STANDARD OPERATING PROCEDURE FOR PROTHROMBIN TIME TEST 1. Put on PPE . Label four tubes (two tubes for sample and two tubes for control), nv 3. Then invert the reagent (Thromboplastin calcium chloride combined reagent) gently several times and mix well prior to pipetting any of this reagent at any step in this procedure. 4, Pipette 25041! of thromboplastin/ calcium reagent into a small glass tube. Place in a 37°C water bath for 2 minutes. 5. Using a calibrated micro pipette, add 50 yl of patient plasma and mix and start the stopwatch. 6. Hold the tube in a water bath and tilt the mixture back and forth looking for a clot formation. When a clot forms, stop the stopwatch and record the time in seconds. 2 Find average of the two results from control and patient sample and report with appropriate units. Convert your results to International normalized ratio (INR) given that international sensitivity index (ISI) is .. 10. Report and comments on your results. Ranges. PT: 11.0 16.0 seconds. INR Normal range for a healthy person is 0.9-1.3, and for people on warfarin therapy, 2.0-3.0, INR of 2.0 — 3.0 for patient receiving warfarin therapy + An INR of 1.0 means that the patient PT is normal. + An INR greater than 1.0 means the clotting time is elevated,an of the ox Results are expressed as the me duplicate reading in: ~ Seconds - Prothrombin ratio zed Ratio (INR) + International Normali 125 seconds, PT Control: 12 tient ? Example: the INR for the pa’ If We got these data PT Patient seconds, and ISI: 1.03 What is PT PT potio = pee = 25/12 = 2.08 Control = (2.08)103 = 2.13 INR = (PT patio) Principle of the test: Plasma or capillary blood is added to a thromboplastin and calcium chloride reagent at 37°C and the time taken for a clot to form is measured. The prothrombin time: is therefore the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added. It measures the function of the Extrinsic Pathway. Reagents: Thromboplastin calcium chloride combined reagent Materials: Water bath, test tube rack, glass test tube, wire hook and stop watch. Capillary blood: This avoids the need to collect venous blood when monitoring patients being treated with warfarin. It should not be used however when a patient is anaemic or polycythaemic (An increase above the normal in the number of red cellsin the blood.) Plasma should then be used. Blood specimen: Use free flowing capillary blood or collect and centrifuge venous blood. Do not refrigerate the venous blood or plasma. Perform the test as soon as possible. Control: Mostly of controls are available within the test kits or alternatively, use rabbit brain capillary reagent.J Ye wep no PROCEDURE FOR TESTING IF PATIENT BLOOD CELLS ARE SENSITIZED Put on PPE Label two test tubes — one for Test and another for Control. Wash patient red cells 3 times Prepare 2-5% in both patient’s cells and control cells. Add 2 drops of sensitized patient’s red cells into the test tube marked ‘Test’ and 2 drops of Sensitized control cells into the tube marked ‘Control’. NOTE. Sensitized control cells ean be prepared in this way (Put 4 drops of incomplete Anti-D in a test tube, Add 1 drop of 50% suspension of washed pooled Group O cells, mix and incubate at 37°C for 30 - 60 minutes, wash the cell 3 times in saline) Add one drop of Anti human Globulin serum into both tubes. Mix well both tubes Centrifuge both tubes gently at 3000 rpm for 30 seconds Re-suspend the cell buttons and examine for agglutination.f PROCEDURE FOR CROSSMATCH/COMPATILITY TEST Label tube with patient and unit identification. Prepare a 2-5% suspension of donor cells in saline. Add 1 drop of donor's 2-5% red cell suspension to the tube. Add two drops of patient serum to the tube. Centrifuge at 1000 rpm for 30 seconds (Immediate spin) Examine for agglutination macroscopically by tilting the tube gently. If there is agglutination then report. If there is no agglutination then proceed to step 9 PEN Awe E De Incubate at 37°C for 30-60 minutes. Wash the cells 3 times with saline and decant supernatant completely after = last wash. 11. Add one drop of antihuman globulin reagent to the dry cell button. Centrifuge at 1000 rpm for 30 seconds and examine for agglutination macroscopically and microscopically. 