reporting a result then each figure given should have a meaning. A blood sugar is not a significant part of the result would not usually be reported as 100.2 mg/100 ml. As 0.2 mg is not a significant part of the result for most blood sugar methods. Thus result should be rounded off to the nearest significant figure. before reporting. What is or is not significant must be established for each test. Use of zeros after a decimal point can give misleading information if they are not significant. e.g. 7.0 g means between 6.95 and 7.05 g 7 g means between 6.5 and 7.5 g. Methodology of its teaching :- lecture with few C.D. demonstration, Evaluation of the session ;- Asking queries regarding the lecture.Chapter 3 Quality Control Objective of the session : To teach the extemal and internal quality control programme Introduction :- Variation in results Biochemical measurements vary for two reasons. There is analytical variation and also biological variation. Laboratory analytical performance A number of terms describe biochemical results. these include: precision and accuracy © Sensitivity and specificity Concentration is always dependent on two factors: the amount of solute and the amount of solvent. The concentration of the sugar solution in the beaker can be increased from 1 spoon/beaker (a) to 2 spoons beaker by either decreasing the volume of solvent (b) or increasing the amount of solute (c). © quality assurance © reference ranges. Precision and Accuracy Precision is the reproducibility of an analytical method. Accuracy defines how close the measured value is to the actual value. A good analogy is that of the shooting target figure 3 shows the scatter of results which might be 12obtained by someone with little skill, compared with that of someone with good precision where the results are closely grouped together. Even when the results are all close, they may not hit the centre of the target. Accuracy is therefore poor, as if the ‘sights’ are off. It is the objective in very biochemical method to have good precision and accuracy. Imprecise Precise but Precise and inacenrate accurate Fig. 3 Precision and accuracy Sensi ivity and specificity Sensitivity of an assay in a measure of how little of the analyte the method can detect, As new methods are developed they may offer improved detection limits which may help in the discrimination between normal results and those in patients with the suspected disease. Specificity of a assay related to how good the assay is at discriminating between the requested analyte and potentially interfering substances. Quality assurance Every laboratory takes great pains to ensure that the methods in use continue to produce reliable results. laboratory staff monitor performance or assay using quality control samples to give reassurance that the method is 13performing satisfactorily with the patients’ specimens. These are internal quality controls which are analysed every day or every time an assay is run The expected values are known and the actual results obtained are compared with previous values to monitor performance. In external quality assurance schemes, identical samples are distributed to laboratories; results are then compared. in this way, the laboratory's own internal standards are themselves assessed. Reference ranges Analytical variation is generally less than that from biological variables. Biochemical test results are usually compared to a reference range considered to represent the normal healthy state (fig. 3) Most reference range are chosen arbitrarily to include 95% of the values found in healthy volunteers, and hence, by definition, 5% of the population will have a result out with the reference range. In practice there are no rigid limits demarcating the diseased population from the healthy; however, the further a result is from the limits of the range, the more likely it is to represent pathology. In some situations it is useful to define ‘action limits’, where appropriate intervention should be made in response to a biochemical result. There is often a degree of overlap between the disease state and the ‘normal value’ (fig. 4) A patient with an abnormal result who is found not to have the disease is a false positive. A patient who has the disease but has a ‘normal’ result is a false negative. 14Biological factors affecting the interpretation of results The discrimination between normal and abnormal results is affected by various physiological factors which must be considered when interpreting any given result. These include: Sex of the patient. Reference ranges for some analytes such as serum. creatinine are different for men and women, Age of the patient. There may be different reference range for neonates, children, adults and the elderly. Effect of diet. The sample may be inappropriate if taken when the patient is fasting or after a meal. Time when sample was taken. There may be variations during the day and night. Stress and anxiety. They may affect the analyte of interest. Posture of the patient. Redistribution of fluid may affect the result. Effects of exercise. Strenuous exercise can release enzymes from tissues, Medical history. Infection and/or tissue injury can affect biochemical values independently of the disease process being investigated. Pregnancy. The alters some reference ranges. Menstrual cycle. Hormone measurements will vary through the menstrual cycle Drug history. Drugs may have specific effects on the plasma concentration of some analytes. 15Others factors When the numbers has been printed on the report from, they still have to be interpreted in the light of a host of variable. Analytical and biological variations have already been considered. Other factors relate to the patient. The clinician can refer to the patient or to the clinical notes, whereas the biochemist has only the information on the request from to consult. The cumulation of biochemistry results is often helpful in patient management. Technical contents :- kits of calibrators, kits of normal and elevated quality control samples. Methodology of its teaching :- lecture with few C.D. demonstration. Evaluation of the session :- Asking queries regarding the lecture. 