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
NEU EO
LYM
BASO
MONO
NRBC
PLT
RBC

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, lysing1.
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
& HGB

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!