Diagnostic Biochemistry.(Ms 1st &2nd Sem.3rd Year)Part-3new | Renal Function | Glycated Hemoglobin

Practicals

Lab. Practical – 1 Lab. Practical – 2 Lab. Practical – 3 Lab. Practical – 4 Lab. Practical – 5 Lab. Practical – 6 Lab. Practical – 7 Lab. Practical – 8 Lab. Practical – 9 Lab. Practical – 10 Lab. Practical – 11 Lab. Practical – 12 Blood Glucose Estimation Oral Glucose Tolerance Glycated Hemoglobin Estimation Measurment of Triglycerides Measurement of Total Cholesterol , HDL and LDL Renal Function Tests: BUN and Creatinine estimation Creatinine Clearance Estimation Liver Function Tests –1: Bilirubin ; Total and Direct Liver Function Tests –2: Enzymes; ALT, AST and GGT Albumin and Total Protein Estimation Bone profile Testes Cardiac profile testes

Lab. Practical 13 – 15 Tutorials

Introduction To Applied Biochemistry

General Comments about testing There are so many different methods used to analyze different chemical compounds that to state one method over another is unfair. Another issue is that your body’ chemistry changes throughout the day in response to external conditions such as exercise and internal conditions such as kidney function. This makes comparisons among various tests difficult to do. One method to lessen these variables is to try to have your tests done by the same laboratory so that comparisons of test values are possible. It is also beneficial then to have your tests drawn under the same conditions (fasting/non fasting, early morning/late afternoon, etc.) so that you can eliminate these interferences when you look at your results.

Practices of Clinical Biochemistry – Part II: Estimation of Blood Glucose
Introduction: The importance of testing the blood glucose level comes from the fact that the brain cells are very dependent on the extracellular glucose concentration for their energy supply; hypoglycemia is likely to impair cerebral functions as well as do the hyperglycemia especially of rapid onset, which can cause cerebral dysfunction by affecting extracellular osmolarity. Objectives: -To know the different methods for estimation of blood glucose -To know the precautions needed to get accurate results and better interpretation of glycemic status in relation to disease condition. Methods: Many methods were developed to estimate the glucose level in body fluids among which the commonly used nowadays, the enzymatic methods. These methods can be summarized and categorized into A) Reduction methods: These methods depend on the reductive property of glucose(aldose) 1-Ferriccyanide( Hoffman’s) method: using ferricyanide which is reduced by the glucose . Fe+++ Fe++ (color change from yellow to colorless solution that will diminish the absorbance measured photometerically ) 2-Copper sulfate methods: Benedict: The reagent contains Na-citrate &Na carbonate with CuSO4. It gives color acc. To conc. of glucose (green-----yellow----brown-----red). Fehling : using KOH &Na/K tartrate with CuSO4 Folin- Wu : Alkaline Cu SO4 +Phosphomolybdic acid molybdenum blue by reducing Cu2O CuO2 3-Smogi-Nelson method: using Arsenomolybdate

-Kinetic method: by measuring the increase in absorbance through increase in NADH+H -Polarigraphic method: using O2 electrode to detect O2 utilization. 3. Polarigraphic determination with post-reaction elimination of H2O2 by: ethanol & catalase or Iodide & molybdate. . B) Aromatic amines method: O-toludine +glucose (aldhyde) heat &acidity glucosamine (colored ) C) Enzymatic methods: 1-Hexokinase methods(The reference method). using GOD/POD method in urine with modification like . GOD/POD method can not used for detection of urine glucose because the urine contains interfering substances for peroxidase (POD) .Glucose Dehydrogenase Method: Glucose +NAD at 340) GDH Gluconolactone +NADH+H (measured . N.N. The reduction methods need alkaline medium &heat These methods are qualitative & semi-quantitative.To use this method treatment of the urine sample either by Somogi Nelson filtrate or Ion Exchange Resin is taken before running .-Dye Colorimetric ) method: which is colorimetric either by spectrophotometer or refractrometer (refractrometeric methods either in a film form [kodak Ectachem] or a strip form [Dry chemistry] ). With pre-deproteinization of sample or without.B. Also. Glucose +ATP +HKADP+G6P G6P +NAD +G6PD 6 Pgluconolactone +NADH+H (measured at 340) 2.B.Glucose oxidase methods: -Trinder’s (Enz.

2. is detected by a chromogenic oxygen acceptor. Cap and mix gently to dissolve contents. it should integrate clinical and other laboratory data. Instrumentation: -Photometer adjusted on wavelength 540 nm . Clinical diagnosis should not be made on a single test result. The formed hydrogen peroxide (H2O2).Presence of particles and turbidity.oxalates/NaF). Requirements: *Samples: -Blood samples Whole blood Serum Plasma (with Ca.? -CSF collected in sterile clean container and to be done immediately or centrifuged to get cell free fluid.Glucose Oxidase for blood glucose estimation (Experiment #1) PRINCIPLE OF THE METHOD Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid. CLINICAL SIGNIFICANCE Glucose is a major source of energy for most cells of the body.10. PREPARATION Working reagent (WR): Dissolve the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer. Diabetes is a disease manifested by hyperglycemia. patients with diabetes demonstrate an inability to produce insulin. Signs of reagent deterioration: 1. phenol-aminophenazone in the presence of peroxidase (POD): Principle: (Trinder’s method ) α -D-glucose Mutarotase β -D-glucose Glucose oxidase β -D-glucose +H2O+O2 D-gluconic acid+H2O2 Peroxidase H2O2+ 4-aminophenazone+phenol +4 H2O Quinonemine The intensity of the color formed is proportional to the glucose concentration in the sample. insulin facilitates glucose entry into the cells. which is the preferred sample -Fresh urine by double void collection technique…….Blank absorbance (A) at 505 nm 0. The reagent is stable 1 month after reconstitution in the refrigerator (28ºC) or 7 days at room temperature (15-25ºC).

. . . . . . . Of the Sample Normal Range: . The colour is stable for at least 30 minutes. . . 505 nm (490-550) Cuvette: . . . .. .. . .-Cuvette (light path) 1 cm -Water bath at 37 ºC -Automatic pipettes. . . . . CALCULATIONS (A) Sample x 100 (Standard conc. Read the absorbance (A) of the samples and standard. PROCEDURE 1 1. . racks and disposable tips for the dispensers. . against the Blank. . . . . 1 cm light path Temperature. 23. . . . . Adjust the instrument to zero with distilled water. . . . Mix and incubate for 10 min at 37ºC or 15-20 min at room temperature (15-25ºC). . .0555= mmol/L. . . disposable test tubes . .3 As the concentration of glucose standard = 100 mg/dl The Glucose concentration in the sample = 333 X Abs. . . . . . .) = mg/dL glucose in the sample (A) Standard Conversion factor: mg/dL x 0. Assay conditions: Wavelength: . . .0 -- -- (WR (mL Standard ((µL Sample ((µL 14. . Of the Standard ~ 0. . . . 15. H2O) Result: Abs. . . . *Linearity of the test = 400 mg/dl (Samples give higher level must be retested with dilution by suitable buffer or dist. 37ºC / 15-25ºC 12. Pipette into a cuvette: Bla nk 1.

Reactive Hypoglycemia b.B.140 mg/dl Urine glucose . Postprandial = 110 . Alimentary Hyperinsulinism .Excessive Insulin Release: a. < detectable limit (Nil) CSF glucose ~ 60 .Blood glucose… Fasting= 70 . To express the result in mmol/L divide by 18 ( MW of Glucose =180) Interpretation: I -Hypoglycemia : The patient considered critically hypoglycemic if: Whole Blood glucose level < 40mg/dl Serum/Plasma glucose level < 45mg/dl A..90 mg/dl N.110 mg/dl & 2 hrs.Well Fed State Hypoglycemia: 1.

Galactose -1.Glycogen Storage Disease b.Fed Status Functional Hypoglycemia: B.Hyperglycemia : .Induced by Exogenous Agents: 1-Alcohol Intake 2-Excessive Insulin Administration 3-Excessive Sulfonylurea Administration II .Hyperthyroidism .Anesthesia .Fasting Hypoglycemia: 1-Organic Hypoglycemia: a-Pancreatic B-Cell disease/CA b-Non-Pancreatic Tumors c-Anterior Pituitary Hypo-function d-Adrenocortical Hypo-function e-Ingestion of Akee Fruit 2.Stress .Inherited Enzyme Defect: a.Diabetes Mellitus .Genetic Deficiency or Delayed Maturation of Enzymes in Premature Babies 2.On specific hepatic enzyme deficiency: 1.c.Phosphate 3. Leucine Hypersensitivity 2.Hemochromatosis .Functional Fasting Hypoglycemia ِ a.Pregnancy .Pheochromocytoma .Hypokalemia .Phosphate b. Fructose -1.

Hyperthyroidism …. disease . Insulin action and other hormonal effects on glucose in the human body. the response of the body regarding the absorption and metabolism of glucose said to be tolerant on meeting the normal elevation and return. Oral . Cushing *Pathological & Disease Correlation: Diabetes Mellitus. Whereas abnormal and improper glucose metabolism is termed glucose intolerance.Cushing disease .Hyperpituitarism (gigantism) Discussion: *Physiological & Biochemical Background: Glucose metabolism... When does a person considered hypoglycemic? What are the types of hypoglycemia ? Give an account on the principle of glucose oxidase method for glucose estimation. This used to diagnose diseases where the glucose metabolism is impaired as in Diabetes mellitus. ORAL GLUCOSE TOLERANCE TEST Introduction: On standard oral glucose dose.etc Questions: 12345What is the basis of reduction methods for glucose estimation ? Give short notes on Trinder’s method for glucose estimation.

After delivery for those was suffering from GDM. Don't drink coffee or alcohol for 8 hours before the test. for the detection of diabetes in pregnant women. Diet--Eat a high-carbohydrate diet (> 150 g/day) for 3 days. and 9 A. However.M. Objectives: It is to practice the OGTT and knowing the uses and interpretation regarding the diagnostic benefits of this laboratory test. 3. It has low reproducibility and is very expensive. * General description of test Test usually takes 3 hours but can last as long as 6 hours (extended OGTT). Lately. Operator gives a test load of glucose. as under taking these drugs the test results may differ (contraceptives to be stopped one cycle before the performance of OGTT). usually 75 – 100 gram dextrose / 300 ml water.Diagnosis of Gestational Diabetes (GDM) at 24 – 28 weeks of gestation especially for those have a family history of diabetes. Drink water frequently during the test (the only allowed fluid to drink). Indications: 1. after you have fasted for 12 hours. .. it is still recommended. thought. OGTT (Experiment # 2): *Patient preparation (Perquisites) . The test must be performed at daytime (morning). the use of OGTT in primary care has been questioned for several reasons. then fast for 10 to 12 hours before the test.M.Borderline fasting blood sugar for >2 times (~ 110 – 125mg/dl) 2. The first blood sample and the first urine sample are collected between 7 A. Activity--Don't smoke or exercise strenuously for 8 hours before the test or during the test. Drugs (medicines)-Inform the person performing the test to omit any medications listed.glucose tolerance test (OGTT) has been widely used as the golden standard for diagnosing diabetes mellitus in clinically doubtful cases.

30min. It is to be dissolved in 250 – 300 ml lemon flavored water. Urine samples . + 90 min. 120min. and 3 hours and sometimes immediately after drinking oral glucose solution. Blood and urine samples are collected at 30 min. Calculation: there are different methods to calculate and interpret the glucose levels (mg/dl)in OGTT: Glucose sample Fasting 30 min. 60 min. Dose of Oral Glucose: Dextrose: 1 – 1. (for adults0 and not exceeds 100 g. first fasting urine and the hourly collected urine samples.lemon flavored . Drink the entire solution in 5 minutes. Fortical : 113 ml completed to 300 ml water Lucozade: 350 ml.1 ½ point  Suspect Zero  Non diabetic Fajan-conn criteria >190  +1 > 165  +1 > 140  +1 3  Diabetic 1 – 2 Suspect Zero  Non diabetic Revised Summation *If Σ of results (F + 60min.120 min. fasting(basal) sample. + 120 min.75 g/kg. 120 min.. 90 min.) > 600 mg/dl = Diabetic *If Σ of results < 600 = non diabetic Results and Diagnosis: Glucose tolerance tests may lead to one of the following diagnoses: Normal Response . 60min. body wt. (ready to use) *Samples: Blood samples .. 60 min. 90 min. (in extended OGTT another 2 samples will be taken at 2½hour and 3 hours). 90min. 2 ½ hour Calculation of Results Wilkerson Criteria > 130 1 point >190  ½ point >140  ½ point >130  1 point 2 – 3 point Diabetic ½ . after oral glucose load..

A person is said to have a normal response when the 2-hour glucose level is less than or equal to 110 mg/dl. they are said to have impaired fasting glucose. There has recently been discussion about lowering the upper value to 180 mg/dl to diagnose more mild diabetes to allow earlier intervention and hopefully prevention of diabetic complications. This is also considered a risk factor for future diabetes. This must be confirmed by a second test (any of the three) on another day. Diabetes A person has diabetes when oral glucose tolerance tests show that the blood glucose level at 2 hours is equal to or more than 200 mg/dl. There has recently been discussion about lowering the upper value to 180 mg/dl to diagnose more people with mild diabetes to allow earlier intervention and hopefully prevention of diabetic complications. and will likely trigger another Impaired Glucose Tolerance A person is said to have impaired glucose tolerance when the 2-hour glucose results from the oral glucose tolerance test are greater than or equal to 140 but less than 200 mg/dl. This is considered a risk factor for future diabetes. or following this normal levels. 60 & 90 minutes 120 minutes Pregnancy <100 <200 <145 Other Adults <110 <200 <140 Child <130 <200 <140 Impaired Fasting Glucose When a person has a fasting glucose equal to or greater than 110 and less than 126 mg/dl. Time Fasting 30. Gestational Diabetes A woman has gestational diabetes when she is pregnant and has any two of the following: a fasting .