13. Report and interpretPROCEDURE FOR DIFFERENTIAL WBC COUNT |. Take a clean slide. Label the slide with the question number or sample mark by writing with a Pon pencil on the frosted edge of the slide, 3. Place a small drop of blood near frosted edge and prepare a thin blood film on a clean slide and let air dry, . allow to dry ona flat surface Stain with leishman stain by flooding the smear on the slide for 2min 4, 5. 6. Double the volume with water 7 min 7. wash in water, wipe at back of the slide 8. Allow to dry. 9. Place the slide on the microscope stage. 10.Scan briefly using the lowest power objective available (x 10 and x40 objective lens) to assess the quality of the film and staining technique. 11.Place a drop of immersion oil on the dried stained film towards the tailed edge. 12.Use x 100 objective lens count each type of WBC in 100 wbcs consecutively. 13.Report the result in percentage (%) and interpretPe ee. PROCEDURE FOR DIRECT COOMB’s TEST put on PPE Label two test tubes — one for Test and another for Control. ‘Wash patient red cells 3 times Prepare 2-5% in both patient’s cells and control cells. Add 2 drops of patient’s red cells into the test tube marked ‘Test’ and 2 drops of Sensitized cells into the tube marked ‘Control’. Add one drop of Anti human Globulin serum into both tubes. Mix well both tubes Centrifuge both tubes gently at 3000 rpm for 30 seconds Re-suspend the cell buttons and examine for agglutination.RATING FOR PROCEDURE FOR EOSINOPIIL COUNT Fs siet and place in a rack, # Fiptte 380 nt of dilating Feagent into two test tubes. nipette 201tL of whole blood sample (El enous 2 ir i a ae iple (EDTA venous or capillary) Expel the blood into the Repeat pipetting 20 tL whole blood, expelling for the second test tube. Mix each tube well and leave for about 2 minutes at room temperature. Clean the improved neubauer chamber and cover glass by flooding them with 70% alcohol. Dry thoroughly with gauze or tissue, do not allow the alcohol to dry on the neubauer chamber. Be sure to remove all lint, 7. Place the cover glass in position over the rulled area of the improved neubauer chamber. 8. Following incubation, mix diluted blood thoroughly by inverting the tube by tapping the bottom of the tubes a few times to re-suspend the cells. Charge the improved neubauer chamber as follows. a) By use of micro pipette place draw the diluted blood and the place the pipette tip on the edge of the ruled and expel the contents until the chambers are properly filled. b) Repeat the procedure to charge the other side of the improved neubauer chamber. ©) Place the improved neubauer chamber on moisten filter paper in a Petri dish and allow standing for 10 minutes to permit the cells to settle. Use the same procedure, charge the a second improved neubauer chamber for Control. Carefully place the improved neubauer chamber on the microscope stage. Perform cell count d) as follows. 1a) With the low power (x10) objective, locate the ruled area and upper left large square. Eosinophil upper bright orange-red b) Eosinophils are counted in the entire ruled area following standard counting procedure. Repeat this counting procedure forthe other side ofthe improved neubauer chamber. Records the counts for each side of the improved neubauer chamber Count the eosinophils on the second improved neubauer chamber following the above counting procedure. °) 4) °) Calculation. The same general formula used to calculate the leukocyte count is also applied for the Eosinophils count. Calculate the Eosinophils count for each improved neubauer chamber and the average results is (x 10% or /mm? Eosinophils /L = number of eosinophils counted x 50 = number of eosinophil /mm? Reference Interval Adults 0- 0.45x x 10°L Birth 0- 0.9 x 10°7/L 6 Months 0-0.9 x 10° /L. 4 Years 0- 0.7 x 10°/Lorl UNT STANDARD OPERATING FOR PROCEDURE FOR EOSINOPHIL CO" 9. Label a test tube and place in a rack. ipette 380 wl of diluting reagent into two test tubes. Pipette 2011 of whole blood sample (EDTA venous or capillary) Expel the blood into the diluting fluid in one test tube. n. 12. Repeat pipetting 20 iL whole blood, expelling forthe second test tube. 13, Mix each tube well and leave for about 2 minutes at room temperature. "4 Clean the improved neubauer chamber and cover glass by flooding them with 70% aleohol. Dry thoroughly with $IuZE oF tissue, do not allow the alcohol to dry on the neubauer chamber. Be sure to remove all lint 18: Place the cover glass in positon over the rlled area ofthe improved neubauer chamber. 