16Chapter No.4 Photometery Objective:- To give the brief idea about the principles applied in several kinds of analytical measurements. Introduction of the topic: Photometery is the most common analytical technique used in clinical biochemistry. The principle of photometery is base on the physical laws of radiant energy or light. In this method, the intensity absorbed transmitted or reflected, light is measured and related to the concentration of the test substance. Photometeric principles are applied in several kinds of analytical measurements. 1. Measurement of absorbed or transmitted light: Colorimetery, spectrophotometery, atomic absorption, turbidometery. 2. Measurement of emitted light, Flame emission photometery Flourometery. Colorimetr, Many methods for quantitative analysis of blood, urine and other biological materials are based upon the production of a coloured compounds in solution, the intensity of which is used as a measure of concentration. Laws of Absorption of Light: There are two common methods of expressing the amount of light absorbed by a solution 1. By% transmittance.2. By optical Density (O.D) Absorbancy (A) or Extinction (E) of the solution. OPTICAL DENSITY-LOG T.%=Log 100/1 Lambert Beer's Law: The Lambert Beer law governing these relationships states that light absorption is proportional to the number of molecules of absorbing material through which light passes. Absorbance, therefore changes with the thickness of the solution and with the concentration of absorbent in a manner characteristic for each absorbing material of a given wavelength. The mathematical expression at a given wavelength is Where- I; = Intensity of emergent light. 1, = Intensity of incident light. K= A constant. C = Concentration of coloured substance. t = Thickness of the layer of solution. e = Base of natural logrithim (2.718) Is/Io is knows as transmittance. By assuming that the cell which is used to measure absorbance, is of constant thickness, it is possible to simplify the mathematical expression for this law in logarithmic form, The mathematical expression at a given wavelength is absorbance / Opticaldensity / Extinetion [E]-Lo (1/T) = Log 100%T so that E=2-Log%. T. Beer Lambert's law is applied fora) Only monochromatic Light b) There should be no changes in ionization, dissociation association or solvation of the solution with concentration. When A (absorbance) is plotted against C (concentration) straight line passing through the origin should be obtained because absorbance is directly proportional to concentration. With the aid of a standard curve the concentration of an unknown solution can then be readily determined. Parts of photo colorimeter A) Light Sources :- This usually on tungsten lamp (420-760m) for U.V. range Hydrogen lamp is used. B) Monochromator/Filters :- Complimentry filters should be used in order to increase the sensitivity of photometeric instrument Table: Colour of solution Filter used Peak transmission range (nm) Bluesh Green Red 580 - 680 Blue Yellow 520 - 580 Purple Green 490 - 520 Red Blue-green 470 - 490 Yellow Blue 430 - 470 Yellowish-green Violet 400 - 430 In the spectrophotometers grits are used. These provide narrow spectrum of light. Grits have practically replaced by prism as they are inexpensive. C) Cuvettes :- Cuvette holds the solution whose absorbance is to be measured. The cuvette must be optically-transparent, scrupulously clean deoid of any scratch and free form contamination. the optical 19path in the cuvette is always one cm. Glass cuvettes are used in visible range colorimetery while quartz/silea cuvwettes are used in UV. range Glvanometer :- This is used for measuring the output of the photosensi element. In most of the instruments a sensitive galvanometer is used. Prepration of solutions for measurement: 4, Blank :- This used to set the pnotometer to zero absorbance (A) or 100% transmittance (%T). 2 Standard :- These are solutions of known concentration which range within limits found in the specimen (normal and test). 3. An unknown solution. Determination of absorpition maxima : For cone/O.D. relation to be linear the wavelength chosen to measure the coloured solution should be that, at which, light is maximally absorbed. This is determined by using fixed concentration of coloured solution and measuring it at different wave lengths. Plot the graph taking O.D. on Y axis and wavelength on X axis & show results Graphically. Verification of Beer-Lambert's Law : To study Lambert Beer's law different concentration of dye, Bromophenol blue are taken. The optical density (O.D.) of these samples are measured in colorimeter. The corrected O.D. (after substracting the blank) readings are plotted on Y axis against the concentration of the dye on the X axis, It gives a liner graph. (ie. straight line passing through origin) up to certain concentration. 20Reagents: Procedure :- Blue standard solution — 1.Smg%- Take 7 test tubes and number them B,S;,S2,S3,S4,Ss, and Test- Tube No. 1. Blank 2. Si 3S 4. 83 5. Sq 6 Ss 7. Test Vol of dye Distilled water (in ml) (in ml ) 0 5 1 4 2 3 3 2 4 1 5 0 Sml (unknown) 0 Now mix tubes & take O.D. using a suitable filter or wavelength as determined by absorption maxima experiment, Plot a graph of O.D. against concentration of dye. From the graph determine the amount of dye present in unknown solution & express as mg% of the dye in solution. Calculations : If a suitable standard is prepared the extinctions of this and test are read, then. Concentration of unknown Concentration of standard and therefore, concentration of unknown = This can be expressed as :- concentration of unknown = Concentration of standard Reading of standard Extinction of unknown Extinction of standard Extinction of unknown X cone. of standard Reading of unknown X cone. of standard 2It SOURCE OF LIGHT FILTER CUVETTE PHOTOCELL = GALVANOMETER Fig (diagrammatic) photoelectric colorimeter Technical contents :- Photoelectric colorimeter Methodology of its teaching :- lecture with few C.D. demonstration. Evaluation of the session ;- Asking queries regarding the lecture. 