Agile - affordable - accurate What happens in the aperture? Basics of Hematology cell counting.

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What happens in the aperture?

Basics of Hematology cell counting

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Blood cells

Name RBC WBC PLT

Population 4.5-5.5 million/µl 5-10.000/µl 150-300.000/µl

Shape, diameter

Biconcave shape

Diameter 8 µm

Thickness 2 µm

NE,EO,BA 15 µm

LYM 8-15 µm

MON 15-25 µm

Fragments

Diameter 2-4 µm

Mean volume

90 fl various 12 fl

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Blood cells – under microscope

NEU EO

LYMBASO MONO

NRBC

PLT

RBCRBC

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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

(pcs/l, pcs/µl)

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

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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

and

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

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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.

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Blood sample

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Impedance cell counting principle

dR/R = 1 / 20 000 = 50ppm,

RBC = 1mV, min PLT= 40µV

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Aperture clogging effect

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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: 10 x 10^6/ul

PLT: 1000 x 10^3/ul

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Sample preparation: diluting, lysing

1.

Blood

3. Hemolyser

1:200

2.

1:32.000

a b c

1. a + diluent → b2. b + diluent → c → RBC/PLT3. b + Lyser → WBC/HGB

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Differential Lysing process

PLT & RBC LYM MON GRAN

RBCs destroyed WBCs selectively shrank to nuclei

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3-part Differential WBC Histogram

& HGB

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Hemoglobin measuring principle

• Specific wavelength light source – green LED @ 560 nm

• Light to frequency converter

• Ambient light compensation with U/D counting, and light chopping

LED SAMPLE DETECTOR LED switching:

Frequency output

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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 = 10.000

CNTsample = HGB count on sample = 4.000

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Thank you for your attention!