16. Following incu B ibation, mix diluted blood : ‘thoroughly by inverting the tube by tapping the bottom of the tubes a few times to resuspend the cells. ‘Charge the improved neubauer chamber as follows, By use of micro pipette ° place draw the diluted blood and the place the pipette tip on the edge of the ruled and ‘expel the contents until the chambers are properly filed Repeat the procedure to charge the other side ofthe improved neubauer chamber. Place the improved neubauer chamber on moisten filter paper ina Pet dish and allow standing for 10 minutes to permit the cells to settle, ‘Use the same procedure, charge thea second improved neubauer chamber for Contr Carefully place the improved neubauer chamber on the microscope stage. Perform cell count as follows. 5 With the low power (10) objective, locate the ued area and upper lef large square, Eosinophil upper bright orange-red ») 8) Eosinophils are counted inthe entire ruled area following standard counting procedure, 1) Repeat this counting procedure forthe other side ofthe improved neubauer chamber 4) Records the counts for each side of the improved neubauer chamber 3 Count the eosinophils on the second improved nevbauer chamber following the above counting, Procedure, Calculation. ‘The same general formula used to calculate the leukocyte count is also applied for the Eosinophils count. ‘The variation wil be in the dilution and volume factor (or Fuchs- Rosental counting chamber) For eosinophils counts the dilution is 1:20 and the volume counted is 0.4, Calculate the Eosinophils count for each improved neubauer chamber and the average results is (x 10" or from? Eosinophils /L = number of eosinophils counted x 50 = number of eosinophil / mm? Eosinophils /L =number of eosinophils counted x 50 number of eosinophil X 10°/L, Reference Interval Absolute count above (400/mm?) of blood is Eosinophilia, Absolute count between (100 to 400/mm?) of blood is normal Absolute count less than 90/mm’) of blood Eosinopenia Or Adults: 0- 0.45 x 10% Birth: 0-1.5 x 107, 6months: 0-0.9 x 10%. 4 years: 0-0.7 x 10°7LSOP LOR G6 PD Dn 1 LOR G6 , HMICIENCY TEST (METH AEMOGLORIN- REDUCTION TI NIQUE) Aettont Poca URL fi —______ pecdoue the Hollow ing procedures us indicated in the table below: . tube Nowmat control | Positive control ‘Test control “Patient's blood onus. 2mls 2mls; GORD reagent O.tmis 0.1mis : Otis: | Fate Methylene btue “/ Stopper the tubes and mix gently, and then incubate al 37°C for 90 minutes xed in step 7. Then pipette TOmls of T Take theve large tubes (15 | exch tube j $ Transter OL ml of well n ample from the Normal, Deficient and Test tubes into the large tubes | respectively, | ° | Wait tor 2-10 minutes TF Bramine the color of the solution in each tube. Calculation/Interpretation Normal: Clear red Deficient: — Brown NOTES: GEPD REAGENT PREPARATION 1, Dissolve 0.5g of Sodium nitrite and 2g Glucose in 40mls Distilled water. METHYLENE BLUE REAGENT 1. Dissolve 0.1g in 100mls of water.PROCEDURE FOR INDIRECT COOMB’S TEST Put on PPE Prepare 3% cells suspension of washed O cells and 5 drops Arrange two test tubes, one marked test (T) and another marked control (C). Add 4 drops of patient serum in tube marked T and 4 drops of Incomplete Anti-D into the tube marked C Add one drop of 3% suspension of washed O cells into both tubes ° Incubate both tubes at 37 C for 30 minutes Wash both tubes three times by using saline and remove the supernatant Add one drop of Antihuman Globulin antisera (AGS) in to both tubes Spin immediately, mix and report your findings.tANDARD OPERATING . ‘OUNT FOR PROCEDURE FOR RETICULOCYTE, 1, Put on PPE iver t aaa Deliver two or three drops of stain into a tube, and add approximately equal p volume of patient's blood. 