22Chapter - 5 Automation Objective :- Advances in diagnostic methodologies and instrumentation have been impressive but most of these are deleted in the teaching programme this chapter tries to cover the automation required for the analysis of samples. Introduction During the past few year, in clinical biochemistry there has been a considerable increase in clinical demand for laboratory investigations. When the volume of work increased, there arose a need for work simplification. Monostep methods are introduced to replace multistep cumbersome and inaccurate methods like Folin-Wu's blood sugar determination. The efficiency of monostep methods was further increased by the introduction of automatic dispensers and diluters. For the common test like blood glucose, blood urea etc., however, most large laboratories found this approach still inadequate to deal with work load and instruments designed to handle the whole analytical process in mechanized fashion have become common place in last decade. This procedure is called automation. It is a self regulating process, where the specimen is accurately pipetted by a mechanical probe and mixed with a particular volume of the reagent and results are displayed in digital forms and also printed by a printer. There is an element of feed back which detects any tendency to malfunction. The function of autoanalyser is to replace with automated devices the steps of pipetting, preparation of protein free filtrated, heating the colour forming reagents in a waterbath and increase the accuracy and precision of the methods.The automated instruments not only save the labour and time but allow reliable quality control, reduce subjective crrors and work economically by using smaller quantities of samples and reagents. Following are the different types of autoanalysers used in clinical chemistry laboratories. Continuous flow analysers The early form of automation was introduced by Technicon Instrument Corporation. It was based on continuous flow analysis. In these systems, the samples and reagents are passed sequentially through the same analytical pathway and separated by means of air bubbles. The relative proportions of sample and reagents were determinated by their individual flow rates Missing occurred when tubes joined to form a common pathway. The disadvantage of these automated systems is that the physician may not be interested in all the test reported by the instrument, and the may be interested in some other tests which the instrument does not report. discrete analysers The various types of discrete autoanalysers used in the clinical chemistry laboratories are (A) Batch analysers and (B) ‘Stat’ (means immediate reporting or emergency determination analysers. (A) Batch analysers These are convenient to analyse specimen in batches such as of sugar, urea creatinine etc. state testing may not be conveniently carried out on these analysers. The batch analysers can be further differentiated as (1) semiautomated and (2) fully automated. 24(1) Semi automated (batch) discrete analysers In the case of these analysers the initial part of the procedure ie. pipetting of reagent and specimen, mixing and incubation is carried out by the technician. Rest of the procedure i.e. setting of incubation temperature (for kinetic determinations), zero setting, photometric readings, result display, automatic printing and data management and processing is carried out by the analyser. Advantage of semiautoanalysers 1) The semiautoanalysers are cheap and compact, compared to other fully automated analyses. 2) Specimen analysis 510 1.0ml. s cheap, since volume of reagent used is 3) Enzyme determinations by kinetic methods are performed accurately in 1 to 3 minutes. 4) The enzymatic reagents are not corrosive and involve monostep testing. Fully automated batch analysers These analysers carry out all the function of a semiautomated analyzer, in addition to the pipetting of specimen and reagents and also the mixing of the reaction mixtures, The basic working stages of these analysers, after selecting general system parameters are as follows 1) the specimen cups are placed on the sampler, 2) The required quantity of reagent is dispensed by a reagent probe, in the reaction cups 253) 4) 5) 6) (@B) The respective specimens from the sampler are pipetied into the appropriate reaction cups by another sample probe. The reaction cups are shaken mechanically to mix the contents. After observing the required incubation time (for delay time in the case of kinetic determinations) the reaction mixture is aspirated by a probe for photometric readings. The resulted values are printed and displayed in appropriate units by digital display. Stat Analysers (Random access analysers) In the case of these analysers many reagents (8 to 20 or more) can be pipetted one after another, so that various biochemical determinations can be performed on one specimen, according to the number of tests ordered for the patient. Hence, these are patient (or specimen) orientated autoanalysers. For examples, if serum specimen No. | requires following tests to be performed; 1) Urea nitrogen 2) Serum creatinine 3) Total proteins 4) Albumin 5) SGPT and 6) SGOT The analyzer is programmed for these tests with respective system parameters. The reagents for urea nitrogen, creatinine, total proteins, albumin, SGPT and SGOT are pipetted automatically by a reagent probe in the respective reaction cups. The required specific serum quantities are added to the respective reaction cups by a specimen probe. The analyzer identifies various reagents and specimen, The photometric determinations are carried out by the autoanalyser. 261) 2) 3) 4) 5) 6) The values of the respective tests are displayed on the computer screen as well as printed on a paper, afler the specific test incubation periods. The advantages of a fully automatic ‘stat’ (or random access) analyzer are as follows The advantages the various chemistry tests from the file. It performs a single test, a profile, an organ panel or a ‘stat! determination. Tt reduces the cost per test by utilization of micro-volumes of a reagent. It performs automatic monitoring of specimen and reagent volumes. It can perform various methodologies such as End point, kinetic, initial rate and bichromatic (readings at two different wavelengths) to eliminate errors which may arise due to haemolytic, icteric or lipemic sera. The analyser can perform repeatation of tests with or without automatic dilution, Technical contents :- semi autoanalyser and fullyautomatic analyser. Methodology of its teaching ;- lecture with few C.D. demonstration. Live demonstration of the semi and fully automatic analyser. Evaluation of the session :~ Asking to demonstrate the basic fimetioning of the above instruments. 