.plasma glucose of more than 105 mg/dl. a 2-hour glucose level of more than 165 mg/dl. a 1-hour glucose level of more than 190 mg/dl. or a 3-hour glucose level of more than 145 mg/dl.

Arginine. Diazoxide. Phenolphthalein. Lithium. Beta-adrenergic blockers. Corticosteroids. Insulin.Discussion: Drugs may affect OGTT results Amphetamines. Clofibrate. Epinephrine. Furosemide. MAO inhibitors. . Chlorthalidone. Dextrothyroxine. Nicotinic acid (large doses). Glucose I. Oral hypoglycemics. Benzodiazepines. Oral contraceptives (estrogen-progestogen combination).V.

Cushing's disease.Phenothiazines. Phenytoin. hypothyroidism. Caffeine. QUESTIONS: a. Draw a graph of a normal glucose tolerance. Triamterene. hypopituitarism. malabsorption. Fever. Pregnancy. Recent infection. Addison's disease. Reduced carbohydrate intake for several days before the test. What are the prerequisites of OGTT ? c. Thiazide diuretics. hemochromo-cytosis. which may cause conflicting results. What are the indications of OGTT ? b. Acute illness. Failure to follow dietary and exercise restrictions. injury to central nervous system. Other factors that may affect test results Ethanol. . People over age 50 tend toward decreasing carbohydrate tolerance. tumor of pancreas islet cells. pheochromocytoma.

glycosylated hemoglobin) is a generic term for hemoglobin bound irreversibly (ketoamine form) to glucose.chain) Using GHb : Monitoring blood glucose is a key component of successful diabetes management. glycated hemoglobin. Total glycated hemoglobin is usually determined by affinity chromatography or immunassays. With the availability of self-monitoring and HbA1C testing. however. Total glycated hemoglobin (Total GHb) refers to all the glycated hemoglobins. Types of Glycated hemoglobins: HbA1a1 = Hb + Fructose-1. Laboratory measurement of glucose. Objectives: It is to know the importance of glycated hemoglobin as a long term monitoring test which may be used to help controlling the treatment of diabetes mellitus.a. . Often. Hemoglobin A1c (HbA1c) is the major subfraction of the glycated normal hemoglobin (HbA1). of α/β. may be useful to verify the accuracy of home glucose monitoring equipment or when there has been a loss of diabetic control. including glycated hemoglobin variants. Determination of HbA1c is usually achieved by ionexchange HPLC or gel electrophoresis.Glycated Hemoglobins Introduction: Glycohemoglobin (GHb. laboratory testing for fasting glucose and 2-hour post-75g glucose load should no longer be used routinely to assess glucose control.6-bisphosphate (FBP) HbA1a2 = Hb + Glucose-6-phosphate HbA1b HbA1c HbA1d = Hb + Pyruvic acid = Hb + Glucose (N-terminal of β-chain) = Hb + Glucose ( internal a. and sometimes to mean hemoglobin A1c. the term is used to mean total glycated hemoglobin.

4. while the other hemoglobins separate (elute) after by sodium chloride solution. glucose levels. Experiment # 3: Estimation of HbA1c by using affinity chromatography column Principle: In a chromatography column. Blood Glucose level reflects the previous few hours glycemic state. glycated Albumin reflects 10 – 14 days glycemic state. thereby complementing day-to-day monitoring. the hemoglobins in a hemolysed sample is bound by different affinity to dihydroxyborate.B.75 X A2) Reference ranges: Degree of glucose control Normal (non-diabetic) Total GHb < 7% Hb A1c < 6% . N. a fraction of hemoglobin will become covalently bound to glucose and other sugar molecules. Gel electrophoresis.separates total glycated hemoglobins by binding to solid-phase dihydroxyborate. Over the life of a red blood cell (which averages 120 days).based on binding to specific antibodies. Elution of HbA1c is carried out by phosphate buffer. Affinity chromatography . HbA1C is the largest single component of these glycated hemoglobins. Cation-exchange chromatography . while HbA1c reflects the longest (2-3 months) glycemic state.c. HbA1C levels are a better (and less expensive) measure of long-term glucose control than repeated fasting and p.HbA1C measurement provides a quantitative and reliable measure of glycemic status and control over an extended period of time. Methods: There are currently four main techniques for determining glycated hemoglobins: 1. 3. 2. Immunoassay . This reaction occurs non-enzymatically and at a rate which is proportional to the concentration of glucose in the blood. Procedure: Calculation: % HbA1c = A1 X 100 /A1 + ( 4.separates hemoglobins using HPLC based on net charge as a result of glycation.

MBG mg/dl = (33.5%).3 X HbA1c) . Various formulae have been proposed to demonstrate the correlation between the mean blood glucose (MBG) and Hemoglobin A1c (HbA1c). or it may be used to indicate the degree of longterm diabetic control in diabetic patients. In contrast.7 times the upper limit of normal (13.86 Or. Annual HbA1c > 1. ↑ HbF ↑ Triglycerides Lead toxicity ↓ iron anemia Splenectomy CRF ± Hemodialysis Causes of decreased HbA1c: . Correlation with Mean Blood Glucose Levels A single fasting blood glucose measurement only gives an indication of the patient's immediate past (last 1 to 2 hours) condition.M. suggesting more likely occurring complications. suggesting less likely occurring complications. Annual HbA1c < 1.8%). The significance of a low glycated hemoglobin level has not been established.1 times the upper limit of normal (8. MBG mg/dl = 10 ( HbA1c +4 ) Discussion: Causes of elevated HbA1c: Uncontrolled D.Near normoglycemic Diabetes Control and Compliance Trial (DCCT) therapeutic goal In good control Actions suggested Not in control 7 to 8% 6 to 7% Less than 7% 8 to 9% 9 to 11% > 11 % 7 to 8% 8 to 9% > 9% The determination of a glycated hemoglobin level may assist in the initial diagnosis of diabetes. the level of glycated hemoglobin is directly related to the average glucose concentration over the life-span of the hemoglobin in the circulation. and may not represent the true status of blood glucose regulation.

pregnancy) Questions: Give the objectives of glycated hemoglobin estimation. Write down different methods for the determination of glycated hemoglobin.- Causes of ↓ RBCs life span ( hemolytic or hemorrhagic) Hemodilution (e.g. What is the principle for the determination of glycated hemoglobin by chromatography ? .

Experiment # 4:Glycerol-Phosphate Oxidase method for Triglycerides PRINCIPLE OF THE METHOD Sample triglycerides incubated with lipoproteinlipase (LPL). In the last reaction. particularly determinations of HDL.The available diagnostic studies and their use. and how often . as well as the need to test for other cardiovascular risk factors .The contribution of hypercholesterolemia to coronary heart disease (CHD) risk. Preparation for having blood collected for lipid testing should include a 12-14 hour overnight fast.The classification of dyslipidemias.Lipid Profile Introduction: Some beneficial aspects of lipids include the following: energy course. Glycerol-3-phosphate (G3P) is then converted by glycerol phosphate dehydrogenase (GPO) to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2). HDL cholesterol. hydrogen peroxide (H2O2) reacts with 4-aminophenazone (4-AP) and p-chlorophenol in presence of peroxidase (POD) to give a red colored dye: Principle: Triglycerides + H2O lipase Glycerol + FFA . liberate glycerol and free fatty acids. ratio of total to HDL cholesterol. including the importance of elevations in total cholesterol. Objectives: . function and structural components of cell membranes. With evidence of a link between elevated lipids and atherosclerosis (also known as arteriosclerosis or atherothrombosis). . and precursor compound to many important substances such as vitamin D and steroid (sex) hormones. Glycerol is converted to glycerol-3-phosphate (G3P) and adenosine-5-diphosphate (ADP) by glycerol kinase and ATP. there is increase interest from both the medical and lay community in the battery of tests commonly ordered as a lipid profile. including who to screen. LDL cholesterol. LDL and total cholesterol.

0 Blan k 1.Glycerol + ATP ADP glycerol kinase Glycerol-3-phosphate + Glycerol-Phosphate Oxidase Glycerol-3-phosphate + O2 DHAP + H2O2 peroxidase H2O2 + 4. . . PREPARATION Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes into one bottle of R 1 Buffer. . 3. 37ºC / 15-25ºC 2. CLINICAL SIGNIFICANCE Triglycerides are fats that provide energy for the cell. 1 cm light path Temperature . . For example liver dysfunction resulting from hepatitis. . . extra hepatic biliary obstruction or cirrhosis. . . . . . . . . . it should integrate clinical and other laboratory data. . Like cholesterol. A diet with a lot of saturated fats or carbohydrates will raise the triglyceride levels. . WR stability: 6 weeks at 2-8ºC or 1 week at room temperature (15-25ºC). . . . . . Stability of the sample: 5 days at 2-8ºC . . . . . 505 nm (490-550) Cuvette: . . . . Adjust the instrument to zero with distilled water. PROCEDURE 1. . . . . . . . . Assay conditions: Wavelength: . . . . diabetes mellitus is associated with the increase Clinical diagnosis should not be made on a single test result. . . . .Aminoantipyrine + Chlorophenol Quinoneimine The intensity of the color formed is proportional to the triglycerides concentration in the sample. . . . . . . . SAMPLES Serum or heparinized or EDTA plasma1.. Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in 10 mL of R 1 Buffer. Cap and mix gently to dissolve contents. . . they are delivered to the body’s cells by lipoproteins in the blood. Pipette into a cuvette: Stand ard 1. . .0 (WR (mL . . . . . The increases in serum triglycerides are relatively non-specific.

10 -- --- Standard ((µL (Sample (µL 4. Read the absorbance (A) of the samples and Standard. at 37ºC or 10 min. Mix and incubate for 5 min. against the Blank. Give the different methods for the determination of triglycerides. c. CALCULATIONS A Sample x 200 (Standard conc. Write a short note on hypertriglyceridemia. Give the upper cut off value of triglyceride for a diagnosis of hypertriglyceridemia.0113= mmol/L. at room temperature. 5. b.) = mg/dL triglycerides in the sample A Standard Conversion factor: mg/dL x 0. . Reference ranges: Normal Fasting blood triglycerides = 60 – 160 mg/dl It is considered normal as long as it is below 200 mg/dl Discussion: -Types of hyperlipidaemias QUESTIONS: a. The colour is stable for at least 30 minutes.

according to the following reaction: The intensity of the color formed is proportional to the cholesterol concentration in the sample Principle (Experiment #5): Cholesterol esters + H2O + FA Cholesterol + O2 + H2O2 H2O2 + 4-AAP + Phenol Cholesterol esterase Cholesterol Cholesterol Oxidase Cholesterol-3-one Quinonimine Peroxidase CLINICAL SIGNIFICANCE Cholesterol is a fat-like substance that is found in all body cells. Cholesterol lowering medications prescribed by physicians inhibit the synthesis of cholesterol by the liver. . it should integrate clinical and other laboratory data. The determination of serum cholesterol is one of the important tools in the diagnosis an classification of lipemia. The largest percentage of synthesized cholesterol is made in the liver. The recommended daily allowance for dietary cholesterol intake is 300 milligrams. PREPARATION Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer. Clinical diagnosis should not be made on a single test result. Cap and mix gently to dissolve contents. thereby reducing the level in the blood stream. High blood cholesterol is one of the major risk factors for heart disease5. The liver makes all of the cholesterol the body needs to form cell membranes and to make certain hormones. OBJECTIVES: The estimation of cholesterol along with other parameters of lipid profile is necessary for the classification and diagnosis of lipemias PRINCIPLE OF THE METHOD The cholesterol present in the sample originates a coloured complex.6.DETERMINATION OF CHOLESTEROL: INTRODUCTION: Cholesterol is a waxy substances used in every cell membrane you have and as a base for several hormones. Most cells have some capacity to synthesize cholesterol.

. . . . against the Blank. . . CALCULATIONS A (Sample) x 200 (Standard conc.2: Stability of the sample for 7 days at 2-8ºC or freezing at –20ºC will keep samples stable for a few months.. . . .0 -10 Stand ard 1. .(WR) is stable: 4 months at 2-8ºC or 40 days at 15-25ºC. . 505 nm (500-550) Cuvette: . . . .. Assay conditions: Wavelength: . . . . . 25. . . . . . 13. Read the absorbance (A) of the samples and Standard.0 10 -- Blank 1. . . . . . . at 37ºC or 10 min. .) = mg/dL cholesterol in the sample A (Standard) Conversion factor: mg/dL x 0. . Adjust the instrument to zero with distilled water. The colour is stable for at least 60 minutes. PROCEDURE 1. .0 --(WR (mL Standard ((µL Sample ((µL 14. . . . . . . SAMPLES Serum or plasma1. . . . *Normal range: Desirable blood cholesterol level < 200 mg/dl Suspect to vascular and CHD 200 – 240 mg/dl High risk group for CHD > 240 mg/dl Risk evaluation: Normal Less than 200 mg/dL Borderline mg/dL 200-239 High mg/dL and above 240 . 1 cm light path Temperature . .0258= mmol/L. . . . . . . . . at room temperature. Mix and incubate for 5 min. . ..37ºC /15-25ºC 2. Avoid direct sunlight. . Pipette into a cuvette: Sam ple 1. .

The assay takes place in two steps. .LDL –Cholesterol PRINCIPLE OF THE METHOD Direct determination of serum LDLc (low-density lipoprotein cholesterol) levels without the need for any pre-treatment or centrifugation steps. 1º Elimination of lipoprotein no-LDL Cholesterol esters + H2O Cholesterol + O2 + H2O2 H2O2 catalase Cholesterol esterase Cholesterol + FA Cholesterol-3-one Cholesterol Oxidase 2 H2O + O2 2º Measurement of LDLc Cholesterol esters + H2O Cholesterol + FA Cholesterol + O2 + H2O2 H2O2 + 4-AAP + Phenol H2O2 Cholesterol esterase Cholesterol Oxidase Cholesterol-3-one Quinonimine + 4 Peroxidase The intensity of the color formed is proportional to the LDLc concentration in the sample.

