Agile - affordable - accurate What happens in the aperture? Basics of Hematology cell counting.
-
Upload
sean-mcdonald -
Category
Documents
-
view
218 -
download
0
Transcript of Agile - affordable - accurate What happens in the aperture? Basics of Hematology cell counting.
agile - affordable - accurate
What happens in the aperture?
Basics of Hematology cell counting
agile - affordable - accurate
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
agile - affordable - accurate
Blood cells – under microscope
NEU EO
LYMBASO MONO
NRBC
PLT
RBCRBC
agile - affordable - accurate
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
agile - affordable - accurate
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
agile - affordable - accurate
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.
agile - affordable - accurate
Blood sample
agile - affordable - accurate
Impedance cell counting principle
dR/R = 1 / 20 000 = 50ppm,
RBC = 1mV, min PLT= 40µV
agile - affordable - accurate
Aperture clogging effect
agile - affordable - accurate
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
agile - affordable - accurate
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
agile - affordable - accurate
Differential Lysing process
PLT & RBC LYM MON GRAN
RBCs destroyed WBCs selectively shrank to nuclei
agile - affordable - accurate
3-part Differential WBC Histogram
& HGB
agile - affordable - accurate
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
agile - affordable - accurate
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
agile - affordable - accurate
Thank you for your attention!