What happens in the aperture Basics of Hematology

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 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 90 fl various 12 fl Shape, diameter Mean volume agile - affordable - accurate

Blood cells – under microscope RBC NRBC PLT NEU BASO RBC MONO EO LYM 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 Number of erythrocytes. RBC = RBCcal x (pcs/l or pcs/µl) (pcs/l, 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 Average volume of individual erythrocytes derived from the RBC histogram. (fl=10 -15 l) Haematocrit – HCT (percentage, absolute) Mean Corpuscular Haemoglobin – MCH (pg, fmol) Calculated from the RBC and MCV values. HCTpercentage = RBC x MCV x 100, HCTabsolute = RBC x MCV 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 The distribution width of the erythrocyte or platelet population derived from the histogram at 20% of peak (absolute) x. DW-SD = RDWcal x (P 2 – P 1) (fl), x. DW-CV = RDWcal x 0. 56 x (P 2 – P 1) / (P 2 + P 1) 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) Mean Platelet Volume – MPV (fl) Number of thrombocytes (platelets) PLT = PLTcal x (pcs/l or pcs/µl) 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: 1. 2. 3. 1. : RBC-LYM discriminator 2. : LYM-MID discriminator 3. : MID-GRA discriminator Percentages calculated from the absolute WBC value. agile - affordable - accurate

Blood sample agile - affordable - accurate

Impedance cell counting principle d. R/R = 1 / 20 000 = 50 ppm, RBC = 1 m. V, 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 (80 um): 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 3. Hemolyser 1. 2. 1. a + diluent → b 2. b + diluent → c → RBC/PLT 3. b + Lyser → WBC/HGB a b c Blood 1: 200 1: 32. 000 agile - affordable - accurate

Differential Lysing process PLT & RBCs destroyed LYM MON GRAN 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! agile - affordable - accurate