7. . . . . PREPARATION . . . . . HDLc/LDLc CAL: Dissolve the contents with 1 mL of distilled water. . . . . light path Temperature . . .conc. . . . Clinical diagnosis should not be made on a single test result.R 1 and R 2: Are ready to use. . . against the Blank. . at 37ºC. . .5. . . . Adjust the instrument to zero with distilled water. . .CLINICAL SIGNIFICANCE The LDLc particle is lipoproteins that transport cholesterol to the cells. PROCEDURE . . diabetes and nephrosis 1. .6. . = mg/dL of LDLc in the sample . . . . Mix and incubate for 5 min. . Assay conditions: Wavelength: . . SAMPLES Serum : After sampling. Read the absorbance (A). 3.. . Stability of the sample: 7 days at 2-8ºC . . . . . . 5. Cap vial and mix gently to dissolve contents. Repeated freezing and thawing should be avoided.. at 37ºC. . . Add: R2 (µL) 100 100 100 6. it should integrate clinical and other laboratory data. . . . . . . . CALCULATIONS (A) Sample x Standard. . . . Often called “bad cholesterol” because high levels are risk factor for coronary heart disease and are associated with obesity. . . . . . .1 cm. . Pipette into a cuvette: R1(µL) Standard (µL) Sample (µL) Bla nk 300 ----------- Standa rd 300 4 -------- Sampl e 300 -----4 4. 600 (590-700) nm Cuvette: . .37ºC 2. . . the test should be performed without delay. Mix and incubate for 5 min. .

A low HDL cholesterol levels. REFERENCE VALUES Levels of the risk Desirable < 100 mg/dL Medium 130-160 mg/dL High > 160 mg/dL HDL cholesterol PRINCIPLE OF THE METHOD The very low density (VLDL) and low density (LDL) lipoproteins from serum or plasma are precipitated by phosphotungstate in the presence of magnesium ions. HDL is known as “good cholesterol” because high levels are thought to lower the risk of heart disease.02586 .(A) Standard Conversion factor: mg/dL x 0. is considered a greater . After removed by centrifugation the clear supernatant containing high density lipoproteins (HDL) is used for the determination of HDL cholesterol CLINICAL SIGNIFICANCE HDL particles carry cholesterol from the cells back to the liver.

m.heart disease risk. Pipette into a centrifuge tube: 12. allow to stand for 10 min at room temperature. for 20 min or 2 min at 12000 r. Procedure : PTA + B. 34. A546 nm Sample x 475 = mg/Dl HDLc in the sample 10 0 1. sample min. Stability : HDL Cholesterol is stable for 7 days at 2-8ºC .p. Clinical diagnosis should not be made on a single test result. it should integrate clinical and other laboratory data.Oxidase reagent - HDL.m. SAMPLES Serum or plasma1: Free of hemolysis. Removed from the blood clot as soon as possible. 0 (R (µL Sample ((mL . at 4000 rpm RT incubation for 10 min Centrifugation for 10 Supernatant cholesterol + Chol. PROCEDURE Precipitation 11.Chol. Collect the supernatant and test HDLc. CALCULATIONS .With Factor: A505 nm Sample x 320 = mg/Dl HDLc in the sample.. Conc. Mix well. Test Following the Cholesterol reagent instructions. 23. Centrifuge at 4000 r.p.

c. Total Chol. Laboratory Renal Function Tests I. b.Triglycerides -HDL cholesterol 5 Questions: a. Which one of the two – HDL/LDL is more dangerous to health and give reason.Urea Estimation & Blood Urea Nitrogen (BUN) Introduction: .& LDL. Which diet can cause increase in HDL-Chol ? Write the equation for HDL-cholesterol calculated from TG. Write a short note on Hyperlipidemia.Calculation of LDL-cholesterol According to the Friedewald Formula: LDL cholesterol = Total cholesterol .

Essentially all seriously sick patients will need their kidney function evaluated during the course of their illnesses. you may check (4) ability to concentrate urine. the initial steps in checking patients' kidneys are performing the following tests: (1) urinalysis (2) serum creatinine (3) serum urea ("blood urea nitrogen".Kidney problems are very common in clinical medicine. After history and physical exam are complete. Next. "BUN"). or dipstick. Objectives: Methods: 1. You can check the ability to concentrate urine using a hygrometer. refractrometer. Both creatinine and BUN are included on the common chemical profiles.Chemical (direct) method: Urea + Diacetyl monoxime(DAM) Diacetyl-Urea + Fe3+ +acidic pH (measured at 540) 2-Enzymatic (indirect) method: Urea + H2O Yellow Orange Urease Diacetyl-Urea Yellow Diazine CO2+ NH3 (at 540 nm) ( HgI2 +KI) NH3 pH indicator dye (dry chemistry) Berthlot’s Kinetic Multienzymatic method Conductivity difference between non-ionized urea and ionized ammonia Ammonia detecting electrode .

. . 13. . ..R 2 NaClO is ready to use : SAMPLES 1. Cap and mix gently to dissolve contents. . Stability: 4 weeks in the refrigerator (2-8ºC) or 7 days at room temperature (15-25ºC). heart failure. Elevated urea can appear in blood (uremia) in: diets with excess of proteins. . . .Urine: Dilute sample 1/50 in distilled water. . . . . . . . renal diseases. it should integrate clinical and other laboratory data. . Principle: Urea + H2O Urease CO2+ NH3 NH3 + Na-salicylate + Na-hypochlorite +Na-nitoprusside Indophenol PREPARATION . . . .Serum or heparinized plasma: Do not use ammonium salts or fluoride as anticoagulants. . . . Adjust the instrument to zero with distilled water. . . . . . PROCEDURE 11. Mix. Pipette into a cuvette: . .Experiment # 6 (Modified Berthlot’s Reaction): PRINCIPLE OF THE METHOD Urea in the sample is hydrolized enzymatically into ammonia (NH4+) and carbon dioxide (CO2). . . . Clinical diagnosis should not be made on a single test result. Preserve urine samples at pH < 4. . . . 2. . . . 1 cm light path Temperature. 580 nm Cuvette: . dehydration or renal obstruction1. . . . . Urea is stable at 2-8ºC for 5 days.. . Ammonia ions formed reacts with salicylate and hypochlorite (NaClO). . . . . Multiply results by 50 (dilution factor).7. . Assay conditions: Wavelength: . gastrointestinal hemorrhage. 37ºC / 15-25ºC 12. . . . . to form a green indophenol: The intensity of the color formed is proportional to the urea concentration in the sample CLINICAL SIGNIFICANCE Urea is the final result of the metabolism of proteins. .6. . . . .Working reagent (WR): Dissolve one tablet R 3 Enzymes in one bottle of R 1 Buffer. in presence of the catalyst nitroprusside. . . . ... . . it is formed in the liver from its destruction.

CALCULATIONS (A) Sample x 50 (Standard conc. high fever.1665 = mmol/L.466 = 21 mg/L urea = 0.49 mmol/L) Urine : 20 . by definition. The colour is stable for at least 30 minutes at 15-25ºC. Mix and incubate 5 min at 37ºC or 10 min at room temperature (15-25ºC). azotemia. 25. *Increased protein catabolism results from:    a really heavy protein meal (Kebda. against the Blank. though there are a few things to remember.) severe stress (myocardial infarction. Read the absorbance (A) of the samples and calibrator.36 mmol/L urea.45 mg/dL (2.0 -- -- (WR (mL Standard ((µL Sample ((µL 14.49-7.35 gr/24 h.) = mg/dL urea in the sample (A) Standard 10 mg/L urea BUN divided by 0.) upper GI bleeding (blood being digested and absorbed) . Mix and incubate 5 min at 37ºC or 10 min at room temperature (1525ºC). REFERENCE VALUES1 Serum : 15. It is due either to increased protein catabolism or impaired kidney function. #Increased BUN is. Conversion factor: mg/dL x 0. etc. etc. El-Bek. Pipette: 0 R2 ((mL 16. 27. Blood Urea Nitrogen (BUN) 8 – 25 mg/dl Interpretation: Interpretation of the BUN is usually straightforward.Bla nk 1.

   Prerenal azotemia results from underperfusion of the kidney: dehydration. some patients with nephrotic syndrome) Severe liver disease (end-stage cirrhosis. #Decreased BUN    Lack of protein (celiac disease. chronic interstitial nephritis. . really bad hepatitis. stones. Mention causes of hyperazotemia.B. In acute renal failure. yellow atrophy. congestive heart failure Renal azotemia has several familiar causes: acute tubular necrosis. "renal". BUN increases around 20 mg/dL each day (*estimates vary. tumors N. etc. halothane or acetaminophen toxicity. glomerulonephritis. or "postrenal".*Impaired kidney function may be "prerenal". psychogenic water-drinking) Discussion: -Physiological & Biochemical Background: -Pathological & Disease Correlation: Questions: Write the different methods for estimation of blood urea? Calculate BUN on estimation of blood urea. shock. Postrenal azotemia results from obstruction of urinary flow: prostate trouble. hemorrhage. surgical mishaps. range of increase is 10-50 mg/dL daily). enzyme defects) Overhydration (iatrogenic.

II. Methods: 1.Plasma Creatinine Estimation Introduction: Creatinine is the end product of muscle metabolism.Direct Chemical methods: a) Jaffe’ method : See the principle and procedure (Experiment ) b) DNB method (used in dry chemistry: Creatinine +Dinitrobenzoic acid +alkaline pH rose 2.Pyruvate + NADH+H+ nm) LDH Creatinine.ADP + P-enol pyrovate (PEP) +Pyruvate . Amidohydrolase Creatine Creatine kinase Creatine -p Creatine +ADP ATP Lactate + NAD (with diminished absorbance at 340 . this test can be performed on specimens drawn from patients in either the fasting or non fasting state. It is excreted through the kidneys and changes in creatinine are an early indicator of kidney disease as well as being seen in severe muscle damage or wasting diseases or with many medications such as antibiotics.Creatine .Indirect Enzymatic methods: a) Deaminase method (One enzyme step method): Creatinine techniques) Deaminase purplish methyl hydantoin + NH3 (detected by different b) Amidohydrolase method ( multi-enzymatic method): .Creatinine .Creatine-p +ATP .

4. Multiply results by 50 (dilution factor). The time interval chosen for measurements avoids interferences from other serum constituents. The creatinine production depends on the modification of the muscular mass.Presence of particles and turbidity. Creatinine stability: 24 hours at 2-8ºC. The intensity of the color formed is proportional to the creatinine concentration in the sample. Assay conditions: . it should integrate clinical and other laboratory data. Clinical diagnosis should not be made on a single test result. The working reagent is stable for 10 days at 15-25ºC. Mix.6 trinitrophenol (Janovski’s measured at CLINICAL SIGNIFICANCE Creatinine is the result of the degradation of the creatine. PREPARATION Working reagent (WR): Mix equal volumes of R 1 Picric Reagent and R 2 Alkaline reagent. that is a source of high energy for the cells. 1. Principle (Jaffe’ Method): Creatinine + Picric acid + alkaline pH complex) 520 nm 2.Serum or heparinized plasma.Blank absorbance (A) at 492 nm 1.5. Elevate creatinine level may be indicative of renal insufficiency1.4. it can be transformed into ATP. PROCEDURE 11. 2. With progressive renal insufficiency there is retention in blood of urea. Signs of reagent deterioration: 1. creatinine and uric acid.80. Creatinine reacts with alkaline picrate forming a red complex. Creatinine stability: 7 days at 2-8ºC.Urine: Dilute sample 1/50 with distilled water. Is excreted by the kidneys. component of muscles. and it varies little and the levels usually are very stable. SAMPLES 1.PRINCIPLE OF THE METHOD The assay is based on the reaction of creatinine with sodium picrate as described by Jaffé.

.1 . 36. . . . . . . .7)µ mol/L 1.6 5-25 mg/Kg/24 h Interpretation: Causes of renal failure Discussion: Physiological & Biochemical Background: Other Renal Function Tests: . . . . 492 nm (490-510) Cuvette: . .4 = µmol/L. . .7 = (53. . .8 – 123. 37ºC / 15-25ºC 12. 1 cm. Pipette into a cuvette: d Bla nk 1. . .Wavelength: . .0 --- (WR (mL Standard ((µL Sample ((µL 1 4. . . .0. . . . . .2 ) µmol/L 1. . .) = mg/dL of creatinine in Conversion factor: mg/dL x 88. . . . . . . 23. . . . . . . . . .4 . . CALCULATIONS ΔA Sample –ΔA Blank the sample ΔA Standard – ΔABlank x 2 (Standard conc. . . Mix and start stopwatch. Read the absorbance (A1) after 30 seconds and after 90 seconds (A2) of the sample addition. . l REFERENCE VALUES Male Femal e 10 8 Male Femal e or plasma = (61. . . . . . Adjust the instrument to zero with distilled water. . . . light path Temperature. .0. 25. . .0 – 97. Calculate: A= A2 – A1.

but when GFR drops below 30 mL/min. Clearance = (conc. because some is not filtered and some is secreted into the proximal tubule. The clearance of a substance is the volume of plasma "cleared" of that substance per unit time. monitoring body position (kept them lying down) and hydration with body surface area measurement. Creatinine clearance is not a perfect measure of GFR. remember that you don't really need to collect urine for a full 24 hours. These fractions tend to cancel each other out in health. tubular secretion approaches or even exceeds the amount filtered at the glomerulus. You need a timed urine sample and a blood sample. in urine) x (urine volume) (conc. .Tutorial Creatinine clearance : is widely used to approximate glomerular filtration. In deciding how to "time" your collection. lots and lots of red meat (protein and creatinine-rich) can lead to overestimates (maybe 30%) in GFR in renal failure patients.). One group got more reliable results by a controlled collection over 4 hours. in plasma) X time of urine collection (min. *Also.

it is also synthesized in the body from simple compounds as shown in figure. *Formulas to adjust "normal" for body surface area have been devised.7/ body surface area)} *Whether or not "corrections" are applied. For kids. worse for hypertensives .Cl.6. X (1. DETERMINATION OF URIC ACID INTRODUCTION: Uric acid is apurine compound that circulates in plasma as sodium urate and is excreted by kidney. OBJECTIVES: • • METHODS: To know the uric acid level in the body To diagnose a case of Hyperuricemia (Gouts) . values tend to fall by around 0.1 or less is normal.20. Another formula to correct the clearance according to body surface area is {Corrected Cr. GFR for adults can be estimated by various formulas.Reference range for creatinine clearance is 90-120 mL/min for young adults. It is derived from the break down of nucleic acids that are ingested or come from the destruction of tissue cells.5 mL/year over age 20.12 x Creatinine Clearence . when tubular secretion of creatinine become proportionately greater. try 1. a height/creatinine ratio of 2. = Cr.Cl. creatinine clearance is a pretty good estimate of glomerular filtration rate except at very low values. etc.