27Chapter No.6 Estimation of Blood Glucose Diabetes Mellitus: It is a chronic disease due to disorder of carbohydrate metabolism, cause of which is either deficiency or diminished level of insulin resulting in hyperglyeemia (increased blood glucose level) & glucose (presence of glucose in urine). Secondary metabolic defect is also seen. Such as metabolism of proteins & fats. 1. Primary or Idiopathic or Essential Diabetes (a) Juvenile Diabetes or. Type I Diabetes or Insuline dependent Diabetes Mewlitus (IDDM) a Less Frequent a Occures before the age of 15 years. a Due to less production of insulin from b cells of langerhans (Pancrea) (b) Maturity onset diabetes or. Type II diabetes or Non-insulin dependent Diabetes melitus (NIDDM) a More frequent in population. a Occures at middle age. a Ketoacidosis is rare. a B cell is degenerated to some extent but response to glucose load is seen. 2. Secondary It is secondary to some other main disease (a) Pancreatic Diabetes. a Pancreatitis a Hemochromatosis 2 Malignancy of Pancrease. 28(b)Inereased level of antagonistic hormone 2 Hyperthyrodism 2 Hypercorticism — Cushings disease 2 Hyperpitnatarism Clinical Biochemical finding in diabetes 1) Presence of large amount of glucose in urine. 2) Large volume of urine & increased frequency of micturition Polyuria) 3) Polyphagia i.e. eats more frequently. 4) Increased catabolisim of fat so there is increase in free fatty acid level in blood & liver. 5) Increased acetyl coA is seen which further lead to increase formation of cholesterol & hence at formation of atherosclerosis. 6) Increased ketone bodies in blood & its apperance in urine leads to acidosis. 7) Increased catabolism of tissue protein for energy requirement lead to loss of weight & increased level of amino acid in blood & more formation of urea by deamination of amino acid. Objective :- To estimate blood glucose. Introduction :- Estimation of glucose in blood was one of the first biochemical tests to be applied clinically and now it has become a routine in clinical biochemistry lab. In blood quantitative estimation of glucose is done with either whole blood, plasma or serum and several methods have been in use. Whole bloodvalues are 10-15% lower than plasma. Arterial blood values are higher than venous values. The term Blood Sugar is used synonymously with blood glucose but certain other substance like glutation, glucuronic acid, threonine, uric acid, ascorbic acid, fructose etc. give erroneously high values (5-20%) when any reduction method is adopted. a) Fasting blood Sugar (FBS) : The blood sample is collected after the patient fasts for 12 hours or overnight. b) _ Post-Prandial Blood Sugar (P P B S) : After the patient fasts for 12 hours, a meal is given which contains starch and sugar (approx. 100 gms). Blood is collected 2 hours afier the ingestion of the meal. ©) Random Sample : Blood is collected any time without prior prepration of the patient. Collection of Blood Sample : Blood is usually collected from a vein and kept in a bottle containing sodium fluoride (Na F) and potassium oxalate mixed at proportion of 1 : 3 Usually 4 mg, of the mixture is required. Both the substances act as anticoagulant and Na F prevents glycolysis in RBC's by inhibiting the enzyme ‘enolase’. Methods of Estimation : 1. Enzymatic : Measure only glucose in blood. a) Glucose Oxidase Method : Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide. This H; O} is broken down to water and oxygen by a peroxidase in the presence of an oxygen acceptor which itself is converted to a coloured compound, the amount of which can be measured colorimetrically, This method is used in various autoanalyzers. 30Glucose H. Reaction : Glucose Gluconolactone— Gluconie Acid+H,0, Oxidase oO, Perxidase H,0, + Chromogenic ———— Chromogen + H;0 O, acceptor (Measured) Hexokinase b) Hexokinase Method: In this glucose + ATP —————_glu-6-P+ ADP G-6-P G—6-P+NAD —————— 6 phosphogluconate + NADH + H* ‘The rate of formation of NADH is followed spectrophotometrically at 340 mn, II. Reduction Methods 1) Alkaline Copper Reduction Methods. « Asatoor & King's method © Folin & Wu method 2) Ortho toludine method : O-Toludine reacts quantitatively with the aldehyde group of glucose to from a ghcosylamine and schiff base. The colour development is rapid and stable. Estimation of Blood Glucose by method of asatoor and King (modified) Principle : Reducing sugars in hot alkaline medium produce cnediols which are strong reducing agents that convert Cu ** ions Cu’ ions combine with OH ion to form yellow CuOH which on heating gives red Cu,O0, Cu,0 produced is proportional to the amount of reducing sugar Phosphemolybdic: acid is added so that oxidation of Cu" to Cu” is coupled with reduction of acid to molybdenum blue which can be estimated colorimetrically 31Modified method gives values close of true glucose. This is achieved by diluting blood with isotonic CuSO, Na2SO solution to prevent hemolysis of RBC. The glucose diffuses out of cells and is estimated while various non glucose reducing substance remain in the intact cells and are precipitated during deproteinisation and removed by centrifugation. So method used is modification by Asatoor and King in 1954 and involves: Precipitation of blood proteins. Reduction of alkaline CuSo4 solution to cuprous oxide by glucose. Estimation of extent of reduction of blue coloured complex by colorimetric measurement at 6.10 nm. Reagents : i) ii) iii) iv) vy) Isotonic copper sulphate solution : Na,SO4 IOH,O0 — 30 gm and CuSO4 5H, 0-6 gm per | litre of solution. Sodium tungstate 10%. Alkaline Tratrate solution. Dissolve 25g NaHCOs, 20gm anhydrous NasCO; and 18 gm Pot. Oxalate in about 500 ml of water. Add 12 gm of sodium potassium tartrate and make up the volume to | L. Phosphomolybdic acid solution : To 35 gm of molybdic acid add 5 gm. Of sodium tungsta'e, and 200 ml of 10% NaOH. Boil for 30- 40 mins to remove ammonia. Cool. dilute to about 350ml ad add 125 ml of cone. (85%) phosphoric acid. Make volume upto to 500 ml Stock glucose Standard solution ; 1 gm% in saturated benzoic acid. 32vi) Working glucose standard solution : Dilute stock 1 ml to 100 ml so cone. Is 10 mg% with isotonic CuSo; solution. Procedure Place 0.1 ml of blood in a centrifuge tube. Add 3.8 ml of isotonic sodium sulphate copper sulphate solution and mix. Add 1 ml of sodium tungstate