Flow Cytometry The Basics In Hematology Interpretation

The information presented here is purposefully simplistic. An elaborate explanation of flow cytometry is not appropriate for the audience and tone of this text. Flow cytometry is a specialty technique and a recent Google search listed 10 pages of entries referring to certificate programs for this specialty. For additional information, the student is referred to

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Figure 20.21 Cell Dyne technology.

Table 20.15 O Applications of Flow Cytometry

• Immunophenotyping

• Diagnosis and staging of leukemia/ lymphoma

• Lymphocyte screening panel: AIDS patients

• DNA content analysis

• Enzyme studies

• Fetal cell enumeration textbooks and websites solely devoted to the principles of flow cytometry and case studies.1

Overview

Flow cytometry is a technique that has greatly impacted the diagnosis of hematological malignancies. Peripheral blood, bone marrow, lymph nodes, solid tumors, needle aspirates, and splenic tissue are all examined by flow cytometry instruments. Each of these tissues has specific antigen characteristics that can be illuminated through the use of flow cytometry. This technique is usually ancillary to traditional means of diagnosis because of the expense and expertise needed to achieve results. Although flow cytometry has many applications (Table 20.15), its use in immunophenotyping has been particularly beneficial to distinguish hematological neoplasms: lymphomas and leukemias. A flow cytome-ter can analyze up to 10,000 cells, separating them into subpopulations and then "looking for" particular antigenic or epitope markers. This discovery is accomplished through the use of monoclonal antibodies that determine cell specificity and fluorescent dye that will aid in the detection of the particular antigen-antibody combination on the flow cytometer. Cell suspensions are stained with monoclonal antibodies that contain fluorochromes and then the suspension is analyzed by the flow cytometer. Additionally, flow cytometry analysis is replacing several obscure but long established techniques such as sucrose hemolysis for PNH, Klei-hauer-Betke for fetal hemoglobin, and nitroblue tetra-zolium test for chronic granulomatous disease.

Principles

Basic analysis of cell preparations includes light scatter and fluorescence.

'The author wishes to acknowledge Candace Breen Golightly MS, MT (ASCP) and Mark Golightly, PhD for their assistance with this section.

Light scatter (forward angle light scatter/side scatter) is the angle at which light is scattered depending upon the nuclear and cytoplasmic complexity of the cells and the size of the cells

The intensity of fluorescence is related to the antigen density of the markers being investigated; cells are mixed with specific monoclonal antibody probes and the absence or presence of fluorescence provides data relative to the matu-rational stage and phenotype of cells

The flow cytometer is composed of three distinct systems: the fluid system, the optical system, and the electronic system (Fig. 20.22).

The fluid system handles sampling in a single fluid stream surround by a sheath fluid that produces laminar flow. This fluid within a fluid creates a differential pressure that allows cells to enter into the conical nozzle. Here individual cells are analyzed by laser light sources and fluorescence. After analysis, the cell droplets will fall into the waste collection tubes.

The optical system includes gas ion laser, dioded lasers, and dye lasers. The lasers measure light scatter and emission of fluorescent light. The information gathered from this measurement are directed into the photomultiplier tube (PMT), beam-specific dichromic mirrors, and wavelength selective filters.

The electronic system is driven by a personal computer that has sophisticated data storage capabilities. This data can then be analyzed by a variety of software packages from third party distributors.

Data are generated that will display the intensity of the fluorescence of those cells that possess the antigen marker. Therefore, it is the patterns of the reactive cells rather than the numbers of reactive cells that are important (Fig. 20.23).

Data Interpretation: One Role for the Medical Technologist

Diagnosing a leukemia and lymphoma is a difficult undertaking. Bone marrow aspirate smears, blood smear interpretation, hematology results, patient's symptoms, cytogenetics, and cytochemical staining each plays a role in diagnosis. Flow cytometry adds an additional piece of supporting information to this entire process. Not every laboratory has a flow cytome-try instrument used specifically for one of the purposes listed above; yet most laboratory professionals will have

328 PartV • Laboratory Procedures

Light sensors

Optical System

Light sensors

Optical System

328 PartV • Laboratory Procedures

Filters to select violet light is measured

Charged detection plates

Filters to select violet light is measured

Sample flow

Charged detection plates

Sample flow

Sorting station

(cells are sorted here)

Fluid System

Collection vials

Figure 20.22 Internal components of flow cytometer, which includes fluid, optical, and electronic systems.

Sorting station

(cells are sorted here)

Fluid System o

Collection vials

Figure 20.22 Internal components of flow cytometer, which includes fluid, optical, and electronic systems.

some exposure to these data in their laboratory careers. Some will make it their subspecialty. Since this technology is rapidly expanding current lists of CD markers (see Table 20.16), their applications and their interpretation are available in a variety of Internet sources.

Flow Cytometry Case Studies

Case 1

A 67-year-old woman came to her physician's office complaining of flu symptoms. Even though she had

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