. . . . . Pipette into a cuvette: Blank WR (ml) 1. . . it should integrate clinical and other laboratory data. . . 1 cm light path Temperature . . . With progressive renal insufficiency. 37ºC / 15-25ºC 2. Elevate uric acid level may be indicative of renal insufficiency and is commonly associated with gout Clinical diagnosis should not be made on a single test result. . . . . . which under the influence of POD.. .0 Standard ------(µL) Sample (µL) ------Standa rd 1. If urine is cloudy. SAMPLES Serum or plasma: Stability 3-5 days at 2-8ºC or 6 months at –20ºC. . PROCEDURE 1. . . . Assay conditions: Wavelength: . . . 4–aminophenazone (4-AP) and 2-4 Dichlorophenol sulfonate (DCPS) forms a red quinoneimine compound: Uric acid + 2H2O + O2 Uricase Allantoine + CO2 + 2H2O2 2H2O2 + 4-AP + DCPS POD Quinoneimine+ 4H2O The intensity of the red color formed is proportional to the uric acid concentration in the sample. . . . . . . . . . . . . . there is retention in blood of urea. (WR) is stable after reconstitution 1 month at 2-8ºC or 10 days at room temperature.0 ------25 . Multiply results by 50 (dilution factor). Adjust the instrument to zero with distilled water. Dilute sample 1/50 in distilled water. .• • Chemical Method (Phosphotungestic acid Method?) Enzymatic Method PRINCIPLE {Enzymatic Colorimetric (Uricase Method)}: Uric acid is oxidized by uricase to allantoine and hydrogen peroxide (2H2O2). . . . . CLINICAL SIGNIFICANCE Uric acid and its salts are end products of the purine metabolism. . 3. Do not refrigerate. .0 25 --------Sam ple 1. . . . . . . . warm the specimen to 60ºC for 10 min to dissolve precipitated urates and uric acid. . . . . . . . . Mix.Urine (24 h)1: Stability 4 days at 15-25ºC. Cap and mix gently to dissolve contents.520 nm (490-550) Cuvette: . . . . pH >8. PREPARATION Working reagent (WR): Dissolve the contents of one vial R 2 Enzymes in one bottle R 1 Buffer. . . . . creatinine and uric acid.

3. 5.6.5 mmol/24 h CLINICAL SIGNIFICANCE: Hyperuricemia (Gout) DISCUSSION: Causes of elevated uric acidemia QUESTIONS: 1.7. . Write a short note on Uric acid metabolism.6 .) Either could be the cause when a patient has been hypotensive and now is azotemic and oliguric.49 . REFERENCE VALUES: Serum or plasma: Women 2.also "hepatorenal syndrome") from renal azotemia (acute tubular necrosis.4. 2. The colour is stable for at least 30 minutes.7 mg/dL =214 – 458 µ mol/L Urine: 250 . Explain the hyperuricemia. PRERENAL VS. CALCULATIONS Serum or plasma (A) Samplex 6 (Standard conc.8 mg/dL = 149 – 405 µ mol/L Men 3.4. RENAL AZOTEMIA : A very common clinical problem is to distinguish prerenal azotemia (due to shock. CHF -.750 mg/24 h = 1. dehydration. The management is different. "renal shutdown". Mix and incubate for 5 min at 37ºC or 10 min at 15-25ºC. (dL) urine 24 h =mg/24 h uric acid Standard Conversion factor: mg/dL x 59.Give the principle for the determination of serum uric acid by uricase method. against the Blank.5=µ mol/L.5 .)= mg/dL uric acid in the sample (A) Standard Urine 24 h A) Samplex x 6 x vol. Read the absorbance (A) of the samples and Standard.

*A further refinement. currently popular. is to measure the fractional excretion of filtered sodium. Urine Protein/creatinine ratio . urine sodium is higher (the renal tubules are unable to concentrate or dilute the glomerular filtrate effectively. and a marker for ill-health . etc. sodium excretion is low (the tubules are plugged.") In acute tubular necrosis. In prerenal azotemia. Another approach is to measure sodium on a urine specimen. This is normally between 10 and 20. suggest prerenal azotemia rather than acute tubular necrosis. Values over 20. Several other factors can complicate the picture in such patients. while secondary hyperaldosteronism (as in cirrhosis) will decrease sodium excretion. your estimate of urine creatinine will be low because these things absorb creatinine . especially in the elderly.) *Tip: If you obtain urine by squeezing a diaper or the absorptive balls you placed into the diaper. not damaged. a high BUN/creatinine ratio is a common finding. values over 2% indicate acute tubular necrosis.) Urinary sodium under 20 mEq/L suggests prerenal azotemia (or hepatorenal syndrome. High values are also seen postrenal azotemia and upper GI bleeding. In acute tubular necrosis due to myoglobinuria. In fact. Diuretics will increase the excretion of filtered sodium.One older technique is to calculate the BUN/creatinine ratio. approximated by: Values less than 1% indicate prerenal azotemia. urinary sodium over 40 mEq/L suggests acute tubular necrosis.). urine sodium is low (the kidney responds to low blood flow by "trying to retain all the sodium it can.

Amylase and lipase Elevated plasma lipase and amylase levels can be observed in dogs with renal disease. shorter the life span of erythrocytes and bone marrow depression.Other causes of anemia in renal disease include haemorrhage. UP/UCr is greater than 1. Generally with proteinuria.Urine protein/creatinine ratio (UP/UCr) is used to calculate urine protein loss into the urine without a need for 24 hour urine collection.urinary protein value. Blood pH Changes in acid-base balance is observed frequently in renal failure especially when advanced. because these two enzymes are eliminated by the kidneys. 3. • LESS FAMILIAR RENAL FUNCTION TESTS 1. NAG) is a lysosomal enzyme (MW 140. such as nephrotic syndrome. Compared to conventional 24 hour. 6. 4. non-regenerative anemia is commonly observed. UP/UCr is less time consuming and less accurate. Lipids Hyperlipidaemia can occur with renal disease. Increased hepatic lipoprotein synthesis and hypoalbuminaemia is proposed in the pathogenesis.000) found in serum and . N-acetyl-beta-D-glucosaminidase ("glucosaminidase". It is mainly due to a reduced erythropoietin level secondary to the loss of renal parenchyma. Plasma protein Generally the concentration of plasma protein is elevated due to dehydration but can be reduced in primary glomerular diseases such as glomerulonephritis and renal amyloidosis. Total Red blood cell In chronic renal disease. 2. 5.0.

7. Isotope scans exist to compare the function of the kidneys. Urine beta-2-m has found widespread acceptance as an research tool. Serum beta-2-m has been suggested as a measure of glomerular filtration rate. Positron emission tomography is the latest way of measuring renal blood flow. Urinary NAG is a proposed marker for tubular disease. and fully reabsorbed by the proximal tubule. 9. It is very sensitive as an indicator of the latter. Lithium clearance is a researcher's way of estimating delivery to the distal tubule. especially subtle industrial poisoning. 11. which is cheap and portable. Most recent of all. It appears if levels in the serum and glomerular filtrate exceed what the proximal tubule can reabsorb (more than 4. etc. similar to creatinine. More recently. Tubular functions: Urinary amino acids and maximum concentrating ability are sensitive screens for tubular damage.5 mg/L) or if there is renal tubular disease. there's a Tc99 scanner that monitors glomerular filtration minute by minute. Adenosine Deaminase Binding Protein is an enzyme from the brush borders of the proximal tubule. These may prove a valuable supplement to the intravenous pyelogram. Like NAG. and early transplant rejection. Alkaline phosphatase in urine comes from the proximal tubular brush border . 13. 10.urine. its presence in urine indicates tubular disease. (These functions are now largely taken over by beta-2 microglobulin). has proved even more useful than these scans in transplant patients. acute pyelonephritis. SPECIFIC GRAVITY OF URINE . it is freely filtered by the glomerulus. Beta-2 microglobulin (beta-2-m) is the short chain of the HLA class I proteins. In health. 8. suitable for the intensive care . the 12. lymphomas. Obviously this isn't a good idea for patients with tissue necrosis. early acute tubular necrosis. color Doppler sonogram.

or with diabetes insipidus. without regard to the size or weight of the particles. Patients with tubular disease ("renal azotemia". Substances such as glucose. Of course. as there is less water in proportion to solutes in the serum or blood. With a standard measurement of osmoles and of milliosmoles for clinical studies. In severe dehydration serum osmolality will be increased. or enthusiastic water-drinkers (asthmatics. or on diuretics. or dyes increase the urinary specific gravity. the same is true of patients in prerenal azotemia (high urinary specific gravity. is a measurement of the concentration of urine. The osmolality of serum. Serum and Urine Osmolality The term osmolality refers to the osmotic concentration of a fluid. Therefore.010). really bad bilateral pyelonephritis or interstitia nephritis. Sodium. acute tubular necrosis.295 mOsm/kg water. a serum osmolality of 285 mOsm correlates with a urine specific gravity of 1. or with end-stage kidney) will have isosthenuria. Urine osmolality. Urine osmolality reflects the total number of osmotically active particles in the urine. i. Thus "normal" people have fairly concentrated urine (SG greater than 1. proteins. Reference values for osmolality: • Serum osmolality: 282 . like specific gravity. urine osmolality is a more accurate measurement of urine concentration than specific gravity. Patients getting lots of fluid by IV. low or zero urinary sodium). Tests of both serum and urine osmolality can yield important information about a patient's ability to maintain a normal fluid balance status. crazies) will have low urine specific gravity. checking urine specific gravity provides very important information about tubular function and hydration. In laboratory reports. and blood glucose levels are major factors in determining serum osmolality.While not a "blood test".010 . blood urea nitrogen. and urine osmolality can be compared with the serum osmolality to obtain an accurate picture of a patient's fluid balance..e. or any other body fluid depends on the number of active ions or molecules in a solution. osmolality is expressed as "so many" milliosmoles per kilogram of water (mOsm/kg water). People in our culture drink relatively little fluid. urine. the precise concentration of active solutes in the serum and urine can be calculated.

The higher the number of millosmoles in the urine. the urine osmolality should be at least 3 times the serum osmolality Increased serum and urine osmolality (hyperosmolality) levels are seen in: • • • • • • • • • • • Renal disease Congestive heart failure Addison's disease Dehydration Diabetes insipidus Hypercalcemia Diabetes mellitus/hyperglycemia Hypernatremia Alcohol ingestion Mannitol therapy Azotemia Decreased serum and urine osmolality (hypoosmolality) levels are seen in: • • • • • Sodium loss due to diuretic use and a low salt diet Hyponatremia Adrenocortical insufficiency SIADH Excessive water replacement/overhydration/water intoxication Panic values for serum osmolality are values of less than 240 mOsm or greater than 321 mOsm.800 mOsm.• Urine osmolality: extreme range of 50 .1400 mOsm/kg water. the more concentrated the urine. A serum of osmolality of 384 mOsm produces stupor. this is the expected physiological response to dehydration This table shows the relationship between serum and urine osmolality and the clinical significance of laboratory values. but average is about 500 . As the serum osmolality rises. the patient may have grand mal seizures. When the serum osmolality is normal or increased. If the serum osmolality rises over 400 mOsm. Serum Osmolality Normal values: 282-295mOsm Normal or increased Urine Osmolality Normal values: 500-800mOsm Increased Clinical Significance Fluid volume deficit . the kidneys are conserving water. After an overnight fast. Values greater than 420 mOsm are fatal. the urine osmolality should also rise.

pregnant animals or azotaemic animals with diluted urine. ADH stimulates renal water resorption and increases urine SG. They are indicated in animals that show polydipsia/polyuria (PD/PU) without azotaemia and dehydration and are contraindicated in dehydrated animals.Decreased Normal Increased or normal Decreased Decreased Decreased (with no increase in fluid intake) Increased Decreased Urine Concentration tests: Fluid volume excess Increased fluid intake or diuretics Kidneys unable to concentrate urine or lack of ADH (diabetes insipidus) SIADH An increase in plasma osmolarity stimulates ADH secretion by the posterior pituitary gland. . These tests are designed to identify concentrating defects in the kidney.

Too little albumin may result in fluids "leaking" out of the blood vessels into surrounding spaces such as the abdomen.Liver Function Tests (LFT) Albumin estimation =========== Introduction: Albumin is made in the liver and is responsible for maintaining proper fluid balances. 5-Dye binding methods I) Precipitation method *Use serum only *Not applied for automation *Used now for separation & manufacturing albumin . Calculation of Albumin = % Albumin X Total Protein *Difficult method for Automation. Increases in albumin do not occur naturally but can be seen in patients who had received albumin suspensions. II) Electrophoresis =========== *Separation of Albumin from the major classes of protein in an electrical field & the staining %is obtained . scanned by densitometer. Decreased amounts of albumin can occur when the liver is not making enough or if albumin is being lost through the kidneys. Methods 1-Precipitation method 2-Electrophoresis 3-Globulin Tryptophan content method 4-Immunochemical methods.   *Precipitation is done by salting out of globulins &then albumin in the supernatant is measured using a protein estimation method. III) Globulin Tryptophan content method ========== . *It is a quantitative method but tends to over estimate Albumin because albumin is the best binder of staining dyes &the band density of alb.