3. Mix and leave in water bath or incubator at 37°C for 15 minutes. 4, Resuspend the red cells by gentle mixing and make a thin film in the usual way. 5. When dry, the films are examined without counter-staining. The reticular material should be stained deep blue and the non-reticulated cells shades of pale greenish-blue. 6. Using the x100 oil-immersion lens, choose an area of the field where the cells are undistorted and the staining is good. 7. Count the number of reticulocytes seen per 1000 red cells. 8. Report in percentage the results and interpret Reports Normal 0.5-2.5% Low count <0.5% High count >2.5%APTT 1. Pipette 0.2m! of well-mixed kaolin/platelet substitute in a small glass tube. 2. Add 0.1ml of plasma, mix, and incubate at 37°C for exactly 2 minutes (tilting the tube at intervals) 3. Add 0.1 ml 0.025 mol/| calcium chloride, mix and start the stopwatch. 4. Hold the tube in the water bath and tilt the mixture back and forth, looking, for clot formation. When a clot forms, stop the stopwatch and record the time. 5, Report the patient’s APTT (average of the duplicate tests) providing the APTT of the normal control plasma is satisfactory. Normal plasma clots in 36-50 seconds Principle of the test: ©. kaolin (surface activator) and platelet substitute (phospholipid) are incubated with citrated plasma at 37°C for the time specified in the test method. o Calcium chloride (CaCl,) is added and the time taken for the mixture to clot is measured «Blood specimen: Collect 9 mls of venous blood (well taken with minimum of stasis) into a plastic tube containing 1 ml of aqueous tri-sodium citrate anticoagulant (32g/l). Mix the blood well with the anticoagulant, Without delay, centrifuge the blood at 1200 r-2000 r for 15 minutes. + This will provide platelet poor plasma. Immediately remove the plasma into a plastic tube (vial) and stopper. If a delay in performing the APTT is unavoidable, refrigerate the sample at 4-8°C. If not refrigerated, the test should be performed within 1 hour of collecting the blood.Steps in the Group A Subgroups Test 1. Label the test tubes for test and control accordingly. 2. Add 1 drop of anti-sera to the corresponding test tubes 3. Add 1 drop of washed cells into the respective test tubes and mix 4, Incubate at room temperature for 30 to 60 minutes.7 5. Re-suspend and examine for agglutination macroscopically Controls: Known A1 and A2 cells Interpretation of results: ‘Al control cells | A2 control cells | Test 1 Test 2 ‘Anti Al [++ : + 5 rare Anti |- cans z Results | Subgroup Ai | Sub group Sub group Ai | Sub group a2 a2HAEMOLYSIN TEST SOP for Haemolysin test Arrange three tubes and mark them A, B and O cells Put 9 drops of the test serum into each tube accordingly Add one drop of 50% washed A, B and O cells into corresponding tubes. 4, Incubate the tubes at 370C for one hour. 5. Spin at 1000 rpm for 30 seconds. 6. Look for haemolysis + Haemolysis means presence of IgG, haemolytic ABO antibodies + No Haemolysis ~ means absence of IgG, haemolytic ABO antibodiesi ple of the erythrocyte osmotic fragility tes Osmotic Fra; Test: Red cells are placed in varyi ing concentrations of hypotonic solutions 1° test their ability to wi ty to withstand different salt concentrations, One observes when (at which concentrati ion) haemolysis first occurs. Principle: iple: When RBCs are placed in a hypotonic solution, H:0 is drawn into the red cells. As H:0 enters, ;, the cell swells and spheres. When the cell reaches 2 critical volume, the membrane leaks and red cell lysis occurs. SOP 1. Specimen requirements: Must set up 2. Steps: 3. Prepare 14 4, Add imlto NB: In hypertonic solutions: Water is drawn out of the red cell. control and patient within 2 hours of drawing blood. dilutions of NaCl in distilled water starting from 0.20 g/dl (0.20% saline) to 0.85 /¢! (0.85% saline). each test tube and put few drop of well washed blood. 5, Stopper the tubes and mix by inversion, 6, Allow the tubes to stand at room temperature for2 7. Spin tubes it in centrifuge for 5 minutes at 2000 rpm a hours then re-mix them the same way. nd look for hemolysis.