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Hematology Cellcounting - Basics
•
Hematology, is the part of internal medicine, that is concerned with the
study of blood, the blood-forming organs, and blood diseases.
•
The bunch of methods for counting, measuring and evaluating blood
cells suspended in fluid is known as Hematology Cellcounting.
•
A blood cell analyzer, is used to perform one of medicine's most oftenrequested and informative diagnostic tests, the complete blood count
(CBC).
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Blood
Blood is a specialized fluid, „liquid tissue”, that circulates
around the body in blood vessels.
It’s main functions:
- Transport through the body of
– oxygen and carbon dioxide
– nutrient molecules, ions, hormones
– „wastes”
– Heat
- Defense of the body against infections, injuries and other
foreign materials.
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Composition of blood
Liquid Plasma: over 90% of plasma is water, it also contains proteins,
salts, metabolits etc. After centrifuging it is called
serum.
Cells:
Erythrocytes(Red Blood Cells, RBC)
Leukocytes(White Blood Cells, WBC)
Thrombocytes(Platelets, PLT)
Erythrocytes
The most numerous and common type of blood cells.
Non nucleated in mammals, with biconcave shape,
erythrocytes are responsible for the transport of
oxygen. Contain hemoglobin(HGB), iron, and
structures on the cell membrane determine blood
group systems.
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Erythrocytes – cont’d
Reticulocytes:
immature blood cell forms, contain remnants of the
nucleus(ribosomal RNA). Present normally in a
very few% of the total RBC number.
Hemoglobin:
red blood cells contain several hundred HGB
molecules which:
transport oxygen(main function)
contain iron
cause the red colour of blood
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Thrombocytes and other parameters
Thrombocytes: thrombocytes or platelets play an important role in
the cellular part of the hemostazis(coagulation)
trying to prevent excessive blood loss. Non
nucleated cells in mammals and the smallest ones
in the same time. Contain specific granules.
Hematocrit – HCT or Packed Cell Volume – PCV:
Shows the volume of blood in % occupied by cells.
It can be measured after separation in a centrifuge, or calculated
after cell counting.
Shows the degree of anemia.
Mean Corpuscular Volume – MCV: Average volume of individual
erythrocytes
Mean Corpuscular Hemoglobin – MCH: Average hemoglobin content
of erythrocytes, calculated from RBC and HGB values.
MCH = HGB / RBC
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Other parameters
Mean Corpuscular Haemoglobin Concentration – MCHC:
Calculated from the HGB and HCT values. MCHC = HGB / HCTabsolute
Red cell Distribut. Width - RDW-SD
Platelet Distribut. Width - PDW-SD
and
Red cell Distribut. Width - RDW-CV
Platelet Distribut. Width - PDW-CV
The distribution width of the erythrocyte or platelet population
derived from the histogram at 20% of peak
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Mean Platelet Volume – MPV: Average volume of individual platelets
derived from the PLT histogram.
Thrombocrit – PCT: Calculated from the PLT and MPV values.
PCTpercentage = PLT x MPV x 100; PCTabsolute = PLT x
MPV
Hemolysis:
rupture of the cell membrane followed by
destruction of the cell. When RBC-s are lysed
hemoglobin is released.
Hemolytic agents: detergents, PH, osmotic pressure, drugs,
immunsystem, diseases etc.
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Leukocytes - WBC
White blood cells are a very important part of the immune system
defending the body against infectious diseases and foreign
materials.
By complexity of the internal structure :
-
Granulocytes (complex nucleus, granules in the cytoplasm)
Agranulocytes (simple nucleus, non granulated)
By cell size:
-
„Small”
„Medium”
„Big” cells
There are 5 main WBC populations: (see next page… )
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Leukocytes – cont’d

Lymphocytes: mononuclear cell with round, dense nucleus and
slight cytoplasm
Monocytes: mononuclear cells with oval, nucleus and variable
cytoplasm (vacuoles)
Neutrophils granulocytes: polimorphonuclear cells with neutral
cytoplasm & granules
Eosinophil granulocytes: polimorphonuclear cells with basic
(alkaline) cytoplasm & granules
Basophil granulocyte: polimorphonuclear cell with acid cytoplasm &
granules
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Types of blood cells
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Measurement principles
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
Fragments
Diameter 2-4 µm
LYM  8-15 µm
MON 15-25 µm
Mean
volume
 90 fl
various
 12 fl
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Measurement principles
Blood sample:
whole blood mixed with EDTA, anticoagulants are
necessary to prevent forming of clots
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Measurement principles
3. Hemolyser
1.
a
Sample preparation: from the whole
blood a small amount (20-25ul) of
primary sample is aspirated.
2.
b
c
Diluent is added to the sample
first (WBC&HGB) dilution is
obtained
From this first dilution a second
sample(100ul) is aspirated
Blood
1.
2.
3.
WBC & HGB
1:200
a + diluent → b
b + diluent → c → RBC/PLT
b + Lyser → WBC/HGB
RBC & PLT
1:32.000
Diluent is added to the second
sample
RBC&PLT dilution is obtained
Lyse reagent is added to the
WBC&HGB dilution RBCs are
destroyed (see next slide)
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WBC differential Lysing process
PLT & RBC
RBCs
destroyed
LYM
MON
GRA
WBCs selectively shrank to nuclei
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Impedance cell counting principle
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3-part Differential WBC Histogram
&
HGB
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Hemoglobin measurement principle
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