Glycoxylic acid +tryptophan (Globulin)Purple chromogen (measured at 540) • Calculation of Albumin = T. Clinical diagnosis should not be made on a single test result. *Used for serum only. F)-Enzyme immune assay (ELISA) PRINCIPLE OF THE METHOD Albumin in the presence of bromcresol green at a slightly acid pH. skin lesions such as dermatitis and burns or dehydration. PREPARATION . including nutrition. C)-Turbidimetry: The reaction between albumin and its specific antibodies form complexes . D)-Nephelometry:. *Used for serum &CSF.that will decrease the light transmission through the reaction phase more than free albumin (antigen). B)-Radial immune diffusion (RID):By measuring the diameter of precipitin ring between albumin &its antibodies incorporated in agarose gel.Protein .Quantitative &Manual. The intensity of the color formed is proportional to the albumin concentration in the sample . CLINICAL SIGNIFICANCE One of the most important serum proteins produced in the liver is albumin. E)-Radio immune assay (RIA). Takes long time. drugs and steroids. bilirubin. maintenance of oncotic pressure and transport of Ca++. it should integrate clinical and other laboratory data. malnutrition. This molecule has an extraordinarily wide rage of functions.Globulin IV) Immune chemical methods ============== A)-Electro -immune-diffusion (EID): Considered the Reference method . Variation in albumin levels indicate liver diseases.*In this method Tryptophan content of the globulin is fist estimated as following. *Migration of protein fractions in an electrical field through a medium contains specific antibodies to albumin. The height of the Rocket precipitin line is correlated to albumin conc. produces a colour change of the indicator from yellow-green to green-blue. free fatty acid.

.5 to 5. The colour is stable 1 hour at room temperature. CALCULATIONS (A) Sample x 5 (Standard conc. . . free of hemolysis: Stability 1 month at 2-8ºC or 1 week at 15-25ºC. .) = g/dL albumin in the sample (A) Standard Conversion factor: g/dL x 144. . . . . . . .0 --- (R (mL Standard ((µL Sample ((µL 14. . 1 cm light path Temperature . . Pipette into a cuvette: Bla nk 1. . . . . .40. .0 g/dL. . . . against the Blank. . 25. Read the absorbance (A) of the samples and Standard.. Adjust the instrument to zero with distilled water. . .Reagent and calibrator are ready to use Signs of reagent deterioration: 1. . . . . . . . 23.Presence of particles and turbidity. . . . . . . . . Assay conditions: Wavelength: . . Mix and incubate for 10 min at room temperature (15-25ºC). 15-25ºC 12. .Blank absorbance (A) at 630 nm 0. . . . . . 630 nm (600-650) Cuvette: . . . . . PROCEDURE 11. . SAMPLES Serum or plasma. . . .9 = µmol/L REFERENCE VALUES 3. . . 2. ..

Since both albumin and globulin can be assayed individually. Elevations or decreases in a total protein must be investigated to find out which of the two components is causing the problem. It is usually done as a general screening assay since it is composed of two major fractions (albumin and globulin).5. If both are decreased. . the most common culprit is liver function. it is necessary to have the total protein rerun at the time these tests are performed.Total protein estimation Introduction: A Total Protein can be done on either a fasting or non fasting specimen. the range of variation is quite small. Acceptable variation is 1.8.0 gram/dL. Since this is a stable assay. a general reference range is 5. they are sometimes reported as an "AG ratio".0 If both the albumin and globulin are elevated. They should consider having tests performed on urine specimens as this will lessen the clotting problem found in the specimen but still provide adequate answers to the physician. Overall.) Patients with Waldenström’s macroglobulinemia may have total proteins above 8. Since many of the next level tests may be reported as percentages or ratios. (See albumin and globulin for specifics. one possibility is dehydration or a slow down of blood flow.0 ..

gr.015 1.8.of Int.A.5 g/dl Adults = 6.7 g/dl 7months -1yr.007 ) *Can estimate T.tryptophan.P Premature babies = begin from 3.of interest) Total protein(g/dl) = 365(Sp.6 -5.=159/L Sp.gr between 1.3 g/dl 1-2yrs.then serial dilutions are made to get solns.5 g/dl to T.3 g/dl b)Column method (Lowry&Hunter): .P (by extravasation of proteins) 1.: *drops of serum are allowed to fall into "Universal"containers filled with CuSO4 soln. *Interference by free tyrosine. each of known sp. *Using Quartz Cuvette (with no scratches) • On using serum . (Stock soln. 3-Refractrometry.035 At certain specific gravity a drop will not move neither up or down(=Sp.gr. = 5.T.15 mol/L. • This method depends on Tryptophan &Tyrosine content of the protein. ▬ 1.1 -7.P between 3. bil.P. 2-Specific gravity method for Total proteins: a)Phillips et al.&U.1-Ultraviolet absorption method 2-Specific gravity methods for T. of sp.gr.0 g/dl . 4-Kjeldahl nitrogen detection method 5-CuSO4 (Cu-Pr complex) Methods a)Phillips or b)Lowry &Hunter a)Titration or a)Lowry or b) kinetic b) Biuret Normal (Reference)Ranges: .7 -7.1). = 5.gr=1. Dilute 1:1000 with NaCl 0.Ammonia (Plasma on Heparin)= 15-51 ug/dl .0 .Exercise &Ambulatory 0.6 g/dl Newborns = 4.Ultraviolet Absorption: 270 –290 nm 200 -225 nm *Used for Solutions rather than serum.3 ▬10.

5 ▬11 g/dl 4-Kjeldahl Method: *The reference method *using protein free filtrate. *Can estimate T.P can be calculated. *A large drop of serum or urine is allowed to spread between slide &thin film and refracted rays make sharp line dividing the dark & light fields.*Using only single gradient column of mixed organic liquids with CuSO4 jacket (maintaining constant temp. Protein H2SO4+Catalyst +Na-Molybdate NH4+ Alkaline PH NH3 HCl(standard Sol. *It requires only one drop.which will be hanged at certain gradient . *Like in Phillips method T.P between 3.). 3-Refractometry method: *This method is based on the refraction of incident light by total dissolved solids. *Depend on estimation of protein nitrogen.) +2oxoglutarate NADPH .

change Concentration of total protein= detected nitrogen X100/16 = detected nitrogen X 6.N2-NPN) X 6.25 5-Alkaline CUSO4 soln.conc. * Corrected pr. = (pr.) K&NaTartarate(=color stabilizer) .25 is the result of 100/16 as each 100 g prt.( either) (Titration) Neutral PH glutamate (Nisselerization) at 340 nm) NADP + (Monitor abs.25 *factor 6. Copper 6-peptide bond protein complex Folin(Fenol)+ Cio-Calteau(PTA+Ph-Molbdic a.Methods: Sample NaOH + CuSO4.16 g Nitrogen.

Molybdinum blue + Pr.g Alb.sulfa&tetracyclines) Tryptophan by wt. Dependence on e. Tryptophan by wt.Complex Tungesten blue(at 650.2 % Glob.=0. The intensity of the color formed is proportional to the total protein concentration in the . 2-12 g Specificity: specific No. Iodide is included as an antioxidant. of reagents: 2 Reagents One reagent 100 times > Biuret’s Less specific Drug Interference Tryptophan&Tyrosine (salicylates.750nm) 546nm) Violet color of Cu(at LOWRY’s Method BIURET’s Method Sensitivity: good for Pr.=2% PRINCIPLE OF THE METHOD Proteins give an intensive violet-blue complex with copper salts in an alkaline medium.

. . . . widely distributed in the organism.High protein levels caused by hemoconcentration like in the dehydrations or increase in the concentration of specific proteins. . .37ºC / 15-25ºC 12. 1 cm. . . . . Adjust the instrument to zero with distilled water. CALCULATIONS . . . The proteins of the serum are divide in two fractions. .sample CLINICAL SIGNIFICANCE The proteins are macromolecular organic compounds. SAMPLES Serum or heparinized plasma: Stability of the sample: 1 month at refrigerator (2-8ºC). . . Clinical diagnosis should not be made on a single test result. . . . . . . They act like structural and transport elements. . PROCEDURE 11. .0 25 --------Sampl e 1.0 ----------Standa rd 1. . . . . . albumin and globulins The determination of total proteins is useful in the detection of: . . . . . .Blank absorbance (A) at 540 nm 0. 25. . . . light path Temperature . . . . . . Pipette into a cuvette: Blan k 1.. . . . 540 (530-550) nm Cuvette: . . . . . . it should integrate clinical and other laboratory data PREPARATION The reagents are ready to use Signs of reagent deterioration: 1. . . . . . . .Presence of particles and turbidity.22. . . . . . .0 ------25 R (mL) Standard (µL) Sample(µL ) 14. 2. The colour is stable for at least 30 minutes. against the Blank. .Low protein level caused by hemodilution by an impared synthesis or loss (as by hemorrhage) or excessive protein catabolism. Mix and incubate 5 min at 37ºC or 10 min at room temperature. 23. . . . . . . . . . . Assay conditions: Wavelength: . . . Read the absorbance (A) of the samples and Standard.

they may show "neonatal jaundice" which is a build up of bilirubin in the blood stream.Direct Skin Bilirubinometer: . Methods for Bilirubin estimation =============== 1. ineffective erythrpoiesis. genetic errors. genetic errors.1 g/dL Bilirubin estimation Introduction: Bilirubin is the end product of red cell lysis and recycling of hemoglobin which is performed in the liver. Bilirubin determinations are used to study liver function and red cell metabolism CLINICAL SIGNIFICANCE Bilirubin is a breakdown product of hemoglobin. because their serum contains no Carotenes. Many medications. and drugs.L while bilirubin reads at 450 nm. The test quantifies two different forms of bilirubin. Causes of hyperbilirubinemia: Total bilirubin (T): Increase hemolysis. Hyperbilirubinemia results from the increase of bilirubin concentrations in plasma.Direct Spectrophotometry: *Restricted to newborn(< 28 days) up to 3 months. Clinical diagnosis should not be made on a single test result. it should integrate clinical and other laboratory data. Direct bilirubin (D): Hepatic cholestasis.2 – 9. one is the final product while the other is an intermediate form. The build up of bilirubin in the blood stream is called jaundice and is a general sign of liver disease.(A) Samplex 7 (Standard conc. It is transported from the spleen to the liver and excreted into bile.A540 ) *That is because Hemoglobin reads the same at both W. hepatocellular damage1.)= g/dL of total protein in the sample (A) Standard REFERENCE VALUES Adults: 6. *Measuring absorbency of Bilirubin in serum at 2 wave lengths 450 & 540 nm The difference in the absorbance represents bilirubin absorbance (A450 . This should go away as the liver matures. for the first several days.3 g/dL Newborn: 5. gall bladder disease as well as viruses such as infectious mononucleosis and hepatitis will have jaundice. neonatal jaundice.6 – 8. Many infants are born with less than fully mature livers.5.6. As a consequence. 2.

Oxidase Biliverdin (measured at 405 nm) . *Used by KODAK ECTACAM only. The Reference method. Or -Filter Multilayered color glass. *Bil.Benzoate Alkaline(PH=12) 560 nm red Azobilirubin +H2O+Co2+Hcl (Direct Coloumetric Reaction: *Bil. A450 . 5-Colometric methods (Experiment # 8): *The most commonly used methods. 4-HPLC(High Purity Liquid Chromatography): • • Using Normal Silica Chromatography .Glucuronides + DSA Bilirubin) *Bil. +Bil.Spectral shift change method: *Spectral shift through adding hydrophobic cationic polymer.A 540 = Absorbance of bilirubin 3.*Restricted also to newborns up to 3 months. Caffeine &Na.L : *Color : Methanol or Neutral 660 Red purple JENDRASSIL-GROF M. *Accelerator: Urea *PH : *W. *Needs calibration using:-Methyl Orange solution.Glucuronides +DSA +Accelerator Azobilirubin +H2O+Co2+Hcl(Total Bilirubin) 6-Bilirubin Oxidase Method : *Specific for Bilirubin only. • Depend on reaction of bilirubin& Diazotized Sulfanilic acid (DSA) MELLOY &EVELYN M.

Color development in R 2.1 cm light path Temperature . . .k R 1 (D) --((mL R 2 (T) 1.5 1. . . . .. . … … . . . . . PROCEDURE 11. . . . . . . . The intensity of the color formed is proportional to the bilirrubin concentration in the sample PREPARATION All the reagents are ready to use Signs of reagent deterioration: 1.PRINCIPLE OF THE METHOD Bilirubin is converted to colored azobilirubin by diazotized sulfanilic acid and measured photometrically. .Serum or Plasma 2. … . . . the results correspond to total bilirubin. .Storage for 3 days in dark & refrigerator (for months if freezed at – 70˚ C) 5. 25. . . . . . while free bilirubin requires solubilization with dimethylsulfoxide (DMSO) to react (bilirubin indirect). . . . . . bilirubin-glucuromide and free bilirubin loosely bound to albumin. . ..Urine sample either Random or 24 hrs. . .Presence of particles and turbidity. . In the determination of indirect bilirubin the direct is also determined. Of the two fractions presents in serum. . Mix and incubate exactly for 5 minutes at 15-25ºC.5 ((mL 50 -( R 3 (µL Sample 100 100 1 4. . . . . .5 --100 0 00 Total Blan B . Assay conditions: Wavelength: . . 2. 555 nm (530-580) Cuvette: . . Specimen Precautions: =================== 1. . Adjust the instrument to zero with distilled water. . . . . . . . . . . .Avoid light exposure 4. not stored for >24 hrs. Read the absorbance (A).Avoid Hemolysis 3. only the former reacts directly in aqueous solution (bilirubin direct). Pipette into a cuvette: rect Bla nk 5 1. . 15-25ºC 12. . 23. . .

