Manual 4th year Advance 2021.pdf

SHARQ ELNEIL COLLEGE SCHOOL OF MEDICAL LABORATORY SCINCES DEPARTMENT OF CLINICAL CHEMISTRY 4thYear Practical Manual& Application Dr. Zuhair Yagoub Abdelkarim B.Sc, M.Sc, PhD, MLS (ASCPi) Assistant Professor Clinical Chemistry  CARBOHYDRATE D...

SHARQ ELNEIL COLLEGE SCHOOL OF MEDICAL LABORATORY SCINCES DEPARTMENT OF CLINICAL CHEMISTRY 4thYear Practical Manual& Application Dr. Zuhair Yagoub Abdelkarim B.Sc, M.Sc, PhD, MLS (ASCPi) Assistant Professor Clinical Chemistry  CARBOHYDRATE DISORDERS Clinical Chemistry Department.Metabolic Disorders (4th year) Page 2 Date………….. Practical No ( ) 4th year Metabolic disorders Types of investigations and their usefulness (Carbohydrate) Aim To differentiate between types of investigations used for screening, diagnosis and monitoring of carbohydrate disorder (ie Diabetes Mellitus) Investigations are classified into 1. Screening 2. Diagnostic 3. Monitoring  Screening& Diagnostic Tests for carbohydrate Screening test include Urine ,Fasting , 2hrpost Prandial( 2hrs after diet contain 100g carbohydrate or 2hr post 100g glucose load)  Diagnostic test Fasting + 2hrs post Prandial , Glucose tolerance test (GTT)  Monitoring Random blood glucose(RBS), Hemoglobin A 1C(HbA1C)  Some times( RBS) is used as diagnostic test (>200+classical symptoms of diabetes)WHO criteria- repeat in subsequent day Samples  Urine, fluoride oxalate plasma(Fasting+ 2hr postPrandial)  GTT 1 fluoride oxalate plasma( fasting ,1hrs after glucose dose ,2hr after glucose dose ) 2 urine(fasting ,1hrs after glucose dose ,2hr after glucose dose) Reagents 1. Urine strip a. Glucose : impregnated glucose reagent (glucose oxidase peroxidase dry reagent) buffer Chromogen (ie potassium iodide) b. Ketone:Sodium nitroprusside, buffer (dry reagent) 2. Glucose mono reagent: phosphate buffer 100 mmol\l, PH 7.5, glucose oxidase > 10 u/l, peroxidase >1 u/l, 4-aminoantipyrine 0.4 mmol/l, phenol 5 mmol\l. 3. Glucose standard: 100 mg\dl Principles Clinical Chemistry Department.Metabolic Disorders (4th year) Page 3 In the trendier reaction, the glucose is oxidized to D-gluconate by the glucose oxidase (GOD) with the formation of hydrogen peroxide. In the presence of peroxidase (POD), mixture of phenol and 4-amino antipyrine (4-AA) is oxidized by hydrogen peroxide, to form a red quinoneimine dye proportional to the conc. the sample. Beta-D- GLUCOSE + H2O + O2 ----GOD---- → D- GLUCONATE +H2O2 4-A.A + PHENOL + H2O2 ---POD---- → quinoneimine +H2O For urine Glucose the same principle except it’s a dry reagent Ketone reacts with sodium nitroprusside to produce a color change range from light pink with negative results to dark pink or purple with positive results. Procedures For plasma Blank STD Test Working reagent 1ml 1ml 1ml SDT 100mg/dl ----- 0.01ml ----- Sample ------- ------ 0.01ml Mix well; incubate for 10 min at R.T, read the absorbance of test and std against reagent blank using 520 nm (green) filter. For urine 1- insert the strip in the fresh urine 2- take the strip out of urine and wipe out the excess urine 3- Compare the color obtained after 30 – 60 sec with the color given on the strip container. Calculation: O.D of test / O.D of STD × conc of STD = conc. Of test (mg\dl) Unites 1. OLD UNIT: mg/dl 2. S.I.U: mmol\l C.F: 0.055 Sensitivity Plasma glucose 0.23mg\dl Urine glucose 50—100 mg/dl Linearity: Clinical Chemistry Department.Metabolic Disorders (4th year) Page 4 Plasma up to 500 mg\dl For higher values dilute sample ¼ with D.W and repeat measurement DF=4 Interferences: Hb>3g/L, Lipemia Triglycerides>125mg/d l, Bilirubin 10mg/dl Reference values F.B.S: Normal 70-----110mg\dl Impaired 110----126mg/dl (this need confirm by GTT) Diagnostic more than 126mgldl 2 Hours Post Prandial B.S: Normal: 80-----120mg\dl Suspicion 120----140mg/dl (this need confirm by GTT) Diagnostic more than 140mg/dl GTT: Normal patient response; FBS as normal (70---110mg\dl); 1 hour < 180 mg\dl; 2 hours < OR = fasting level. (Urine sugar is negative) Diabetes Mellitus response according to severity of disease as fasting> OR = 140 mg \dl, 1 hour > 200 mg \dl 2 hours > OR =200 mg\dl.