solution and mix, Centrifuge at 2000 pm for 10° Use clear supernatant for test. Solutions T Si S S B ml. Supernatant 10 - = = Std. Glucose = 02 04 06 7 Isotonic CuSO, ~ 08 0.6 04 1.0 Alkaline Tartrate 1.0 1.0 1.0 1.0 1.0 Mix well. Plug the tubes with cotton wool and put in boiling water bath for 10 mins. Cool and 3 add mol of phosphomolbdie acid and 8 ml of distilled water to each tube, Mix, take O.D. at 610 nm. Plot a graph of standard against O.D. Calculation : OD of Test Amt. of Std. Blood glucose (mg/dl) =————— x ————__ X 100 OD ofStd Vol. of Blood Interpretation : The normal fasting blood glucose varies from 60-100 mg/dl and post prandial from 100-140 mg/dl. The appear limit increase with age and during pregnancy.Hyperglycagmia : Causes are : 1 Diabetes mellitus — Fasting blood sugar raised. Values of 140 mg/dl on more than one occasion or post prandial level of 200 mg/dl confirms the di gnosis. If the value is below 100 mg/dl it excludes diabetes mellitus Values in uncertain range between these figures calls for oral GTT to diagnose the condition. Hyperactivity of thyroid, pituitary, or adrenal gland. Surgical removal of pancrease, pancreatitis, carcinoma of pancrease, fibrocystic disease or baemachromatosis of pancrease. Intracranial diseases like meningitis, encephalitis, tumors and haemorrhage show a moderate hyperglycaemia. Drug induced eg : thiazide diuretics, steroids, ACTH, thyroid extracts. Diabetes mellitus is the commonest cause of hyperglycaemia and its carly detection is of vital importance Hypoglycaemia : When blood glucose falls below 60 mg/dl. i Ce Aw ew Most commonly seen due to overdosage of insulin in treatment of diabetes mellitus. Hypothroidism — cretinism, myxoedema. Insulin secreting tumours of pancrease — rare. Hypoadrenahsm (Addison's disease) Hypopitruitism, Severe exercise. Starvation. Chronic alcoholism Congenital disease like — glycogen storage disorders 34normally the blood sugar levels are well maintained due to action of various hormones, Along with estimation of blood glucose levels urine must be tested for: 1. Reducing Sugar ~ Commonly in diabetes mellitus. 2. Albumin — Diabetic nephropathy. 3. ketone bodies — Diabetes ketoacidosis. Recently quick measurement of blood glucose can be done by using only drop of blood from a finger prick using glucometer or dextrostix. Technical contents :- kit of sugar, colorimeter or semi autoanalyser. Methodology of its teaching :- Demonstration and estimation of sugar. Evaluation of the session :- Asking to perform the test and taking readings. 35Chapter No.7 Glucose Tolerance Test Object :- To estimate the glucose tolerance. Introduction : Glucose Tolerance is defined as the capacity of the body to tolerate an extra load of glucose. Normally the blood glucose level remains relatively constant the fasting being 63-100mg% which returns to normal within 2 hours. The definitive diagnostic test for DM is the G.T.T. Oral G T T : Procedure : After an overnight fast of 12-16 hrs. fasting blood sample is taken, Then 75gm of glucose dissolved in 250-300ml of water is given orally. Blood samples are collected at 30 mins interval for 2-3 hours but 2 hours sample is most important for interpretation of result according to WHO criteria. Corresponding urine samples can also be collected and presence of reducing sugar tested by Benedicts qualitative test. Blood sugar in cach sample is estimated by King and Asatoor method. A curve between time and blood glucose concentration, is plotted. Precaution: 1) The patient should be on a diet of 300 gm of carbohydrate per day for at least last 3 days. 2) Fasting should not be less than 10 hrs. and not more than 16 hours. Only water is permitted after dinner, 3) The patient should not be taking drags that affect carbohydrate metabolism. 364) Durings the test patient activity should be normal (mild to moderate) and he should abstain from smoking. The patient should be at rest mentally. Factors affecting GIT : 1) Starvation/Ingestion of high fat diet. 2) Exercise. 3) Pregnancy-tolerance is decreased. 4) Illness-stress causes decreased tolerance, so patient recovered from surgery, burns or child birth should be allowed 2 weeks time before the test is carried out. 5) Physiologically decreased tolerance with age. 6) Endocrine disorders. 7) Drugs-certain drugs must be withdrawn before the test eg-Oral contraceptives, thiazide diuretics, insulin, oral hypoglyan mic agents, salicylates etc. 8) Liver diseases. Interpretation: The following important type of response are seen commonly : a) Normal Response : Fasting blood sugar is normal. After 1 hour level rises, but remain below the renal threshold of 180%. It returns to normal fasting level within 2 hours. b) Diabetic curve : Fasting level are 7.8 mmol/L (140mg dL) and 2 hour venous blood glucose of 200mg/dl (11 Immol/L) or more are diagnostic of diabetes. Glycosuria is usually seen. 37c) Impaired GTT: 2 hours values of blood glucose between 140mg/dl and 200mg/dl are not abnormal and must be followed up for DM 4d) Renal Glycosuria : Curve is normal Due to lowered renal threshold one or more samples of urine contain glucose. e) Lag storage/Alimentary Type: Fasting blood glucose is normal. Due to rapid absorption, maximum level is found at 30 mins which crosses 180mg/dl (80 glycosuria seen) and hypoglycaemic levels may be reached at end of 2 hours. ) Flat curve of enchanced glucose tolerance : the fasting blood glucose level is normal. Throughout the test the level does not vary + 20me%. Extended GTT: In this blood samples are taken for 4-5 hrs after giving glucose to see how the curve behaves below the normal fasting glucose limits. Done in some conditions causing hypoglycaemia. Cortisone Stressed GTT : Can be used for detecting latent Diabetes mellitus. Intrevenous GTT : In is done if oral glucose is not tolerated or oral GTT curve is flat. In these cases 20% glucose as 0.5¢ glucose/Kg body weight is infused. Blood samples are collected hourly. Usually peak occurs within 30 mins after infusion and returns to normal after 90 mins. Methodology of its teaching :- Plotting the glucose tolerance graph. Evaluation of the session :- Asking queries regarding the above test. 38Chapter No.8 Glycated hemoglobin Object : To estimate Glycated hemoglobin. Introduction : Human Hemoglobin inside erythrocytes undergoes a non enzymatic chemical