25 mg/dL=4.10 mg/dL=18. Bilirubin (D) = 14 Conversion factor: mg/dL x 17. REFERENCE VALUES Bilirubin Total Up to 1. measured photometrically. It is an exceptionally sensitive indicator of stress in these sites. Alcohol consumption (even a little) and many medications are the chief causes of these swings. gall bladder. according to the following reaction: γ--L-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine γ-GT γ-L-Glutamyl-glycylglycine + 2-Nitro-5-aminobenzoic acid The rate of 2-nitro-5-aminobenzoic acid formation.27µ mol/L Questions: Write causes of jaundice What are the commonest methods of estimating serum bilirubin in neonates? Mention the different causes of elevated direct and indirect bilirubins. As a consequence. and pancreas. Catalytic (Enzymatic) activities of Liver (ELFT) 1-Gamma Glutamyl Transferase (GGT) Introduction: This enzyme used to metabolize materials in the kidney.1 =µ mol/L. liver. variations in results may be quite common.CALCULATIONS With Factor: ((A) Sample .81 µmol/L Bilirubin Direct Up to 0. liver or pancreatic function PRINCIPLE OF THE METHOD (Kinetic test (Szasz) Gamma-glutamyl transferase (γ-GT) catalyses the transfer of γ-glutamyl group from γ-glutamyl-p-nitroanilide to acceptor glycylglycine.1 .(A) Sample Blank) x Factor* = mg/dL bilirubin in the sample : Theoretical factor: Bilirubin (T) = 19. is proportional to the catalytic concentration of γ-GT present in the sample CLINICAL SIGNIFICANCE . This test is used to follow kidney.

. . . . 3. . . 405 nm Cuvette: . . . . . Pipette into a cuvette: 14. . CALCULATIONS Mean A= (ΔA)/min = (A1+A2+A3) / 3 Enzyme activity (U/L) = Δ A X Factor . PREPARATION Working reagent (WR): Dissolve one tablet of R 2 Substrate in one vial of R 1 Buffer. . . . . . start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. . . . Assay conditions: Wavelength: . . . . . . . . . . . . . . Measurements of gamma-glutamyl transferase (γ-GT) activity are used in the diagnosis and treatment of hepatobiliary diseases such biliary obstruction. . 1 cm light path Constant temperature . Read initial absorbance (A) of the sample. . . . Stability: 21 days at 2-8ºC or 5 days at room temperature (15-25ºC). 25. primarily in the kidney. . . . Signs of reagent deterioration: 1. . . . it should integrate clinical and other laboratory data. γ−GT is stable for at least 3 days at 2-8ºC. . . . . . . .Presence of particles and turbidity. liver and prostate. . pancreas. Mix. .25ºC /30ºC / 37ºC 2. 8 hours at 15-25ºC and 1 month at – 20ºC. . . . cirrhosis or liver tumours Clinical diagnosis should not be made on a single test result.20. Adjust the instrument to zero with distilled water or air. 2. . wait for 1 minute. . . Cap and mix gently to dissolve contents. . .Gamma-glutamyl transferase (γ-GT) is a cellular enzyme with wide tissue distribution in the body. . . PROCEDURE 1. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min). .Blank absorbance (A) at 405 nm ≥ 1. 36. . . . .. SAMPLES Serum. .

The concentration is expressed in units per litre of sample (U/L).Alanine Transaminase (ALT) PRINCIPLE OF THE METHOD Alanine aminotranferase (ALT) o Glutamate pyruvate transaminase (GPT) catalyses the reversible transfer of an amino group from alanine to αketoglutarate forming glutamate and piruvate.∆A/min x 1190 = U/L of γ-GT Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute. The piruvate produced is reduced to lactate by lactate dehydrogenase (LDH) and NADH: . REFERENCE VALUES C 25ºC 4-18 U/L 6-28 U/L Wom en Men 2. in standard conditions.

its better application is in the diagnosis of the diseases of the liver. . . . . . . . . . . PROCEDURE 1. .00. . . 1 cm light path Constant temperature . . . incubate for 1 minute. Mix. . Stability: 21 days at 2-8ºC or 72 hours at room temperature (15-25ºC). .Blank absorbance (A) at 340 nm < 1.. . since the value of the ALT stays within the normal limits in the presence of elevated levels of AST Clinical diagnosis should not be made on a single test result. . . 3. . . . . . . . SAMPLES Serum or plasma: Stability 7 days at 2-8ºC. When they are used in conjunction with AST aid in the diagnosis of infarcts in the myocardium.. . . . . . .. . .α-ketoglutarate + L-Alanine Pyruvate Pyruvate + NADH+H+ L-Lactate + NAD ALT (GPT) L-Glutamate + Lactate dehydrogenase (LDH) The rate of decrease in concentration of NADH. it should integrate clinical and other laboratory data. Signs of reagent deterioration: 1. . . . . 2. . . . Adjust the instrument to zero with distilled water or air. is proportional to the catalytic concentration of ALT present in the sample CLINICAL SIGNIFICANCE The ALT is a cellular enzyme. . . measured photometrically. Cap and mix gently to dissolve contents. . Pipette into a cuvette: 14. . Assay conditions: Wavelength: . . . .Presence of particles and turbidity.25ºC / 30ºC / 37ºC 2. . . 340 nm Cuvette: . . . . . . . . . . . . . . . . . . diseases of muscles and traumatisms. PREPARATION Working reagent (WR): Dissolve one tablet of R2 Substrate in one vial of R1. . High levels are observed in hepatic disease like hepatitis. . found in highest concentration in liver and kidney.

25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1-minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min). CALCULATIONS ΔA (mean difference of readings) = (A1 +A2+A3) / 3 ALT enzyme activity (U/L) = ΔA X Factor (F1) ∆A/min x 1750 = U/L of ALT Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L). REFERENCE VALUES 25ºC 30ºC 37ºC Men up to 22 U/L 29 U/L 40 U/L Women up to 18 U/L 22 U/L 32 U/L Normal newborns have been reported to show a reference range of up to double the adult, attributed to the neonate’s hepatocytes. These values decline to adult levels by approximately 3 months of age.

3- Aspartate Transaminase (AST) Introduction: Aspartate Transaminase (AST) is also known by its older name, SGOT, this enzyme is needed in the utilization of energy sources. It is found in high concentrations in muscle (cardiac and others), liver, and other organs. This test usually is ordered to follow cardiac and muscle disease . This test can be performed on specimens from patients who are either in a fasting or non fasting. Adult reference ranges vary widely with different instruments. PRINCIPLE OF THE METHOD Aspartate aminotransferase (AST) formerly called glutamate oxaloacetate (GOT) catalyses the reversible transfer of an amino group from aspartate to α-ketoglutarate forming glutamate and oxalacetate. The oxalacetate produced is reduced to malate by malate dehydrogenase (MDH) and NADH: AST (GOT) α-ketoglutarate + L-Aspartate L-Glutamate + Oxaloacetate Malate+ NAD The rate of decrease in concentration of NADH, measured photometrically, is proportional to the catalytic concentration of AST present in the sample.
Malate dehydrogenase (MDH)

Oxaloacetate + NADH+H+

CLINICAL SIGNIFICANCE The AST is a cellular enzyme, is found in highest concentration in heart muscle, the cells of the liver, the cells of the skeletal muscle and in smaller amounts in other weaves. Although an elevated level of AST in the serum is not specific of the hepatic disease, is used mainly to diagnostic and to verify the course of this disease with other enzymes like ALT and ALP. Also it is used to control the patients after myocardial infarction, in skeletal muscle disease and other Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data. PREPARATION Working reagent (WR): Dissolve one tablet of R.2 Substrate with one vial of R1 Buffer. Cap and mix gently to dissolve contents. Stability: 21 days at 2-8ºC or 72 hours at room temperature (15-25ºC).

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 340 nm < 1.00. SAMPLES Serum or plasma: Stability 7 days at 2-8ºC.. PROCEDURE 1. Assay conditions: Wavelength: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 nm Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm. light path Constant temperature . . . . . . . . . . . . . . .25ºC /30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette:

14. Mix, incubate for 1 minute. 25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min). CALCULATIONS ΔA (mean difference of readings)/min. = (A1 +A2+A3) / 3 AST enzyme activity (U/L) = ΔA X Factor (F1) ∆A/min x 1750 = U/L of AST Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L). REFERENCE VALUES 25ºC Men up to 19 U/L Women up to 16 U/L 30ºC 26 U/L 22 U/L 37ºC 38 U/L 31 U/L

ISE PRINCIPLE OF THE METHOD The measurement of calcium in the sample is based on formation of color i. Changes in calcium are used to assess bone function. Flame photometer iv. Atomic absorption Spectrophotometry (AAS) iii.and γ-globulins.Bone Profile Testes Calcium Determination Introduction: Calcium is required for cell function overall and for bone metabolism. primarily to albumin. -To know the status body calcium (Tetany) METHODS: Chelation with o-Cresolphthalein Complexone(Colorimetric) ii. Higher blood levels usually mean lower bone levels. but also to some extant to the α-. the unionized diffusible form constitutes 5 % approx. and about 43 – 47 % of the total plasma calcium is protein bound. . β. Too little calcium gets you either a loss of tissue function or soft bones (osteoporosis) while too much gives you tetni ( cardiac arrest and/or lock jaw is from over clenching of muscles) or over brittle bones. OBJECTIVES: -Ionized calcium constitutes 48 to 52 % of the total calcium. Usually performed in conjunction with Phosphorous determinations.

vitamin D deficiency. . . . Approximately 99% of body calcium is found in bones. Pipette into a cuvette: Blank R1 (mL) R2 (2 drop) Standard (µL) 2. . Assay conditions: Wavelength: . The intensity of the colour formed is proportional to the calcium concentration in the sample CLINICAL SIGNIFICANCE Calcium is the most abundant and one of the most important minerals in the human body. PREPARATION All the reagents are ready to use.0 1 ----Standa rd 2. . Among causes of hypercalcemia are cancers. . osteoporosis. pseudohypoparathyroidism. . it should integrate clinical and other laboratory data. large intake of vitamin D. . . SAMPLES Serum or plasma: Separated from cells as rapidly as possible. . . . . mix according to this proportion: 50 vol. . . . . . . . The collecting bottles should contain 10 ml of diluted Nitric acid (50% v/v). . sarcosidosis. . . hyperparathyroidism. . . Multiply results by 2 (dilution factor). enhaced renal retention. . . Blood anticoagulants with oxalate or EDTA are not acceptable since these chemicals will strongly chelate calcium. . . . . . Record the volume. 3. . . . . Clinical diagnosis should not be made on a single test result. . Stability of the samples: Calcium is stable 10 days at 2-8ºC. Mix. . . Low levels of calcium are found in hypoparathyroidism. . . Dilute a sample 1/2 in distilled water. malnutrition and intestinal malabsortion.0 1 ----- . . . .Urine: Collect 24 hour urine specimen in calcium free containers. .complex between calcium and o-cresolphtalein in alkaline medium: Ca++ + o-Cresolphtalein OH Colored complex O-Cresolphthalein Complex one gives violet color in alkaline medium. 1 cm. . . To prepare monoreagent. .. 570 nm (550-590) Cuvette: . of R1 and 1 vol. of R2. .. 37ºC / 15-25ºC 2. A decrease in albumin level causes a decrease in serum calcium. PROCEDURE 1. . thyrotoxicosis. . light path Temperature . . .0 1 20 Sampl e 2. . Adjust the instrument to zero with distilled water.

REFERENCE VALUES Serum or plasma: Adults 8. Vitamin D intoxication 5. Mix and incubate for 5 min at 37ºC / 15-25ºC.25= mmol/L.Sample (µL) ----- ------ 20 4. Vitamin D deficiency 2.3.3 mmol/L Newborns 8 -13 mg/dL = 2 . Thyrotoxicosis 4.5 mg /dL = 2.25 mmol/L RESULTS: CLINICAL SIGNIFICANCE: * HYPOCALCEMIA 1.5 . 5.) = mg/dL calcium (A) Standard Conversion factor: mg/dL x 0. CALCULATIONS Serum and plasma (A) Sample x 10 (Standard conc.6 mmol/L Children 10 -12 mg/dL = 2. Hyperparathyroidism 2. against the Blank. Read the absorbance (A) of the samples and calibrator. Hypoparathyroidism 3. Alkalosis (Alkalemia) * HYPERCALCEMIA 1. The color is stable for at least 40 minutes. Malignancy of bone 3.1-2.5-10. Idiopathic DISCUSSION: .

Low levels of phosphorus. The intensity of the color formed is proportional to the inorganic phosphorus concentration in the sample CLINICAL SIGNIFICANCE Phosphorus is an essential mineral for tissue bone formation and is required by every cell in the body for normal function. What is HYPOCALCEMIA? Write a short note on Tetany. Give the principle for the determination of serum calcium by colorimetric method. Quantitative determination of phosphorus PRINCIPLE OF THE METHOD Inorganic phosphorus reacts with molybdic acid forming a phosphomolybdic complex. Avoid venous stasis (Increase protein & calcium) 2. . primary hyperparathyroidism. 3. Lipemic Samples (Prepare blank 0. can be caused by hypervitaminosis 0. renal tubular disorders. Enumerate different methods for the determination of serum calcium.TETANY PRECAUTIONS: 1.05 ml sample + 2.W) QUESTIONS: 1. antacids or malabsortion. 2. Approximately 85% of the body phosphorus is found in bone and in teeth.5 D. Its subsequent reduction in alkaline medium originates a blue molybdenum colour. Do not use contaminated glass ware (Increase calcium) 3.

.5 . it should integrate clinical and other laboratory data.37°C 1 15-25°C 2. SAMPLES . . Stability: 10 days at 2-BoC.5 50 -Blank (WR (mL 1. PROCEDURE 1.. bone metastases. liver disease..Adjust the instrument to zero with distilled water. light path Temperature . diarrhea and vomiting Clinical diagnosis should not be made on a single test result.. Adjust to pH 2.(Sample (µL . 3.Serum: Free of hemolysis.5 -50 Standard 1.High levels of phosphorus can be caused by diet. Dilute the sample 1/10 with distilled water...(Standard (µL -.. Stabilitr 7 days at 2-8C.Urine· (24 h): Collect the specimen into a bottle containing 10 mL of 10% v/v hydrochloric acid (HCI) to avoid phosphate precipitations. Mix.1 cm. alcohol ingestion. protected from light.Pipette into a cuvette: Sample 1. Serum should be removed from the clot as quickly as possible to avoid elevation of serum phosphorus from hydrolysis or leakage of phosphate present in erythrocytes. Multiply the result by 10 (dilution factor).Assay conditions: Wavelength: 710 nm (620-750) Cuvette: . PREPARATION Working reagent (WR): Mix equal volumes of R 1 (Molybdic) and R 2 (Catalyzer) Stability: 24 h at 2-8C..

