Chapter 5

HematologyHematology

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. )

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.

R eservo ir

D iaphragm(ins ide neck)

P ipe tte

P ipe tte sh ie ld

O verflowcham ber

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. )

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 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. )

0.1 mm depth Cover slip

Ruled area

V slash Moat

W W

W W

R R

R

R R

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.

RBC/L = (total cell count in all five squares)(volume counted)(0.02 L)(dilution ratio)(106)

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.)

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. )

Time(a)

Re

sist

an

ce

(c)

(b)

Resistive cell number

10

5

00

Cell volume (fl)100 200

Conventional aperture

Hydrodynamic focusing aperture

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 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.)

Cell typeCell type Percentage of Percentage of WBCWBC

Absolute number (Absolute number ( 10 1099/L)/L)

Neutrophil Neutrophil 35 – 71 35 – 71 1.5 – 7.4 1.5 – 7.4

BandBand 0 – 60 – 6 0.0 – 0.70.0 – 0.7

Lymphocyte Lymphocyte 24 – 44 24 – 44 1.0 – 4.4 1.0 – 4.4

MonocyteMonocyte 1 – 10 1 – 10 0.1 – 1.0 0.1 – 1.0

EosinophilEosinophil 0 – 40 – 4 0.0 – 0.40.0 – 0.4

Basophil Basophil 0 – 20 – 2 0.0 – 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 typeCell type Size (fL)Size (fL)

LymphocytesLymphocytes 35 – 90 35 – 90

MonocyteMonocytess 90 – 160 90 – 160

GranulocytesGranulocytes 160160 – 450– 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.