reaction with glucose. The rate and extant of this reaction are thought to be depended on the average blood glucose concentration during the life time of the erythrocytes there are several reaction procedure, “Glycated hemoglobin”, which collectively Hb Ay. The most abundant of these is Hb A,, the ratio of Hb Alc or HbA1 to the total HbA concentration has been suggested as a reliable measure of the degree of metabolic control in diabetic patients. Technical Contents :- Using kit of Glycated hemoglobin and its measurement by semi autoanalyser or by using Ghb analyser. Principle and procedure: 1. Using kit of Glycated Hemoglobin an its measurement by semi autoanalyser. A hemolysed preparation of whole blood is mixed continuously for 5 minutes with a weak binding cation-exchange resin during this time the non glycated hemoglobin, which consists of the bulk of the hemoglobin (Hb,o) binds to the resin. After the mixing period a filter is used to seprate the supernatant containing the glycohemoglobin from the resin. The percent glyeohemoglobin is determined by measuring the absorbance at 415 nm (405-420nm acceptable) of the glycohemoglobin fraction and the total hemoglobin fraction. The ratio of 39the two absorbances gives the percent glycohemoglobin. Normal range is 6.0% to 8.3% 2. Using Ghb analyser. Tt uses low pressur cation exchange chromatography in conguction with gradient elution to separate human hemoglobin sub types and variants from hemolysed whole blood. The separated hemoglobin fraction are monitored by means of absorption of light at 415nm the chromatogram obtained is recorded and stored by the onboard computer. The analyser performs the analysis of the chromatogram and generates a printed report, Expected range of Hb Atc Sugar - 90-150 5-0% to 7.0% Sugar — 150-180 7.0% to 8.0% Sugar — 180-360 9.0% to 14.0%. Method of teaching : Using kit of Ghb flowing its procedure and giving demonstration. Evaluation of session: To calculate the percent of glycated hemoglobin after doing the experiment. 40Chapter No.9 Estimation Of Blood Urea Kidney function tests : Since the kidneys perform a multitude of functions, a single test cannot give information about the entire range of renal functions. A group of tests is required to evaluate the different renal functions. Abnormal results may sometimes be obtained due to a temporary renal dysfunction. Hence the test should be performed repeatedly and interpreted on the basis of a series of results. Moreover, the results of renal function tests may some-times be affected by extra-renal factors. Therefore, the results must always be interpreted in conjunction with the clinical picture. A large number of tests have been introduced during the past decades to assess the renal functions. Only a few of them have stood the test of time. ‘The more important and commonly employed tests will be discussed below. 1) Blood urea 2) Serum creatinine Object : To estimate blood urea. Introduction : Urea is main end product of protein catabolism. It is formed in the liver and is excreted through urine. Urea represents about 45-50% of the non-protein nitrogen of blood and 80-90% of the total urinary nitrogen exeretion. The urea concentration in the glomerular filtrate is same as that in plasma, Tublar reabsorption of urea varies inversely with the rate of urine flow ad hence is not a useful measure of GFR. Blood urea nitrogen varies directly with protein intake and inversely with the rate of excertion of urea. 4lEstimation : Various methods are use : a qi) Enzymatic using urease : a) Neseler's Method : Urea is converted to ammonia by the enzyme urease. Ammonia is made to react with Neseler's reagent (Potassium mercurie iodide) giving rise to a brown coloured compound which is read at 450nm the enzyme acts optimally at 55°C pH 7.0 to 8.0 and is inhibited by ammonia and fluoride. Disadvantages are turbidity, colour instability and non linear, calibration beside susceptibility to contamination with NH3 from the laboratory and endogenous ammonia in the specimen. b) Berthelot Reaction : In this NH; reacts with phenol in presence of hypochlorite to form an indophenol which gives a blue coloured compound in alkali. Nitroprusside acts as a catalyst increasing the rate of reaction, intensity of colour obtained and its reproducibility. ©) In the urease/glutamate dehydrogenose method, glutamate production from ammonia and 2.0 x 0- glutarate is measured by the absorbance change at 340nm. Owing to concomitant conversion of NADH’ to NAD". Each molecule of urea hydrolysed causes the production of two molecules of NAD’. Kinetic Method: GLDH method Urea is hydrolysed in presence of urease to produce ammonia and CO2 the ammonia produced combines with alfa oxoglutarate and NADH in presence of GLDH to yield glutamate and NAD" aii) The decrease in extintion due to NADH in unit time is proportional to the urea concentration. Normal range of serum urea = 15 to 45 mg/dl Colorimetric Method : Diacetyl! Monoxime Method Principal : Urea reacts with diacetyl monoxime in acidic conditions at nearly 100°C to give a red coloured product which is measured colorimetrically at 520nm. Thiosemicarbazide and ferric ions are added to catalyse the reaction and increase the intensity of colour. This method is linear only upto 300mg% urea. For higher values if expected, the blood sample should be diluted. Reagents 1) 3) 4 5) 6) 72 8) Reagent A : Dissolve 5g of ferric chloride in 20ml of water. Transfer this to a graduated cylinder and add 100ml of orthophosphoric acid (85%) slowly with strring. Make up the volume to 250ml with water. Keep in brown bottle at 4°C. Reagent B : Add 200 ml conc, H:SO, to 800 ml water in 2L flask slowly with stirring and cooling Acid Reagent : Add 0.5 ml of reagent A to | L of reagent B. keep in brown bottle at 4°C Reagent C : Diacetyl monoxime 20g/L of water. Filter and keep in brown bottle at 4°C. Reagent D : Thiosemicarbazide Sg/L of water, Colour Reagent : Mix 67 ml of C with 67 ml of D and make up the volume to 1000 ml with D.H,0 keep in brown bottle at 4°C. Stock urea standard : 100mg/100 nl water. Working urea standard : Dilute | ml stock to 100ml with DH,O so cone. is | mg/100ml.Procedure : 0.1 ml if serum/plasma is diluted to 10 ml. set up the test tubes as follows : BT S S: Ss S Ss Serum (ml) > = = = = = = (dil 1:100) Std (ml) - : 0.2 04 0.6 