The colour is stable for at least 2 hours.8 mmo/lL) 5 (Calibrator cone.5. 5.) = mg/dL of phosphorus in the Quantitative determination of alkaline phosphatase (ALP) . Mix and incubate for 10 min at 37°C or 30 min at room temperature (15-30°C).0 mg/dL = Adults (1. REFERENCE VALUES Serum: Children : 4.3 .2. against the Blank. CALCULATIONS Serum (A) Sample x sample (A) Calibrator Conversion factor: mg/dL x 0.0 mg/dL = (O.323 = m m ol/L.4.1.8 .7.0 .5 .2 mmol/L) : 2.Read the absorbance (A) of the samples and calibrator.

SAMPLES Serum or heparinzed... according to the following reaction: p-Nitrophenylphosphate + H20 p-Nitrophenol + Phosphate The rate of p-Nitrophenol formation.. .405 nm . liberating p-nitrophenol and phosphate. Both increases and decreases of plasma ALP are of importance clinically. obstructive liver disease.. Causes of decreased plasma ALP: Cretinism and vitamin C deficiency1. intestine and kidney.. .. Clinical diagnosis should not be made on a single test result.5. liver. placenta. hepatotoxicity caused by drugs or osteomalacia.serum.. Stability: 3 days at 2-8°C. being particularly high in bone. separated from the clot as soon as possible. PROCEDURE 1-Assay conditions: Wavelength: ...... plasma _Use unhemolyzed . . measured photometrically..6. PREPARATION working reagent (WR): Dissolve one tablet of R 2 Substrate in one vial of R 1 Buffer. hepatitis.PRINCIPLE OF THE METHOD Alkaline phosphatase (ALP) catalyses the hydrolysis of p-nitrophenyl phosphate at pH 10. is proportional to the catalytic concentration of alkaline phosphatase present in the sample CLINICAL SIGNIFICANCE Alkaline phosphatase is an enzyme present in almost all weaves of the organism. Causes of increased plasma ALP: Paget's disease of bone. it should integrate clinical and other laboratory data.4.

. . 6-Calculate the difference between absorbances and the average absorbance differences per minute (ΔA/min). CALCULATIONS (ΔA/min) x 3300 = U/L de ALP Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute.. 3-Pipette into a cuvette: WR (mL) (µL)Sampl e 4. . . .. . . .207 U/L 98 . The concentration is expressed in units per litre of sample (U/L). . . .170 U/L 73 . start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. .25°C 30°C 37°C 1 cm light path 2-Adjust the instrument to zero with distilled water or air. . . Constant temperature . . Mix.279 U/L Factors affecting ALP activities in a normal population include exercise. periods of repaid growth in children and pregnancy. REFERENCE VALUES1 25°C Children (1-14 years) U/L Adults 60 .. in standard conditions. . . . 30°C 37°C < 480 U/L < 645 < 400 U/L 1. .2 20 . 5-Read initial absorbance (A) of the sample. .. . incubate for 1 minute. .Cuvette: . .

. is proportional to the catalytic concentration of CK present in the sample PREPARATION Working reagent (WR): Dissolve 1 tablet of R 2 Substrate with one vial of R 1. Stability: 5 days at 2-8ºC or 24 hours at room temperature (15-25ºC). it should integrate clinical and other laboratory data. Do not use reagents over the expiration date. Its physiological role is associated with adenosine triphosphate (ATP) generation for contractile or transport systems. Elevated CK values are observed in diseases of skeletal muscle and after myocardial infarction Clinical diagnosis should not be made on a single test result. Cap vial and mix gently to dissolve contents.Cardiac profile Testes Quantitative determination of creatin kinase (CK) CLINICAL SIGNIFICANCE Creatine kinase is a cellular enzyme with wide tissue distribution in the body. protected from light and contaminations prevented during their use. measured photometrically. STORAGE AND STABILITY All the components of the kit are stable until the expiration date on the label when stored tightly closed at 2-8ºC. Do not use the tablets if appears broken. This reaction is coupled to those catalysed by hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH): Phosphocreatine + ADP CK Creatine + ATP ATP + Glucose HK ADP + Glucose-6-phosphate G6P + NADP + G6P-DH 6-Phosphogluconate + NADPH + H + The rate of NADPH formation. PRINCIPLE OF THE METHOD Creatine kinase (CK) catalyses the reversible transfer of a phosphate group from phosphocreatine to ADP.

. . . . . . . . The creatin kinase activity decreases 10% after 1 day at 2-5ºC or after 1 hour at 15-25ºC.. . . . . . REFERENCE VALUES 25ºC Men. . . . .25ºC / 30ºC / 37ºC 2. Read initial absorbance (A) of the sample. . 3. Pipette into a cuvette: WR (mL) Sample (µL) 14.. . . . up to 70 U/L 30ºC 130 U/L 110 U/L 37ºC 195 U/L 170 U/L . . . . . up to 80 U/L Women. . . 25. 2. . . . . 36. . . . . . . CALCULATIONS ∆A / min x 4127 = U/L CK ∆A / min x 8095 = U/L CK Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute.Blank absorbance (A) at 340 nm ≥ 1. . Mix.Presence of particles and turbidity. SAMPLES Serum or plasma: Stability 7 days at 2-8ºC. . in standard conditions.1 cm light path Constant temperature . . . . PROCEDURE 1. incubate for 2 minutes. . . . Assay conditions: Wavelength: . . . . .Signs of reagent deterioration: 1. . . The concentration is expressed in units per litre of sample (U/L). . . . . . protected from light. . . . 340 nm Cuvette: . . . .60. . start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. Adjust the instrument to zero with distilled water or air. . . . Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min). . . .

Stability: 8 days at 2-8ºC or 24 hours at 15-25ºC. it should integrate clinical and other laboratory data. Cap vial and mix gently to dissolve contents. in skeletal muscle damage. CK-MB is usually present in serum at low concentration. The activity of the non-inhibited CK-B subunit is then assayed by the following series of reactions: Phosphocreatine + ADP CK Creatine + ATP ATP + Glucose HK ADP + Glucose-6-phosphate G6P + NADP + G6P-DH 6-Phosphogluconate + NADPH + H + The rate of NADPH formation. is proportional to the catalytic concentration of CK-B present in the sample CLINICAL SIGNIFICANCE CK-MB is an enzyme formed by the association of two subunits from muscle (M) and nerve cells (B).Working reagent (WR) Dissolve one tablet of R 2 in one vial of R 1. PREPARATION . . measured photometrically. Also is increased.Quantitative determination of creatine kinase MB (CKMB) PRINCIPLE OF THE METHOD An antibody to the anti CK-M inhibits completely CK-MM and subunit (M) of the CK-MB. it is increases after an acute infarct of myocardium and later descends at normal levels. rarely. Clinical diagnosis should not be made on a single test result.

PROCEDURE 1. . . . Read initial absorbance (A) of the sample. . Assay conditions: Wavelength: .. . CK-MB activity decreases a 10% after 24 hours at 4ºC or 1 hour at 25ºC. . . Percentage of CK-MB activity in sample: CK-MB Activity % CK-MB Activity = CK total Activity x 100 REFERENCE VALUES Heart infarct probability is high at the following conditions: 25ºC 30ºC 37ºC CK-MB > 10 U/L > 15 U/L > 24 U/L CK-MB activity is between 6 and 25% of total CK activity. 1 cm light path Constant temperature . (WR (mL 0 40 Sample ((µL 4. protected from light.Blank absorbance (A) at 340 nm≥ 1. . The concentration is expressed in units per litre of sample (U/L). . . . . . Calculate the difference between absorbances : ΔA= A2 – A1. . . . . . .Presence of particles and turbidity. Incubate for 10 minute. 6. . . 5. . .60. . 340 nm Cuvette: . . . . .25ºC / 30ºC / 37ºC 2.Signs of reagent deterioration: 1. . . . . . . SAMPLES Serum or plasma: Stability 7 days at 2-8ºC. . . . . . . . Pipette into a cuvette: 1. . . . in standard conditions. 2. . . . . . . . 3. . . Adjust the instrument to zero with distilled water or air. . . . Mix. . . . CALCULATIONS A x 1651 = U/L de CK-MB ΔA x 825 = U/L de CK-B Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per minute. .. start the stopwatch and read again after 5 minutes (A2). . . . .

Tests Blood Tests Glucose: Glucose is the primary blood sugar test and indicates blood sugar level at the time blood was drawn.Appendix (1) Collective Knowledge of Most Common Lab. Fructosamine: Indicates blood sugar levels over the past one to three weeks. High values are seen in diabetics. Glucose may be altered by diet and medication. . Normal fasting value is 70-110. In addition to pancreatic functions.

kidney disease and lack of Vitamin D. Normal range is 1. Globulin: Globulin helps to combat infection on a normal level. ASAT/ALT: Material found in the liver cells and muscle (heart) cells. Normal range is 0. Slight increases are sometimes seen without significance.0.5. Abnormal values occur in liver disease and poor nutrition. Total Protein: This is a combination of albumin and globulin.0. This test measures kidney function.2.4. which are proteins. BUN: BUN stands for Blood Urea Nitrogen and is a waste product which should be removed from the blood by the kidneys. Normal range is 2. Some people normally have isolated elevations of bilirubin called Gilbert's disease. Alkaline Phosphorus: A material found in the blood related to liver and bone. LDH: LDH is a material found in blood cells and liver cells.3-4. SGPT: Two measures of liver function.0-1. Creatinine: Creatinine is a waste product which should be removed from the blood by the kidneys. Normal range is 6-20. Normal range for adult males is 20-125. A/G Ratio: Albumin value divided by the globulin value. Normal range is 0. Normal range is 10-60. SGOT. Calcium: The most abundant mineral found in the human body.7-8. Total Bilirubin: The level of pigment in the blood. Normal range is 8. It is the total protein value minus albumin value. Breakdown of the blood cells as in heart disease or liver damage may increase values. Abnormalities are found in loss of bone. This test measures kidney function. .5-10. normal range for adult females is 42-124. Normal range is 6. Normal range is 91-180. GGTP: The earliest liver function to become abnormal.5-1.HGB A1C (Glycohemoglobin): Indicates blood sugar activity for the past three months.2. occasionally affected by muscle injury.8-2. Damage to these cells will increase values. Elevations can be associated with liver disease or breakdown or red blood cells.

6-5. Abnormal levels are found in pancreatitis. etc.0-6. normal range for feales is 35-135. . Lipoproteins: Proteins combined with lipids that serve as carriers of cholesterol. if in excess. in addition to diet and exercise. it usually follows the same pattern as sodium. Normal range is 101-111.0. Uric Acid: A material which. Magnesium: An element absorbed in the intestine. Values of 180 or less are associated with least risk of heart disease. beef.5-4. LDL ("Bad" Cholesterol). liver. Normal range is 21-31. the less likely that cholesterol deposits are in the blood stream and the less likely the chance of coronary heart disease. alcoholism and Addison's disease.0-7. High levels can impair circulation and lead to hardening of the arteries. Triglycerides: A blood fat related to calories and starch (sweets) in the diet. pork. Cholesterol/HDL ratio measures the coronary risk factors. normal range for females is 2. Alcohol also will increase the value.Phosphorous: Related to bone activity and usually follows exact opposite of calcium.6. Increased values may indicate a tendency to have hardening of the arteries.0. Normal range is 3.8-2. Normal range for males is 40-160. Cholesterol: A blood fat related in part to eating animal fats such as eggs. "Water pills" may lower potassium and increase kidney damage. cream. Normal range for males is 4.4. Socium: A body salt. Normal range is 135-145. Co2: Buffer system which assists in the transport of carbon dioxide from the tissue to the lungs. HDL ("Good" Cholesterol). Kidney disease and some diseases of the adrenal gland and dehydration can cause abnormal results. can deposit stones in the kidney or in the joints and cause gout. Normal range is 2. Potassium: A body salt or electrolyte found mostly inside of cells. Fast overnight test for accurate test results. also termed electrolyte. Chloride: A body salt/electrolyte.0. The higher the value. HIV antibody: Presence of antibody is associated with having been infected by the virus known to cause AIDS (Acquired Immune Deficiency Syndrome). Normal range is 1. cheese.

It helps combat infection. HGB/HCT: Hemoglobin is an iron-bearing protein which is the red coloring matter found in blood. normal range for females is 12-16. Protein: Possible kidney infection or disease.1. possibility of glucose intolerance or low renal threshold. Complete Blood Count WBC (White Blood Cells): White blood count is the number of white blood cells. Urine Tests WBC (White Blood Cells): Indicates possible infection of urinary tract.7-6. red blood count number and hemoglobin concentration. Normal range for males is 4. Casts: Possible kidney infection or disease. kidney infection or tumor. Normal range is 130-400.810. including prostate cancer. normal range for females is 4. malignant and benign prostatic tissue. Glucose: Sugar in the urine.2-5. . clinical discrimination is based upon its serum level. Normal range is 4. Platelets: Platelets deal with hemostasis and blood coagulation. Because PSA is found in normal.PSA: Abnormal levels in the serum are associated with clinical abnormalities of the prostate. It relates to anemia and oxygen transport. MCH/MCV/MCHC: Mathematical relationship between red blood count size.8. bladder or kidney. RBC (Red Blood Cells): Possible kidney stone. Normal range for males is 14-18.4. RBC (Red Blood Cells): Red blood count is the number of red blood cells.