( Urine sugar is positive in most of specimens) ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. Clinical Chemistry Department.Metabolic Disorders (4th year) Page 5 ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Clinical Chemistry Department.Metabolic Disorders (4th year) Page 6 ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ………………………………………………………………………………………………… Clinical Chemistry Department.Metabolic Disorders (4th year) Page 7 Date………….. Practical No ( ) 4th yearMetabolic disorders HEMOGLOBIN A1C Chromatographic - Spectrophotometric ION EXCHANGE Aim To measure hemoglobin A1C concentration in human blood Reagents 1 Reagent Potassium phthalate 50 mmol/L, detergent 5 g/L, pH 5.0, sodium azide 0.95 g/L 2. Reagent Phosphate buffer 30 mmol/L, pH 6.5, sodium azide 0.95 g/L 3. Reagent Phosphate buffer 72 mmol/L, pH 6.5, sodium azide 0.95 g/L 4. Microcolumns Contain a pre-weighted amount of resin equilibratedwith phosphate buffer 72 mmol/L, pH 6.5, sodium azide 0.95 g/L Samples Whole blood collected by standard procedures. Hemoglobin A1C is stable for 7 days at 2-8ºC. Heparin or EDTA may be Used as anticoagulants. Principle After preparing the Hemolysate, where the labile fraction is eliminated, hemoglobin’s are retained by a cationic exchange resin. Hemoglobin A1C (HbA1c) is specifically eluted after washing away the hemoglobin A1a+bfraction1 (HbA1a+b), and is quantified by direct photometric reading at 415nm. Procedure  Hemolysate Preparation and Labile Fraction Elimination 1. Bring the columns and reagents to room temperature (21-26ºC)(Note 1). 2. Pipette into a test tube: Blood 50 µLReagent (1) 200 µL 3. Shake thoroughly and let it stand at room temperature for 10-15minutes. This Hemolysate will be used in steps 6 and 11.  Column Preparation (Notes 2 and 3) 4. Remove the upper cap of the column and then snap the tip off thebottom. Clinical Chemistry Department.Metabolic Disorders (4th year) Page 8 5. Using the flat end of a pipette, push the upper disc down to the resinsurface taking care not to compress it Let the column drain completelyto waste  Separation and Reading of HbA1c fraction 6. Carefully pipette on the upper filter:Hemolysate 50 µL Let the column drain to waste 7. In order to drain any sample residue left above the upper disc pipette:Reagent (2) 200 µL Let the column drain to waste 8. Pipette:Reagent (2) 2.0 mL Let the column drain to waste 9. Place the column over a test tube and add:Reagent (3) 4.0 mL Collect the elute (HbA1c Fraction) 10. Shake thoroughly and read the absorbance (A) of the HbA1c fraction at415 nm against distilled water (AHbA1c). The absorbance is stable for atleast one hour.  Reading of HbTOTAL 11. Pipette into a test tube: Reagent (3) 12.0 mLHemolysate 50 µL 12. Shake thoroughly and read the absorbance (A) at 415 nm againstdistilled water (HbTOTAL) The absorbance is stable for at least onehour. Calculation The concentration of Hb A1c in the sample can be calculate using the following formula 𝐴 𝐻𝑏 𝐴1𝑐 × 𝑉 𝐻𝑏𝐴1𝑐 × 100 = %𝐻𝑏𝐴1𝑐 𝐴𝐻𝑏 𝑇𝑜𝑡𝑎𝑙 × 𝑉 𝐻𝑏 𝑇𝑜𝑡𝑎𝑙 Volume of HbA1c (V HbA1c)= 4ml and Volume of Hb Total (V Hb Total)= 12ml The Formula become 𝐴 𝐻𝑏 𝐴1𝑐 100 × = %𝐻𝑏𝐴1𝑐 𝐴𝐻𝑏 𝑇𝑜𝑡𝑎𝑙 3 Reference Values 4.4 - 6.7 % Non-Diabetic 6.7 - 7.3 %Goal 7.3 - 9.1 %Good Control > 9.1% Action suggested Sensitivity Clinical Chemistry Department.Metabolic Disorders (4th year) Page 9  Lower than 4.3 %. Linearity: At least 17.0 %   Interferences: Bilirubin (20 mg/dL) and lipemia (triglycerides 10 g/L) don’t interfere. Some drugs and other substances may interfere In the ionic exchange chromatographic methods, the presence ofhemoglobin C or S in the sample may slightly alter results, butdifferences are not clinically significant. Other hemoglobin variants likeHbE, HbF, carbamyl-Hb and acetyl-Hb can interfere5 …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. Clinical Chemistry Department.Metabolic Disorders (4th year) Page 10 ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. …………………………………………………………………………………………………….. ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… …………………………………………………………………………………………………….. Clinical Chemistry Department.Metabolic Disorders (4th year) Page 11 Date………….. Practical No ( ) 4th year metabolic disorders Case Study ( ) Carbohydrate Disorders ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Clinical Chemistry Department.Metabolic Disorders (4th year) Page 12  Assessment OF RENAL function Assessment of Tubular Function (B2 microglobulin) Assessment of GFR (Clearance Test) Assessment of Nephrotic Function (Protein Excretion, Albumin/Creatinine Ratio& Urea/Creatinine Ratio) Clinical Chemistry Department.Metabolic Disorders (4th year) Page 13 Date............. Practical No( )4th year Metabolic disorders Renal Function Assessment Aim To assess renal function  ESTIMATION OF BLOOD UREA Reagents: 1/ R1 (Buffer): Phosphate pH 6.7, EDTA, Sodium salcylate and Sodium nitroprusside. 2/ R2 (NaCIO): Sodium hypochlorite and Sodium hydroxide. 3/ R3 (Enzymes)): Urease (tablets) 4/ CAL urea standardUrea 50 mg/dl 8.3 mmol/l Organic matrix based primary standard. Sample: Serum or heparin plasma freeof hemolysis NOTE; if we use urine sample must be diluted 1:50 Principle: Urea is hydrolyzed by Urease into ammonia and carbon dioxide. The ammonia generated reacts with alkaline hypochlorite and sodium salcylate in presence of sodium nitroprusside as coupling agent to yield a blue cromophore color known as endophenol.The intensity of the color formed is proportional to the concentration of urea in the sample. Urea + H2O ----Urease---- → 2NH3 +CO2 NH4 + Salcylate+ NaCIO---nitroprusside and OH---→ Endophenol +NaCL Procedure: Test STD Blank R1 1ml 1ml 1ml Sample 0.01ml STDW.STD 50mg/dl 0.01ml Mix well; incubate for 10 mn at R.T. Mix and incubate for 10 mn at R.T. read the absorbance of sample and standard at 600 nm R2 1 ml. 1ml. 1 ml. against reagent blank. Calculation: Clinical Chemistry Department.Metabolic Disorders (4th year) Page 14 O.D of test / O.D of STD ×Con of STD. = conc. Of test (mg\dl) Units: 1/ OLD UNIT: mg\dl. 3) BUN BUN= mg/dl/2.14 2/ S.I.U: mmol\l. mmol/L= mg/dl x10 /M.W or mg/dlx0.17 C.F: 0.17 ReferenceValues: Adults: Blood 15----50 mg\dl Urine: 20-----35 g/24 Sensitivity: 0.002mg\dl. Linearity: Up to 300 mg\dl Interference Factors: Any contamination by ammonia or ammonium salt The working enzyme reagent is not stable at elevated temperatures  ESTIMATION OF CREATININE BY JAFFE REACTION Reagents Composition: 10% sodium tungestate 2\3 N H2SO4 Sodium Hydroxide 0.75 mol/L Picric acid (saturated) Creatinine Standard: (0.5-2mg/dl) Samples: Serum, plasma or urine collected by standard procedures. Dilute fresh urine 1/100 with D.W. Heparin, EDTA, oxalate and fluoride may be used as anticoagulants. Principle: Creatinine in the sample, after removal of proteins, reacts with picric acid in alkaline media (alkaline picrate) forming red- orange chromogen (adduct) which absorbed colorimetrically at (480- 520) nm. Clinical Chemistry Department.Metabolic Disorders (4th year) Page 15 Procedure :( Endpoint) Step 1 Into centrifuge tube, take the followings: 1ml serum, 1ml 10% Na2WO4, 1ml 2/3 H2SO4 and 1ml D. W Mix well, centrifuge for 3 minutes then: Step2 Test STD Urine Blank Supernatant 1.5ml............................. W.STD......... 1.5ml.................... Diluted urine................. 