08 10 D. Water (ml) 2 10 18 16 14 12 1.0 Colour Reagent (ml) 20 2.0 2.0 2.0 2.0 20 20 Acid reagent (ml) 2.0 2.0 2.0 2.0 2.0 20 2.0 Mix all the tube thoroughly. Keep in boiling water bath for exactly 30 mins. Then cool and read absorbance at 520nm. Calculation : O.D. Test Amount of Std. Blood urea (mg%) =—————_ x_ ——_____~ O.D. Std. Vol. of bld. A standard curve can be plotted end value of unknown sample calculated from it, Interpretation : Nonmal blood urea in adults is 15-S0 mg% when expressed as blood urea nitrogen (BON) it is 7-25 mg%. It is somewhat higher in men than women and there is gradual rise with age. The urea concentration varies with the amount of protein in the diet.Serum urea is lower in pregnancy probably due to hoemodilution, usually between 15-20mg/100ml. Increase in urea is significant as a measure of renal function. The causes may be Pre Renal : When there is reduced plasma volume it leads to decreased renal blood flow and hence GFR leading to urea retention. Seen in Reduced plasma volume :- - Acute intestinal obstruction — prolonged vomiting. - Severe diarrhoea. = Pylorie stenosis. - Ulcerative colitis. - Diabetic Keloacidosis. - Shocks, severe bums and haemorrhage. Increased protein catabolism :- - Fevers - Thyrotoxicosis Cardiac failure Renal Disease : Blood urea is increased in all forms of renal diseases like; = Acute glomerulonephritis. - Renal failure = Malignant hypertension = Chronic pyclonephritis = Congenital cystic kidneys Post renal : Due to obstruction to flow of urine there is retention and so reduction in effective filteration pressure at the glomeruli - Enlargement of prostrate. - Stones in urinary tract. 4s- Urethral strictures. Methodology of its teaching :- demonstration of its estimation using kits. Evaluation of the session :- Asking to demonstrate the test. 46Chapter No. 10 Estimation Of Serum Creatinine Object :- To estimate serum creatinine. Introduction: Creatinine is a waste product formed in muscle by creatine metabolism Creatine is synthesized in the liver which then passes into circulation where it is taken up by skeletal muscle for conversion to creatine phosphate which serves as storage form of energy in skeletal muscles. Creatine and creatine phosphate are spontaneously converted to creatinine at a rate of about 2% the total per day. This is related to muscle mass and body weight. Creatinine formed is excreted in the urine. On a normal diet almost all creatinine in urine is endogenous. Its excretion is fairly constant from day to day and has been used to check the accuracy of 24 hours urine collection. It is independent of urine flow rate and its level in plasma is quite constant. Estimation of serum creatinine by Jaffe's Alkaline Picrate Method Principle : Creatinine in alkaline medium reacts with picric acid to form a red tautomer of creatinine picrate the intensity of which is measured at 520nm. The two chief disadvantages with Jaffe's reaction are: - Lack of specificity Only 80% of the colour developed is due to creatinine in serum, other non specific chromogens that react with picric acid are proteins, ketone bodies, pyruvate, glucose, ascorbate ete. - Sensitivity to certain variables like PH temperature ete. 47Reagents: 1) Picric acid - 0,04M (9.16e/L) ay Sodium hydroxide — 0.75N. 3) Sodium tungstate — 10% 4) 2/3 N H2 SO, 5) Creatinine standard stock — 100mg% 6) Working standard — 3mg% Procedure :- In a centrigure tube, take 2ml of serum with 2 ml of distilled water. Precipitate proteins by adding 2ml sodium tungstate and 2ml of 2/3 N sulphuric acid drop with constant shaking, Stand for 10 minutes and filter. Use protein free filterate for test. Make the test tubes as follows and add B Ss Ss Ss. © Filtrate : : : : : 3ml Standard 3mg% - 05 10 2.0 3.0 - D. Water 3ml 25 20 10 - - NaOH Im 10 10 10 1.0 10 Picric acid 1.0 1.0 10 10 1.0 10 Mix well Allow to stand for 15 min. and read absorbance at 520nm. Calculation : OD. Test Amount of Std. Serum creatinine (mg%) x 100 =, OLD. Std. Vol. of Serum a8Interpretation : Normal serum creatinine levels are males: 0.7-1 4mg/100m | Females: 0.4-1.2mg/100m 1 In is higher in males since it is related to body size. Increased serum levels are seen in renal failure and other renal diseases in a manner similar to urea. Creatinine, however, does not increase with age, dehydration and catabolic states (eg fever, sepsis, haemorrhage) to the same extent as urea. It is also not affected by diet. But the Jaffe's reaction for measuring serum creatinine is not as sensitive andreliable as method for urea. It is interfered with by Ketone bodies and glucose and hence false high values may be obtained in diabetes ketoacidosis. How serum creatinine is not significant. It is associated with muscle wasting diseases. Technical Contents: Kit of creatinine. Methodology of its teaching :- demonstration of its estimation using kits. Evaluation of the session :- Asking to demonstrate the test. 49Chapter No. 11 Estimation Total Protein Object : To estimate total protein. Introduction: Serum contains a large variety of proteins. More than hundred different proteins have been identified so far, and perhaps many more are yet to be identified. Albumin and the various globulins constitute the bulk of the total amount of proteins present in serum. The most widely used method is still the biuret method. (the name derives from the reaction between biuret and cupric ions in an alkaline medium. Biuret Method Principle : Cupric ions form chelates with the peptide bonds of proteins in an alkaline medium. sodium potassium tartrate keeps the cupric ions in solution. The intensity of the violet colour that is formed is proportional to the number of peptide bonds which, in turn, depends upon the amount f proteins in the specimen. Reagents (i) Biuret Reagent — 3 mg of copper sulphate is dissolved in 500 ml of water. 9 gm of sodium potassium tartrate and 5 gm of potassium iodide are added and dissolved. 24 gm of sodium hydroxide, dissolved separately in 100 ml of water is added. The volume is made up to 1 litre with water. The reagent is stored in a well-stoppered polythene bottle. 