8. Test Current units Factor SI units Diabetic Screen Glucose.5 mg/dl 0-0.05 mmol/L 3.2 mmol/L >0.57-6.5-8.Appendix (2) Common Blood Profiles Reference values for the more commonly employed laboratory tests are given in the following table.8 65-110 mg/dl 71-180mg/dl 5. The reference values are in the units currently often used and in the International System (SI) of Units.3 mmol/L <5.5 .0259 0.8 gm/dl 2.0113 1 <5.1 17.1 17.0-3.5 mg/dl 0-0.9 mg/dl 6.055 0.9-10.0 mmol/L .9 g/dl 1-2.0.54 .25-1.0259 0.9 mmol/L <2.6 mol/L  0 .3-25.74mmol/L 20-39 g/L 1-2. fasting Glucose .055 3.5 17.8 0.8  mol/L 0-14  mol/L 65-85 gm/L 0.154 10 1 4. random Glycosylated hemoglobin ( Hba1c ) Heart disease risk factors (fasting lipids ) Total Cholesterol HDL cholesterol LDL cholesterol Triglyceride Total cholesterol/HDL ratio Liver function tests Total Bilirubin Direct Bilirubin Indirect Bilirubin Total Protein Albumin Globulin Albumin/Globulin ratio 0.5-4.1 10 0.5 <200 mg/dl >35 mg/dl <150 mg/dl <205 mg/dl <5.5 gm/dl 3.9 mmol/L <3.5% 0.0259 0.

059 1 1 0.35-7.67 X 10-8 0.47 mmol/L 3.5-8.45 80-105 Factor 1 1 0.67 X 10-8 8.9-10.3 mg/dl 4.1 X 10-8 Katal/L Alanine aminotransferase (SGPT / ALT) Aspartate aminotransferase (SGOT/ AST) Renal/Kidney Function Tests Urea (BUN) Creatinine Uric acid Potassium Sodium Total Calcium Free Calcium Phosphate 8. Current units 11-35  mol/L 7.357 88.9-8.67 X 10-8 18-84 X 10-8 Katal/L 7-35 IU/L 12-58 X 10-8 Katal/L 45-125 1.25 0.5-5.133 SI units 11-35  mol/L 7.4 -67 X 10-8 Katal/L 8-25 mg/dl 0.g -Glutamy transpeptidase (GGT) -Male g -Glutamy transpeptidase (GGT) -Female Alkaline Phosphatase 11-50 IU/L 1.4 -58 X 10-8 Katal/L 5-40 IU/L 1.25 0.5 mg/dl 0.5 mmol/L Other Common Serum Chemistries/Enzymatic Activities Test Ammonia (plasma) Blood gases (arterial.57 mmol/L 1.5-4.9 mmol/L 53-150  mol/L 0.323 2.35-7.0 kPa .25 mmol/L 0.3-4.21-0.67 X 10-8 IU/L(up to 1000 IU/L in young children) 5-35 IU/L 1.3-4.9 mmol/L 135-145 mmol/L 2.12-1.0 mg/dl 3.45 10.0 mg/dl 2.6-14.pH Blood gases (arterial.8-1.23-2.4 0. whole blood) .75-2.7 mg/dl 3.6-1.9 mmol/L 135-145 mmol/L 8.

27 1.PCO2 mmHg 35 .58 gm/L 95-105 mEq/L 70-140  g/dl 85-155  g/dl 118226CH50U/ml 81-175 mg/dl 12-34 mg/dl 60-120 ml/min 13-145 ng/ml 25-240 ng/ml 12-130 ng/ml 150-360 mg/dl 1.9  mol/L 95-105 mmol/L 11.7-12.0186 6.7 1 0. whole blood) .Female Complement (total.0-22  mol/L 13. adult Fibrinogen Folate .6  mol/L Immunoglobulin .3  mol/L 0.8-1.23-0.Male Copper .01 0.5-3.37 gm/L 14.whole blood) .157 0.Red cell Haptoglobin 50-300  g/dl 0.6 gm/L 3.01 0.IgA 39-358 mg/dl Immunoglobulin .3-28.5-3.IgG Iron .39-3.45 mmHg 0.01 0.0 kPa 22-31 mmol/L Blood gases (arterial.01 2.179 1.6 ng/ml 153-602 ng/ml 100-300 mg/dl 0.7-6.01 0.00 gm/L 0.Plasma Folate .Children Ferritin -Male.9-5. adult Ferritin .29 gm/L 6.01 0.133 1 4.12-0.157 0.34 gm/L 2.PO2 Blood gases (arterial.75gm/L 0.58 gm/L 0.01 0.25 29-326 pmol/L 56-540 pmol/L 27-292 pmol/L 0.Female.Male 679-1537 mg/dl 80-160  g/dl .10-3. 22-31 mmol/L whole blood) -Carbon dioxide content ß-Carotene Ceruplasmin Chloride Copper .6  mol/L 1.IgM 33-229 mg/dl Immunoglobulin . hemolytic) C3 C4 Creatinine Clearence Ferritin .3-24.33-2.79-15.9-29 nmol/L 347-1367 nmol/L 0.

5-8.Binding capacity Iron .Beta globulin Protein electrophoresis Gamma globulin Vitamin A Vitamin B12 0.05-3.3 mmol/L 0.035 0.5 gm/dl 1 1 0.5 1 10 10.Female Iron .Transferrin saturation Lactate (plasma) Magnesium Osmolality Protein electrophoresis Alpha.7 gm/dl electrophoresis .1.7 gm/dl 10 7-17 gm/L 10 7-17 gm/L 30-95  g/dl 200-800 pg/ml 0.Male Creatine kinase Female Lactic dehydrogenase Lipase 5' .67 X 10-8 SI units 2.739 1.3-1.7-62.7-1.67 X 10-8 1.globulin Protein electrophoresis Alpha -2.7-1.67 X 10-8 1.1 mEq/L 270-290 mOsm/kg 0.1 IU/L 25-115 IU/L Up to 185 IU/L Up to 150 IU/L 90-250 IU/L 4-24 IU/dl 2-16 IU/L Factor 1.globulin 60-135  g/dl 250-350  g/dl 16-57% 0.7-24.7-1.2  mol/L 44.5 X 10-8 Katal/L 42-192 X 10-8 Katal/L Up to 309X10-8 Katal/L Up to 251X10-8 Katal/L 150-417 X 10-8 Katal/L 40-240 IU/L 3-27 X 10-8 Katal/L .2 gm/dl 10 3-12 gm/L Protein 0.3 mmol/L 1.Iron .67 X 10-8 1.1 mmol/L 270-290 mOsm/kg 1-5 gm/L 0.3-1.5-13.3-1.6  mol/L 16-57% 0.67 X 10-8 1.5-2.Nucleotidase Current units 1.1-0.32  mol/L 148-591 pmol/L Other common serum enzymatic activities Test Aldolase Amylase Creatine kinase .67 X 10-8 10 1.

Phosphatase, acid

Up to 0.7 IU/L 1.67 X 10-8

Up to 1.2X10-8 Katal/L

Common Serum Hormone Values Test ACTH, fasting (8 AM) Aldosterone Cortisol (plasma, morning) FSH - Male FSH Female Follicular FSH Female - Luteal FSH Female Midcycle FSH Female Postmenopausal Gastrin, fasting Growth hormone, fasting 17Hydroxyprogesterone - Prepubertal Current units 20-100 pg/ml 10-160 ng/L 8-25  g/dl Up to 20 IU/L Up to 20 IU/L Up to 15 IU/L 15-30 IU/L >40 IU/L Up to 130 pg/ml <5 ng/ml 3-90 ng/dl Factor 0.22 2.77 0.027 1 1 1 1 1 1 1 SI units 4.4-22 pmol/L 28-443 mmol/L 0.22-0.57  mol/L Up to 20 IU/L Up to 20 IU/L Up to 15 IU/L 15-30 IU/L >40 IU/L Up to 130 ng/L <5  g/L

1727-199 ng/dl Hydroxyprogesterone - Male, adult 17Hydroxyprogesterone - Female - Follicular 15-70 ng/dl

1735-290 ng/dl Hydroxyprogesterone - Female - Luteal Insulin, fasting (72 hr) LH - Male LH - Female - Luteal LH - Female Midcycle <10 mU/L Up to 25 IU/L Up to 25 IU/L 50-150 IU/L 1 1 1 1 1 <10 mU/L Up to 25 IU/L Up to 40 IU/L Up to 25 IU/L 50-150 IU/L

LH - Female -Follicular Up to 40 IU/L

LH - Female Postmenopausal Parathyroid hormone Progesterone - Male Progesterone Female -Follicular Progesterone Female - Luteal Progesterone Female -1st trimester Prolactin - Male Prolactin - Female Renin activity (plasma) Testosterone, total Male Testosterone, total Female Testosterone, free Male Testosterone, free Female Thyroxine, total ( T4 ) Thyroxine, free T3 resin uptake Triiodothyronine (T3) T4 index TSH Vitamin D, 1,25 dihydroxy Vitamin D, 25 hydroxy

>30 IU/L 2-10 U/ml Up to 100 ng/dl Up to 150 ng/dl 250-2800 ng/dl 1300-5000 ng/dl 2-12- ng/ml 2-20 ng/ml 0.9-3.3 ng/ml/hr 280-1000 ng/dl 20 -120 ng/dl 52-280 pg/ml 1.1-6.3 pg/ml 5.0-11.0 ug/dl 1.0-2.3 ng/dl 35-45% 100-216 ng/dl 1.75-4.95 Up to 10  U/ml 20-76 pg/ml 10-55 ng/m

1 1

>30 IU/L 2-10 arb units

1 1 0.278 0.0346

2-12 ug/L 2-20 ug/L 0.2-0.9 ng/L.s 10-35 nmol/L 1-4 nmol/L

0.00346

0.18-1.0 nmol/L 4-22 X 10-³ nmol/L

12.9 12.9 0.01 0.0154 1 1

64-142 nmol/L 13-30 pmol/L 0.35-0.45 arb units 1.54-3.23 nmol/L 1.75-4.95 arb units Up to 10 mU/L

Common Urinary Chemistries Test Current Factor SI units

units -aminolevulinic acid  Amylase Calcium Catecholamines Dopamine Epinephrine Norepinephrine Copper Cortisol, free Creatinine - Male Creatinine - Female 1.3-7.0 mg/day 0.04-0.30 IU/min < 250mg/day < 135  g/day 90-440  g/day < 13  g/day 11-86  g/day 15-50  g/day 20-90  g/day 1.0-2.0 gm/day 0.6-1.5 gm/day 5.3 7.63 5.5 5.9 0.0157 2.76 0.0088 Up to 71 nmol/day 65-507 nmol/day 0.2-0.78  mol/day 55- 248 nmol/day 0.009-0.018 mmol/day 0.005-0.013 mmol/day 9.5-31.8  mol/day 191-588  mol/day 7.6 1 0.025 1 9.9-53  mol/day 0.04-0.30 IU/min < 6.25 nmol/day Up to 135  g/day

5Hydroxyindoleacetic 1.8-6 mg/day acid Hydroxyproline, total 25-77 mg/day Metanephrine Oxalate PorphyrinCoproporphyrin PorphyrinUroporphyrin Protein Vanillylmandelic acid (VMA) 0.3-1.0 mg/day Up to 40 mg/day 15-125 g/day < 30  g/day < 150 mg/day 0.5-7 mg/day

7.93 1.53 1.2 0.001 5.05

< 317  mol/day 23-191 nmol/day Up to 36 nmol/day Up to 0.150 gm/day 2.5-35.3  mol/day

Common Hematologic Studies Test Current Factor SI units

0-97.66-2.Female Erythrocyte / RBC count .3-22.36 mmol/L 106 4.7 pg/cell 32.062 190-405 X 109/L 1.62 10.8-17.0 X 10¹²/L 106 3.0 sec Prothrombin time Coagulation studies .0 sec 11.7-35.3-18.1 gm/dl 4.5-5.0X106/ l 3.1-15.56-10.Female Hemoglobin .4-0.6 cu  80.0 mmol/L 80.5 gm/dl 1 1 0.7 X106/ l 3.units Coagulation studies Bleeding time Coagulation studies -Partial thromboplastin time 2.5 min 25-41 sec 60 1 150-570 sec 25-41 sec Coagulation studies .9-5.7X10³/ l 1.7-33.8-9.8-13.Male Erythrocyte / RBC count .8X 10³/ l 1.62 20.5-9.7-50.5 sec 0.50-9.3-18.95.503 arb units 0.11.10.01 0.3% 13.3X 10³/ l 0.5 sec Thrombin time Hematocrit .2 gm/dl 12.8-6.36-0.44 arb units 8.5-5.7 X 10¹²/L 3.8-13.Female Leukocyte count Leukocyte profile Lymphocytes Leukocyte profile Mononuclear cells Leukocyte profile Granulocytes Platelet count Erythrocyte indices Mean corpuscular hemoglobin Erythrocyte indices Mean corpuscular hemoglobin concentration Erythrocyte indices Mean corpuscular volume 40.1-44.8-9.6 fl .0-97.3% 36.Male Hemoglobin .6X 10³/ l 190 -405 X10³/ l 26.10.7 mmol/L 7.8 X 109/L 106 0.2-3.09 fmol/cell 0.Male Hematocrit .

.....…………………….. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………..2-2...... ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………... ………………………………………………………………………………………………… ………………………………………………………………………………………………… .…………………………………………….... ………………………………………………………………………………………………… ……………………… Objectives: …………………………………………………………………………………………………................…………………….... ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………...0% 1 Up to 30 mm/hr Practical Sheet #( ) Practical Experiment: ........ ………………………………………………………………………………………………….8-14..........Erythrocyte indices Red cell distribution width Erythrocyte Sedimentation rate Reticulocyte count 11.…. ………………………………………………………………………………………………….... ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….……………………..6% Up to 30 mm/hr 0.. Principle: …………………………………………………………………………………………………. ………………………………………………………………………………………………….

……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ……………………………………….………………… .…………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….…………………….……………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………….

……………………. ………………………………………………………………………………………………….……………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………….……………………. ………………………………………………………………………………………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….…………………….Results: …………………………………………………………………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… .……………………. ………………………………………………………………………………………………….

………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………….………………………………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….…………………….…………………….……………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ……………………………………… COMMENTS & QUESTIONS ………………………………………………………………………………………………….……………………. ………………………………………………………………………………………………… . …………………………………………………………………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… ………………………………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………… ………………………………………………………………………………………………… …………………………………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………. ………………………………………………………………………………………………….…………………….

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