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Chapter 5
Hematology
Figure 5.1 (a) A double ended needle partially pierces the rubber stopper in an evacuated
tube. (b) After the left needle pierces the vein, the evacuated tube is pushed left so the right
needle pushes though the rubber stopper and blood fills the evacuated tube. (From Turgeon, M.
L. Clinical Hematology: Theory and Procedures. 2nd Edition. Copyright © 1993 by Little, Brown and
Company. Reprinted by permission of Little, Brown and Company. )
Pipette
Diaphragm
(inside neck)
Reservoir
Pipette shield
Overflow
chamber
Figure 5.2 The Unopette system pipette fills by capillary action from the blood collection
tube or free-flowing capillary blood from the lanced fingertip or from blood collection tube.
The pipette covered with the shield punches through the diaphragm, then is removed and the
shield discarded. The reservoir contains a premeasured amount of diluting solution. The
reservoir is squeezed slightly, the pipette inserted on the top and pressure released. This
draws the blood into the reservoir, where it is mixed. Slight squeezing then expels the diluted
blood.
Figure 5.3 In the centrifugal analyzer, venous or
capillary blood fills a capillary tube coated with
acridine orange and oxalate stains and a float is
introduced. After centrifugation, specific gravity
variations separate the blood. An ultraviolet viewer
shows different fluorescent colors for each layer.
(From Stiene-Martin, E. A., Lotspeich-Steininger, C.A.,
and Koepke, J. A. (eds.) Clinical Hematology:
Principles, Procedures, Correlations. 2nd Edition.
Coypright © 1998 by Lippencott-Raven Pubishers.
Reprinted by permission of Lippencott-Raven
Publishers. )
Cover slip
V slash
0.1 mm depth
Moat
Ruled area
Figure 5.4 Neubauer hemocytometer, side and top view. The central platforms contain the
ruled counting areas and are 0.1 mm under the cover slip, which is suspended on the raised
ridges. (From McNeely J. C. and Brown D. 1992. Laboratory evaluation of leukocytes. (From StieneMartin, E. A., Lotspeich-Steininger, C.A., and Koepke, J. A. (eds.) Clinical Hematology: Principles,
Procedures, Correlations. 2nd Edition. Copyright © 1998 by Lippencott-Raven Pubishers. Reprinted by
permission of Lippencott-Raven Publishers. )
W
W
R
R
R
R
W
R
W
RBC/L = (total cell count in all five squares)(volume counted)(0.02 L)(dilution ratio)(106)
Figure 5.5 The Neubauer cytometer ruled counting area is 3 mm  3 mm. The red
blood cell counting area is marked by R and is 200 µm  200 µm. The white blood
cell counting area is marked by W.
Figure 5.6 An automatic analyzer aspirates whole blood, divides it, dilutes it, mixes it and
then analyzes it for hemoglobin and cell characteristics. (From Lotspeich-Steininger, C.A.,
Stiene-Martin, E. A., and Koepke, J. A. (eds.) 1992. Clinical Hematology: Principles, Procedures,
Correlations. Copyright © 1992 by Lippencott-Raven Pubishers. Reprinted by permission of
Lippencott-Raven Publishers.)
(b)
Resistance
Resistive cell number
10
(a)
Time
Hydrodynamic
focusing aperture
5
Conventional
aperture
0
(c)
0
100
200
Cell volume (fl)
Figure 5.7 (a) Non-conducting cells passing through the aperture of the Coulter
counter increase the resistance between the electrodes. (b) A surrounding fluid
sheath forces cells to flow centrally in hydrodynamic focusing. (c) Hydrodynamic
focusing yields a narrower, more accurate cell volume distribution. (From Handin, R.I.,
Lux, S.E. and Stossel, T.P. (eds.) Blood: Principles & Practice of Hematology. Copyright © 1995 by
L.B. Lippincott Company. Reprinted by permission of J.B. Lippincott Company. )
Figure 5.8 Each cell passing through the Coulter counter aperture causes a
resistance change proportional to the cell volume. Thus measuring the height of
each voltage spike yields the cell volume. (From Turgeon, M. L. 1993. Clinical Hematology:
Theory and Procedures. Copyright © 1993 by Little, Brown and Company. Reprinted by permission of
Little, Brown and Company. )
Figure 5.9 Number of cells versus cell volume from a Coulter counter. (a) Nucleated RBCs
(N), lymphocytes (L), mononuclear cells (M), and polymorphonuclear leukocytes (PMN).
(b) Leukocyte differential distribution (WBC), RBC distribution (RBC), and platelet
distribution (PLT). (From Handin, R. I., Lux, S. E., and Stossel, T. P. (eds.) Blood: Principles &
Practice of Hematology. Copyright © 1995 by J.B. Lippincott Company. Reprinted by permission of
J.B. Lippincott Company. )
Figure 5.10 In flow cytometry, a sheath surrounds the sample to hydrodynamically focus the
cells to the center, where they are illuminated by a laser. Forward (low) angle scatter
measures cell volume. Right (high) angle scatter measures cell type. (From Stiene-Martin, E.
A., Lotspeich-Steininger, C.A., and Koepke, J. A. (eds.) Clinical Hematology: Principles, Procedures,
Correlations. 2nd Edition. Copyright © 1998 by Lippencott-Raven Pubishers. Reprinted by
permission of Lippencott-Raven Publishers. )
Figure 5.11 Mia analysis of RBCs measures low-angle and high-angle scattering to yield
corpuscular volume V in fL and corpuscular hemoglobin concentration CHC (HC) in g/dL.
(From Handin, R. I., Lux, S. E., and Stossel, T. P. (eds.) Blood: Principles & Practice of Hematology.
Copyright © 1995 by J.B. Lippincott Company. Reprinted by permission of J.B. Lippincott Company. )
Figure 5.12 Leukocyte differential classifies the five basic leukocyte classes by
forward light scatter versus peroxidase absorption. (From Handin, R. I., Lux, S. E., and
Stossel, T. P. (eds.) Blood: Principles & Practice of Hematology. Copyright © 1995 by J.B. Lippincott
Company. Reprinted by permission of J.B. Lippincott Company. )
Figure 5.13 After cell identification by fluorescent light scattering, the cell sorter charges
each cell droplet, and electrostatically deflects it to separate cells by type. (From StieneMartin, E. A., Lotspeich-Steininger, C.A., and Koepke, J. A. (eds.) Clinical Hematology: Principles,
Procedures, Correlations. 2nd Edition. Coypright © 1998 by Lippencott-Raven Pubishers. Reprinted
by permission of Lippencott-Raven Publishers.)
Cell type
Percentage of
WBC
Absolute number ( 109/L)
Neutrophil
35 – 71
1.5 – 7.4
Band
0–6
0.0 – 0.7
Lymphocyte
24 – 44
1.0 – 4.4
Monocyte
1 – 10
0.1 – 1.0
Eosinophil
0–4
0.0 – 0.4
Basophil
0–2
0.0 – 0.2
Table 5.1 Normal values of white blood cell types (From Brown, B.A. Hematology:
Principles and Procedure. 6th ed. Copyright © 1993 by Lea &Febiger. Reprinted
by permission of Lea & Fehiger.)
Cell type
Size (fL)
Lymphocytes
35 – 90
Monocytes
90 – 160
Granulocytes
160 – 450
Table 5.2 Sizes of white blood cell types (From Brown, B.A. Hematology:
Principles and Procedure. 6th ed. Copyright © 1993 by Lea &Febiger. Reprinted
by permission of Lea & Fehiger.)
Figure 5.14 Suggested squares to use for platelet count.