50(i) Biuret blank — this is prepared in the same way as the buiret reagent with the difference that copper sulphate is not added. (iii) Standard protein solution — the best way is to determine the total protein concentration in pooled human serum by Kjeldahl method, dilute it to bring the protein concentration to the desired level, say 6 gnv100 ml and use it as standard, Alternatively, a 6 gm/100 ml solution of bovine albumin in water may be prepared and used as standard. Procedure : label 3 test tubes ‘Unknown’, ‘Standard’ and 'Blank', Measure 5 ml of biuret reagent into each. Wash 0.1 ml of serum into 'Unknown', 0.1° ml of standard protein solution into ';Standard' and 0.1 ml of water into ‘Blank’. Mix and allow to stand for 30 minutes. Read "Unknown! and ‘Standard! against ‘Blank’ at 540 nm or using a green filter, Calculations Au Serum total proteins (gm/100 ml) = x6 As Note. The above procedure gives reliable results with clear and unhaemolysed specimens, If the serum specimen is lipaemic, icteric or haemolysed, an additional tube (Serum Blank) should be prepared. 0.1 ml of serum should be mixed with 5 ml of biuret blank in this tube and read after 30 minutes against ‘Blank’. Absorbance of 'Serum Blank; should be subtracted from that of 'Unknown before calculations. slInterpretation The normal range of serum total proteins is 6-8 gnv/100 ml in adults, The values, Are lower in neonates, rise gradually in infancy and reach the adult levels in early childhood. The level decreases in pregnancy. A slight change ‘occurs with posture also. The level is a little higher in upright posture than in recumbent posture due to shift of water from the vascular compartment into the interstices. An increase in serum total proteins occurs in dehydration due to haemoconcentration. An increase may also occure in multiple myeloma, macroglobulinaemia, chronic infections, chronic liver disease and auto- immune diseases. A decrease in serum total proteins may results from heavy losses of proteins in urine as in nephritic syndrome. Protein undernutrition, intestinal malabsorption and protein losing enteropathy may also lower the serum total proteins. A decrease may also occur in shock, burns, crush injuries, haemorrage ete. due to haemodilution. Increased breakdown of proteins, as in hyperthyroidism, un-treated diabetes mellitus, wasting diseases etc. may also lower the level of proteins in serum, Technical contents : Using kit of total protein. Method of teaching : Demonstration and its measurement of serum total protein by kit and taking readings. Using a colorimeter or semi auto analyser. Evaluation : Giving exercise of total protein estimation. 52Chapter No. 12 Estimation of Serum Albumin and Globulin Object: To estimate of serum albumin and globulin. Bromocresol Green Method Principle : The method is based on the protein error of indicators. Biding of a protein to an indicator changes its colour. Among serum proteins, only albumin binds to BCG this binding produces a change in the colour of BCG which is measured colorimetrically. The pH is maintained during the reaction by a bufier. Reagents a di) Gii) (iv) Succinate buffer - 11.8 gm of soccinic acid is dissolved in about 800 ml of water. The pH is adjusted to 4.0 with 0.1 N sodium hydroxide, The volume is made up to | litre with water. This solution should be stord in refrigerator. BCG solution - 419 mg of bromocresol green is dissolved in 10 ml of water. The solution is stored in refrigerator. Buffered BCG solution — 250 ml of BCG solutions is mixed with 750 ml of succinate buffer. The pH is adjusted to 4.2 with 0.1 N sodium hydroxide solution. 4 ml of Brij — 35 solution (30%) is added. Standard albumin solution — an aqueous solution of human albumin with a concentration of 4 gm/100 ml can be prepared and used a standard, Sodium azide should be included in this solution (50 mg in every 100 ml) z sodium azide) or a control serum having an albumin concentration of a preservative, Pooled human serum (preserved with 4 gn/100 ml can also be used as a standard.Procedure : Level 3 test tubes ‘Unknown’, ‘Standard’ and ‘Blank’. Measure 4 ml of buffered BCG solution into each. Wash 0.02 ml of serum into ‘Unknown’, 0.02 ml of standard albumin solution into 'Standard' and 0.02 ml of water into 'Blank’, Mix and allow the tubes to stand for 5 minutes. Read "Unknown! and ‘Standard! against ‘Blank’ at 630 nm or using a red filter. Calculations : Serum albumin (gr/100 ml) = x4 Interpretation : The normal range of serum albumin is 3.7-5.3 gnv/100 mi. Serum globulin ranges from 1.8 to 3.6 gm/100 mi. the A:G ratio is roughly 2:1 though it may range from 1.2:1 to 2.5:1 Decrease in serum albumin may occur in protein undermutrition, intestinal malabsorption, protein-losing enteropathy, liver disease, wasting diseases, nephritic syndrome and haemodilution, A severe decrease or near — absence may be seen in analbuminaemia which is a genetic disease with autosomal recessive in-heritence. A rise in serum albumin may occur in dehydration due to haemoconcentration. Serum globulin may decrease due to haemodilution in shock, bums, haemorrhage tc, serum globulin increases in multiple myeloma, macroglobulinaemia, chronic liver disease, chronic infections and auto- immune diseases. A:G ratio may be decreased or reversed in these conditions. 54Since the colorimetric measurement of albumin is much simpler than that of globulin, the concentrations of total proteins and albumin are measured in serum, and globulin is obtained by difference, Technical contents : Using kit of serum albumin. Method of teaching : Demonstration and its measurement of serum albumin by kit and taking readings, Using a colorimeter or semi auto analyser. Evaluation : Giving exercise of serum albumin estimation. 58Chapter No. 13 Estimation Of Aminotransferases (Transaminases) Object :- To estimate aminotransferases. Asparate Aminotransferase (AST) SGOT Alanine Aminotransferase (ALT) SGPT Introduction : The aminotransferases are a group of enzymes that catalyse the inter conversions of the aminoacids and a-Keto acids by transfer of amino groups. Distinct isoenzymes of AST are present in the cytoplasm and mitochondria of cells. Method for the estimation of AST and ALT : Colorimatric Methods : Reitman & Frankel (1957), Tietz (1970), Bergmeyer & Brent (1974 b) Modified Reitman & Frankel Method : Principle :- ‘The amount of oxaloacetate or pyruvate produced by transamination is reacted with 2,4 dinitrophenyl hydrazine (DNPH) to form a brown coloured hydrazone, the colour of which in alkaline solution is read at §20nm. 56