Wintrobe's Clinical Hematology 14e SC

2 Part 1: Laboratory Hematology— SECTION 1

WBC

RBC

basic blood count parameters, but with less agreement for reticulocyte

counts, nucleated RBCs, and WBC differentials, indicating that manual review remains a valuable tool. 9

REL#

V O L U M E

Manual RBC, WBC, and platelet counts are performed using a mi-

croscope after dilution of the sample in a hemocytometer, a specially

50

100

200 300 f

constructed counting chamber that contains a specific blood volume. This

PLT

process is time consuming, requires a great deal of technical expertise,

and has largely been replaced by automated methods. There are a variety

REL#

of automated hematology analyzers available from manufacturers, such

as Abbott, Beckman Coulter, Siemens, Sysmex, Horiba, and others.

2 10

20

30

f

DF 1

Analyzer selection depends on the volume of samples to be tested and

the specific needs of the laboratory and ordering physicians. The ana-

lyzers range in price and workload capacity from those that would be

RBC 4.56 13.5 40.3 88.3 29.5 33.5 13.4 Hg Hct MCV MCH MCHC RDW PLT 202 8.2 MPV

ID# 1

WBC

6.7

appropriate for an individual physician’s office or point-of-care facility

#

ID# 2 Sequence #

%

to those needed in a busy high-volume reference laboratory.

NE LY MO EO BA

59.4 31.6 7.7 0.7 0.6

4.1 2.1 0.5 0.0 0.0

Automated hematology analyzers sample directly from phlebotomy

DATE:

06/21/96 08:55:45

tubes and use volumes as small as 150 µL for a full complete blood count (CBC) analysis. 9 They perform a variety of hematologic measurements in

Normal WBC Pop Normal RBC Pop Normal PLT Pop S TIME: Cass/Pos

addition to basic cell counting, such as Hb concentration, red cell size, and

leukocyte differentials. They may also perform more specialized testing,

such as reticulocyte and nucleated RBC counts, and flagging of blasts, left- shift, and variant lymphocytes. 9-11 Current analyzers utilize combinations

FIGURE 1.2 Histograms and printout generated by the Coulter automated he- matology analyzer utilizing light scatter and electrical impedance. BA, basophil;

of techniques to detect and differentiate specific cells types, including

electrical impedance, radiofrequency conductivity, laser light scattering,

flow cytometry, fluorescence detection, cytochemistry, and monoclonal antibodies ( Figures 1.1 and 1.2 ). 9,11 Using flow cytometric technologies,

EO, eosinophil; Hct, hematocrit; Hg, hemoglobin; LY, lymphocyte; MCH, mean

corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;

some analyzers detect specific blood cell populations by antigen expression,

MCV, mean corpuscular volume; MO, monocyte; MPV, mean platelet volume;

NE, neutrophil; PLT, platelet; RBC, red blood cell; RDW, red cell distribution

such as detection of CD34-positive peripheral blood stem cells or leuke- mic blasts. 9,12-14 Integration of data from various sources of information

width; WBC, white bl od cell; DF1, differential; Rel, relative.

has improved the accuracy of the five-part differential and decreased the

numbers of unidentifiable cells requiring manual review for identification,

RED BLOOD CELL PARAMETERS

although analyzers do still frequently generate flags for abnormalities that require further investigation. 15-18 The International Consensus Group for Hematology Review has suggested criteria that should lead to manual review of a specimen after automated analysis and differential counting. 15

RBCs are defined by three quantitative values: the volume of packed red

cells or hematocrit (Hct), the amount of Hb, and the red cell number per

unit volume (RBC). Three additional indices describing average quali-

tative characteristics of the red cell population are also collected. These

Various Angles of Scattered Light

are mean corpuscular volume (MCV), mean corpuscular hemoglobin

(MCH), and mean corpuscular hemoglobin concentration (MCHC).

All of these values are routinely determined by hematology analyzers.

Volume of Packed Red Cells (Hematocrit)

The Hct is the proportion of the volume of a blood sample that is occu-

pied by red cells. Hct may be determined manually by centrifugation

of blood at a given speed and time in a standardized glass tube with a uniform bore, as was originally described by Wintrobe. 19 The height of

the column of red cells after centrifugation compared with total blood

Sample Stream

sample volume yields the Hct. Macromethods (using 3-mm test tubes)

Focused Laser Beam

with low-speed centrifugation or micromethods using capillary tubes

and high-speed centrifugation may be used.

Manual methods of measuring Hct are simple and accurate means

of assessing red cell status. They are easily performed with little

specialized equipment, allowing adaptation for situations in which

automated cell analysis is not readily available or for office use.

However, several sources of error are inherent in the technique. The

spun Hct measures the red cell volume, not red cell mass. Therefore,

patients in shock or with volume depletion may have normal or high

Sheath Stream

Hct measurements because of hemoconcentration despite a decreased

Sample Feed Nozzle

red cell mass. Technical sources of error in manual Hct determinations

usually arise from inappropriate concentrations of anticoagulants, poor mixing of samples, or insufficient centrifugation. 19 Another inherent

error in manual Hct determinations arises from trapping of plasma in

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. FIGURE 1.1 Optical flow cytometric technology used in automated hematology analyzers. A suspension of cells is passed through a flow chamber and focused the red cell column. This may account for 1% to 3% of the volume in microcapillary tube methods, with macrotube methods trapping relatively more plasma. 20,21 It should be noted that abnormal red cells

into a single cell sample stream. The cells pass through a chamber and interact

(eg, sickle cells, microcytic cells, macrocytic cells, or spherocytes)

with a laser light beam. The scatter of the laser light beam at different angles is

often trap higher volumes of plasma because of increased cellular rigidity, possibly accounting for up to 6% of the red cell volume. 21

recorded, generating signals that are converted to electronic information about

cell size, structure, internal structure, and granularity. (Adapted and redrawn from

Very high Hcts, as in polycythemia, may also have excess plasma

Cell-Dyn 3500 Operator’s Manual . Santa Clara, CA: Abbott Diagnostics; 1993.)

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