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What happens in the aperture?

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Presentation on theme: "What happens in the aperture?"— Presentation transcript:

1 What happens in the aperture?
Basics of Hematology cell counting

2 Blood cells Name RBC WBC PLT Population 4.5-5.5 million/µl 5-10.000/µl
Shape, diameter Biconcave shape Diameter  8 µm Thickness  2 µm NE,EO,BA 15 µm LYM  µm MON 15-25 µm Fragments Diameter  2-4 µm Mean volume  90 fl various  12 fl

3 Blood cells – under microscope

4 Hematology parameters 1
White blood cell – WBC (pcs/l, pcs/µl) Number of leukocytes. WBC = WBCcal x (pcs/l or pcs/µl) Red Blood Cell – RBC Number of erythrocytes. RBC = RBCcal x (pcs/l or pcs/µl) Haemoglobin concentration - HGB (g/dl, g/l, mmol/l) Measured photometrically at 540 nm; in each cycle blank measurement is performed on diluent HGB = HGBcal x (HGBmeasured – HGBblank) Mean Corpuscular Volume - MCV (fl=10-15 l) Average volume of individual erythrocytes derived from the RBC histogram. Haematocrit – HCT (percentage, absolute) Calculated from the RBC and MCV values. HCTpercentage = RBC x MCV x 100, HCTabsolute = RBC x MCV Mean Corpuscular Haemoglobin – MCH (pg, fmol) Average hemoglobin content of erythrocytes, calculated from RBC and HGB values. MCH = HGB / RBC

5 Hematology parameters 2
Mean Corpuscular Haemoglobin Concentration – MCHC (g/dl, g/l, mmol/l) Calculated from the HGB and HCT values. MCHC = HGB / HCTabsolute Unit of measurement is displayed according to the one chosen for HGB result (g/dl, g/l or mmol/l) Red cell Distribut. Width - RDW-SD and Platelet Distribut. Width - PDW-SD (fl) Red cell Distribut. Width - RDW-CV Platelet Distribut. Width - PDW-CV (absolute) The distribution width of the erythrocyte or platelet population derived from the histogram at 20% of peak xDW-SD = RDWcal x (P2 – P1) (fl), xDW-CV = RDWcal x 0.56 x (P2 – P1) / (P2 + P1) by the factor of 0.56 CV is corrected to the 60% cut

6 Hematology parameters 3
Platelet – PLT (pcs/l, pcs/µl) Number of thrombocytes (platelets) PLT = PLTcal x (pcs/l or pcs/µl) Mean Platelet Volume – MPV (fl) Average volume of individual platelets derived from the PLT histogram. Thrombocrit – PCT (percentage, absolute) Calculated from the PLT and MPV values. PCTpercentage = PLT x MPV x 100, PCTabsolute = PLT x MPV White blood cell differential: LYM#, LY% : lymphocytes MID#, MID%: monocytes and some eosinophil granulocytes GRA#, GR%: neutrophil, eosinophil and basophil granulocytes Absolute values counted in the channels determined by the three WBC discriminators: Percentages calculated from the absolute WBC value. 1.: RBC-LYM discriminator 2.: LYM-MID discriminator 3.: MID-GRA discriminator 1. 2. 3.

7 Blood sample

8 Impedance cell counting principle
dR/R = 1 / = 50ppm, RBC = 1mV, min PLT= 40µV

9 Aperture clogging effect

10 Coincidence effect >1 cells in aperture look one big cell
Less counts, distorted histogram Solution: diluting samples, mathematical coincidence correction Linearity range (80um): WBC: 100 x 10^3/ul RBC: x 10^6/ul PLT: 1000 x 10^3/ul

11 Sample preparation: diluting, lysing
1. Blood 3. Hemolyser 1:200 2. 1:32.000 a + diluent → b b + diluent → c → RBC/PLT b + Lyser → WBC/HGB a b c

12 Differential Lysing process
PLT & RBC LYM MON GRAN RBCs destroyed WBCs selectively shrank to nuclei

13 3-part Differential WBC Histogram

14 Hemoglobin measuring principle
Specific wavelength light source – green 560 nm Light to frequency converter Ambient light compensation with U/D counting, and light chopping LED SAMPLE DETECTOR LED switching: Frequency output

15 Hemoglobin calculation – HGB blank
HGB needs blank measurement on clean diluent to compensate temperature drift HGB = C * ln (CNTblank / CNTsample) CNTblank = HGB count on diluent = CNTsample = HGB count on sample =

16 Thank you for your attention!

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