3..Manual CBC.pptx

complete blood count

1. Manual CBC Aliaa Adel EL- Feshawy , MSc Clinical Pathology Department Faculty of Medicine-Menoufia University

2. Manual techniques Despite the widespread use of automated instruments, the manual techniques remain important both as reference methods and in the investigation of blood samples that appear to give discrepant results with automated analyzers.

3. Hemoglobin concentration (1) Cyanmet -hemoglobin method is the Reference Method for determining the Hb concentration of blood. Principle: Dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Hb, Met-Hb & carboxy-Hb but not S-Hb are converted to Cyanmet-Hb . The absorbance of the solution is measured in a spectrophotometer at 540 nm (546 nm Filter).

4. Diluent The modified solution, Drabkin -type reagent pH 7.0–7.4. The diluent should be clear and pale yellow in color. If stored at room temperature, in a brown borosilicate glass bottle, the solution keeps for several months. If the ambient temperature >30°C, the solution should be stored in the refrigerator but brought to room temperature before use. Reagent must be discarded if: Turbid. pH outside 7.0–7.4 range. an absorbance other than zero at 540 nm against a water blank.

5. Method: Make 1 in 201 dilution of blood by adding 20 μl of blood to 4 ml of diluent . Stopper the tube containing the solution and invert it several times. Let the test sample stand at room temperature for at least 5 min. Pour into a cuvette and read the absorbance in a spectrometer at 540 nm against a reagent blank. The absorbance of the test sample must be measured within 6 hours of its initial dilution.

6. Standard The absorbance of a commercially available cyanmet -hemoglobin standard should also be compared to a reagent blank in the same spectrometer as the patient sample. The standard should be kept in the dark and, to ensure that contamination is avoided, any unused solution should be discarded at the end of the day on which the ampoule is opened. Calculation of Hb Concentration Hb (g/l) = A* of test sample / A of standard × Conc. of standard × Dilution factor (201)/1000 A* = absorbance

7. (2) Direct Spectrometry Hb of a diluted blood sample can be determined by spectrometry without the need for a standard, provided that the spectrometer has been correctly calibrated. The blood is diluted 1:251 with cyanide–ferricyanide and the absorbance is measured at 540 nm. Hb is calculated as follows: Hb ( g/l) = (At × 16114 × dilution factor) / ( 11 × d × 1000) Hb ( g/l) = At × 36.77 At = absorbance of test sample 16114 = monomeric molecular weight of hemoglobin dilution = 251 when 10 µl of blood is diluted in 2.5 ml of reagent 11.0 = milli molar coefficient extinction d = layer thickness in cm 1000 = conversion of mg to g.

8. Packed cell volume (Hematocrit) The packed cell volume (PCV) can be used as a simple screening test for anemia, as a reference method for calibrating automated blood count systems and as a rough guide to the accuracy of Hb measurements. The PCV × 100 is about three times the Hb expressed in g/dl e.g. 0.36 × 100 is approximately 12 (Hb) × 3.

9. Microhematocrit Method The microhematocrit method is carried out on blood contained in capillary tubes 75 mm in length and having an internal diameter of about 1 mm. The tubes may be plain for use with anti-coagulated blood samples or coated inside with heparin for the direct collection of capillary blood. The centrifuge used for the capillary tubes provides a centrifugal force of c 12000 g and 5 min centrifugation results in separation of the column of blood into red cells, buffy coat and plasma.

10. Method Allow blood from a well-mixed specimen or from a free flow of blood by skin puncture, to enter the tube by capillarity, leaving at least 15 mm unfilled. Then seal the tube by a plastic seal. After centrifugation for 5 min, measure the proportion of red cells to the whole column.

11. Factors affecting accuracy of micro-hematocrit method: Anticoagulant: K2-EDTA is recommended because K3-EDTA causes shrinking of the red cells reducing the PCV by about 2%. Anticoagulant concentration in excess of 2.2 mg/ml may also cause a falsely low PCV as a result of cell shrinkage. Blood Sample: The test should be performed within 6 hours of collecting the blood sample but a delay of up to 24 hours is acceptable if the blood is kept at 4°C. Failure to mix the blood sample adequately will produce an inaccurate result.

12. Manual cell counts using counting chambers Counting Chamber (hemocytometer) Improved Neubauer ruling : This has nine 1 mm × 1 mm ruled areas which when covered correctly with the special thick cover glass each contain a volume of 0.1 μl of diluted blood . The sample is introduced between the chamber & the cover glass using a pipette and the preparation is viewed using ×40 objective and ×10 eyepieces. With Improved Neubauer chambers, the cells in squares are counted, including the cells that touch the bottom and left-hand margins of the small squares.

13. Improved Neubauer ruling

14. (1) The red cell count Diluent: saline Dilution 1: 200 Calculation: R1+R2+R3+R4+R5× 10,000 = count/µl Red cell indices MCV(fl) = Hct / RBC MCH(pg) = Hb / RBC MCHC(g/dl) = Hb / Hct

15. (2) The white cell count Diluent: 2 % Glacial acetic Acid colored pale violet with drop of Leishman stain. Dilution: 1: 20 ( 0.1 ml of well-mixed blood “lack of adequate mixing is a major source of error” to 1.9 ml of diluent). Calculation: W1+W2+W3+W4 × 50 = count/µl

16. (3) The platelet count Diluent: 1% aqueous ammonium oxalate (To lyse RBCs) Brilliant cresyl blue can be added to the diluent. This stains platelets light blue and facilitates their identification. Dilution 1: 20 Calculation: P1+P2+P3+P4+P5 × 1000 = count/µl

17. Setting up and using a microscope

18. If you need to move or lift the microscope do so using only the arm . Adjust the position of the eye pieces so that they match your inter-pupillary distance and look at the slide through the eye pieces, making sure that the light is at a comfortable level. Use the coarse and then the fine focus. Examine the slide with the × 10 objective, then rotate in a × 40 objective.

19. When you have finished working, rotate back in the lowest power objective and lower the stage. Do not leave the microscope turned on when you are away from your workstation. Keep the microscope clean. Dust can be removed with a small brush. Lenses should be cleaned only with lens tissues. These can be moistened with methanol. Cover the microscope with a dust cover when not in use.