Page 651 - Clinical Hematology_ Theory _ Procedures ( PDFDrive )

CHAPTER 30 ■ Instrumentation in Hematology 635

Clinical Applications of Flow Cytometry Other platelet assays include platelet sur ace receptor

quantitation and distribution or the diagnosis o congeni-
Because single-cell suspensions o peripheral blood and bone tal platelet unction disorders, platelet-associated immuno-

marrow are easy to obtain, most clinical applications o f ow globulin G (IgG) quantitation or the diagnosis o immune

cytometry are in the specialties o hematology and immunol- thrombocytopenias, and platelet cross-matching or trans-

ogy. A number o instruments are currently manu actured usion. Other assays include brinogen receptor occupancy

or various uses. studies or monitoring the clinical e cacy o platelet-directed

Counting Reticulocytes and Platelets anticoagulation in thrombosis. Detection o activated plate-
let sur ace markers, cytoplasmic calcium ion measurements,
Reticulocytes and platelet microparticles or the assessment o hypercoag-

Manual counting o reticulocytes has been conducted ulable states can be per ormed.

since the 1940s. It is tedious and time consuming and ana-

lyzes ewer erythrocytes than do f ow cytometry systems. Other Cellular Applications

Enumeration o reticulocytes by f ow cytometry is more

accurate, precise, and cost-e ective than manual counting. Flow cytometry applications are extended to various areas o

Flow cytometry also provides additional reticulocyte param- specialized study.

eters o the IRF, or reticulocyte maturity index (RMI), and Im m unophenotyping

the measurement o reticulocyte maturity.

Reticulocytes can be counted by using a stain or resid- Monoclonal antibodies, identi ed by a cluster designation

ual RNA in erythrocytes (e.g., new methylene blue, thia- (CD), are used in most f ow cytometry immunophenotyping

zole orange, and oxazine 750); proprietary f uorescent dye (Fig. 30.21; able 30.6). Cell sur ace molecules recognized by

CD4K530 is used by one manu acturer. T e Coulter system monoclonal antibodies are called antigens because antibod-

uses neomethylene blue and sul uric acid as reagents. ies can be produced against them or markers because they

In addition, ully automated f ow cytometers speci cally identi y and discriminate between (“mark”) di erent cell

designed or reticulocyte enumeration by optical light scatter populations. Markers can be grouped into several categories.

have been incorporated into existing hematology analyzers Some are speci c or cells o a particular lineage (e.g., CD4+

(see able 30.5). lymphocytes) or maturational pathway (e.g., CD34+ progen-
itor stem cells), and the expression o others varies according

Platelets to the state o activation or di erentiation o the same cells.

Measurement o platelets provides an estimate o young, Measuring T Cells for Acquired Im m unode ciency

reticulated platelets by counting platelets that stain with Syndrom e Analysis

an RNA dye (e.g., thiazole orange or coriphosphine-O).

Platelets in whole blood are also labeled with PE-conjugated T e quantitation o and B cells using monoclonal sur ace

CD41 antibody to distinguish them rom other small par- markers can be per ormed using f ow cytometry. With the

ticles. CD-41–positive platelets are evaluated or RNA con-

tent. T e nding o elevated reticulated platelets indicates CD34

“stress” platelets rom increased bone marrow production

and is consistent with a diagnosis o immune thrombocyto-

penic purpura.
Stem Cell

Examples of Hematology CD45 CD45 CD45 CD45 CD45

TABLE 30.5 Analyzers w ith a Reticulocyte

Enumeration Feature

Manufacturer Instrument CD15 CD14 CD3 CD19 CD161

Abbott Diagnostics CELL-DYN 3700, CELL-DYN Ruby, Granulocyte Monocyte T-Lymphocyte B-Lymphocyte Thrombocyte

CELL-DYN Sapphire

Beckman Coulter Inc. HmX, LH 500, LH 700/750, LH 785, CD45 CD45

Horiba LH 1500, LH 7801

ABX Diagnostics Pentra DX 120 CD4 CD8

Siemens Healthcare Advia 120, Advia 2120, Advia 2120i

Diagnostics CD3 CD3

Sysmex XE 2100, XE 5000, XE-alpha N, T-Lymphocyte T-Lymphocyte

XT-2000i FIGURE 30.21 CD membrane markers.