1.5ml........... 1/100 D.W.............................. 1.5ml NaOH 0.75N 0.5ml 0.5ml 0.5ml 0.5ml Picric acid 0.5ml 0.5ml 0.5ml 0.5ml Mix well; incubate for 10 minutes at RT, read at 490nm. Calculations: Serum in mg/dl=O.D Test/O.D STD X CONC.STD X DF (4) Urine in mg/dl= O.D Test/O.D STD X CONC.STD X DF (100) Unite Old unit: mg/dl (M.W=113). S.I.U: μmol/L mg/dl x10000/M.W Reference Value: SERUM: Male: 0.9---1.3 mg/dl. (80---115) μmol/L Female: 0.6----1.1mgldl (53---97) μmol/L URINE: 100-----200mg/dl 1------2 g/day Linearity: Up to8 mg\dl if the value exceed this level dilute the sample with D.W repeat the assay and multiply by D.F Interference Factors: Pseudocreatinine (ascorbic acid, acetoacetate, acetone, glucose, pyrvate) Clinical Chemistry Department.Metabolic Disorders (4th year) Page 16 CREATININE BY KINETIC METHOD Reagents Composition: Reagent (A): Sodium Hydroxide 0.4 mol/L Reagent (B): Picric acid 25 mmol/L Creatinine Standard: 2 mg/dl (177μmol/L) Reagents Preparation: Working Reagent: Mix equal volumes of reagent A and reagent B. (0.5ml reagent A + 0.5 ml reagent B) Samples: Serum, plasma or urine collected by standard procedures. Dilute fresh urine 1/100 with D.W. Heparin, EDTA, oxalate and fluoride may be use as anticoagulants. Principle: Creatinine in the sample reacts with alkaline picrate forming a colored complex. The complex formation rate is measured in a short period to avoid interferences. Procedure: 1. Pipette into a cuvette: Working reagent 1.0 ml STD or Sample 0.1 ml 2. Mix and insert cuvette into the photometer. Start stopwatch. 3. Record the absorbance at 490 nm after 30 second (A1) and after 90 second (A2). NOTE The test is time and temperature sensitive it is essential to maintain the reaction timings and temperature during the test procedure Calculations: (A2-A1) Sample/ (A2-A1) STD x Conc. STD x D.F if present= Conc. of sample. Unite: Old unit: mg/dl (M.W=113). S.I.U: μmol/L mg/dl x10000/M.W Reference Value: SERUM: Clinical Chemistry Department.Metabolic Disorders (4th year) Page 17  Male: 0.9---1.3 mg/dl. (80---115) μmol/L Female: 0.6----1.1mgldl (53---97) μmol/L Linearity: Up to20 mg\dl if the values exceed this level dilute the sample with D.W repeat the assay and multiply by D.F Interference Factors: High level of ascorbic acid false increase Drug such as diuretics and Dextran A diet with high roast meat false increase Measurement urine protein Reagents  3% T.C.A  Protein standard 100mg/dl.  Principle: Trichloroacetic acid(T.C.A)denaturise the urine protein as strong acid and so caused turbidity directly proportional with conc. of protein present in urine sample and can be measured using blue filter(430 nm). Sample: 24 hr urine sample collected under certain construction (not diluted). Procedure: REAGENT BLANK STD TEST 3% T.C.A 1.5ml 1.5 ml 1.5 Ml D.W 0.5 ml ------ ------ W.STD (100 mg/dl.) ------ 0.5ml ----- Urine sample ------ ------- 0.5 ml Mix well; incubate for 5 min at R.T. Read the absorption of TEST and STD against BLANK. At 430 nm Clinical Chemistry Department.Metabolic Disorders (4th year) Page 18 Calculation: 𝑂.𝐷𝑇−𝑜.𝐷𝑇𝑐 Urine protein mg/dl=: × 𝐷𝑓 × 𝑠𝑡𝑑𝑐𝑜𝑛 𝑂.𝐷𝑠𝑡𝑑 D f=10 Interferencefactors: Amorphous urate, epithelial cell and casts, for calculation Sensitivity: