The Compendium April 1996 Small Animal

PERSPECTIVES IN VETERINARY MEDICINE V

Flow Cytometry:
The Next Step in Diagnostic Testing

Paula C. Perkins,
MT(ASCP), DVMa
Carol B. Grindem, DVM, PhD
Diplomate, ACVP
Philip B. Carter, PhD
Department of Microbiology,
Pathology and Parasitology
College of Veterinary Medicine
North Carolina State University
Raleigh, North Carolina
available or under investigation
at veterinary institutions using
flow cytometry.
What is Flow
Cytometry?
Flow cytometry is an analyti-
cal method that combines the
two elements of its name to de-
termine characteristics of indi-
vidual cells. Digested solid tis-
sue, blood, or body fluids are
first made into a cell suspen-
sion. After exposure to appro-
ample, are used to stain the nu-
cleic acid in reticulocytes or tu-
mor cells. In this manner, retic-
ulocytes can be differentiated
from mature erythrocytes, and
tumor cell DNA can be quantita-
tively compared with DNA of
normal cells. Monoclonal anti-
bodies conjugated to a fluores-
cent marker can also be used to
label specific cell types. Cells
bound to monoclonal antibod-
ies give off light of known
wavelengths when exposed to
short time. Assuming appropri-
ate monoclonal antibodies and
reagents are available, the
range of test applications for
this technology is limitless.
Immunophenotyping
Numerous monoclonal anti-
bodies directed against hemic
cells have been developed for
dogs, cats, cows, pigs, labora-
tory animals, and chickens.1
Monoclonal antibodies are pro-
duced by first inoculating mice

F
low cytometry is a pow-
erful diagnostic tool that
is used extensively in the
study of many human diseases.
The best known and most
widely used application of flow
priate reagents, the cells in the
suspension are introduced into
a flow cytometer and injected
into a large moving stream of
isotonic carrier fluid. The core
of this stream is then accelerat-
ed, pulling the cells toward the
the flow cytometer’s laser. The with tissue containing the cell
fluorescence distinguishes (antigen) of interest. Canine
them from cells that have not thymus, for example, is used to
■ Flow cytometry is an analytic method that de-
termines characteristics of individual cells in
blood, tissue, and body fluids.
KEY POINTS

cytometry is in the manage-

ment of HIV infection. The abili-
ty to differentiate and enumer-
ate helper T lymphocytes in
HIV-infected persons is critical.
Veterinary applications are also
being developed, and private
practitioners will soon have at
their disposal many diagnostic
tests that until a few years ago
were unavailable or prohibitive-
ly expensive. Blast cell identifi-
cation, lymphocyte subset enu-
meration, DNA ploidy and cell
cycle analysis, antiplatelet anti-
body detection, and evaluation
of reticulocyte maturation are
just a few of the tests currently
aDr.Perkins is currently affiliated
with CDC Technologies, One
Great Hill Road, Oxford, CT.
center of the stream until they
are flowing in a single-cell line.
This focusing of the stream al-
lows cells in the suspension to
travel individually through a
beam of laser light, where de-
tectors determine the relative
size of the cells by their ability
to block the light, and the rela-
tive complexity of the cells by
their ability to refract the light.
Using this technique, cells of in-
terest can be identified and sep-
arated from other cell types in a
mixed population.
The next step in flow cyto-
metric analysis involves the use
of fluorescent reagents to dis-
tinguish subpopulations of
cells. Fluorescent dyes, for ex-
■ Flow cytometry can be used to monitor lymphocyte
subpopulation changes in immunodeficient animals.
■ Flow cytometric assays are available for analyz-
ing the cycling behavior of tumor cells, detect-
ing antiplatelet antibodies, and enumerating
reticulocytes.
reacted to the monoclonal anti- stimulate mice to produce anti-
bodies. Also, by simultaneously body against canine T lympho-
using several fluorescent com- cytes. The mouse develops an
pounds that give off different immune response to the for-
wavelengths of light, multiple eign tissue, and the B lympho-
reagents can be used to analyze cytes that secrete the antibody
different populations in a single are harvested and fused with
specimen or categorize cells myeloma cells in culture. The
that can react to several anti- resulting hybrid cells produce
bodies. The flow cytometer is large amounts of protein con-
therefore able to determine the taining monoclonal antibody
identity and reactivity of a very that will bind to canine T lym-
large number of cells in a very phocytes. 2 Using this tech-
Small Animal
nique, antibodies can be pro-
duced that react with broad
categories of cells, such as T
lymphocytes, or with specific
subpopulations, such as helper
and cytotoxic T lymphocytes.
Fluorescent monoclonal anti-
bodies and flow cytometry can
therefore be used to determine
the lineage of blast cells or enu-
merate and monitor changes in
the subpopulations of lympho-
cytes.
Lymphocyte subpopulation
analysis is vital to the study of
human AIDS, in which the
number of CD4+ (helper) T lym-
phocytes declines over time.
This decline is routinely moni-
tored using flow cytometric
analysis of CD4+ and CD8+ (cy-
totoxic) T lymphocytes. The
cells are identified and enumer-
ated using fluorescent mono-
clonal antibodies, and a ratio of
CD4+:CD8+ cells is calculated.
This ratio declines as the popu-
lation of helper T lymphocytes
decreases. Changes in the ratio
have therapeutic and prognos-
tic implications for HIV-infected
patients.
Feline and bovine immuno-
deficiency viruses are currently
being investigated as models
for AIDS because of similarities
in pathogenesis and lympho-
cyte subset alterations.3 In addi-
tion to their research value,
however, these studies have
identified changes in the
CD4+:CD8+ ratio that have impli-
cations for practicing veterinari-
ans. The absolute CD4+ count
has been shown to be lower in
cats with long-standing FIV in-
fections. Also, in the face of an
immunologic challenge (e.g.,
vaccination), FIV-positive cats
have exhibited a decrease in the
CD4+:CD8+ ratio, with possible
acceleration of the disease and
permanent immunosuppres-
sion.4–6 In FeLV-positive cats,
on the other hand, there is a de-
crease in the number of both
CD4 + and CD8 + cells, which
produces a ratio that is within
normal limits.5 Immunopheno-
typing is therefore an increas-
ingly valuable tool for monitor-
ing immunodeficient cats,
evaluating therapeutic re-
sponse, and determining prog-
nosis.
DNA Ploidy and
Cell Cycle Analysis
Flow cytometric analysis of
DNA is used to differentiate
atypical cells from neoplastic
cells, determine if tumor cells
contain a normal (diploid) or
abnormal (aneuploid) number
of chromosomes, and study the
behavior of neoplastic cells as
they reproduce. This informa-
tion determines the therapy and
prognosis for many types of
human cancer. Ploidy analysis
is accomplished by comparing
the amount of DNA in neoplas-
tic cells to that of normal cells.
A fluorescent dye is used to
penetrate the tumor cells and
bind to the nucleic acid. The
amount of fluorescence pro-
duced when these cells are ex-
posed to laser light is directly
proportional to the quantity of
DNA present in the cells. The
quantity of DNA is then com-
pared with a normal standard.
Cells exhibiting greater or lesser
fluorescence than the normal
standard are labeled aneuploid.
Cells with fluorescence similar
to normal cells are termed
diploid.
The DNA content of neoplas-
tic cells is also used to study
cycling behavior of the tumor.
Strand condensation, duplica-
tion, and other mitotic activities
are interpreted by the flow cy-
tometer as subtle differences in
the quantity of DNA present in
the cell. In this way, DNA analy-
sis identifies the stage of mito-
sis for the neoplastic cells and
determines the relative propor-
tions of cells in each stage. Tu-
mor behavior can then be stud-
ied by comparing the cycling
behavior of the neoplastic cells
with that of normal cells.
Limitations do exist with flow
cytometric analysis of DNA
ploidy because the information
obtained reflects only quantita-
tive changes. A normal amount
of DNA does not guarantee a
normal karyotype. Transloca-
tions and other genetic manipu-
lations that do not alter the
quantity of DNA will not be de-
tected by flow cytometric analy-
sis. Aneuploidy, however, does
indicate abnormal mitotic activi-
ty.7 DNA analysis of neoplasias
in animals is currently being
studied at many major institu-
tions, and the relationships be-
tween abnormal ploidy and tu-
mor prognosis and therapy are
rapidly being derived.
Antiplatelet Antibody
Determination
Immune-mediated thrombo-
cytopenia is a common disease
in veterinary medicine. Testing
for the presence of immuno-
globulin on the surface of platelets
is critical for the correct diag-
nosis and treatment of this dis-
ease. Current tests for antiplate-
let antibody have problems with
sensitivity and specificity, how-
ever, and flow cytometry is
providing a more accurate
means of analysis. Before being
introduced into the flow cy-
tometer, patient platelets are
exposed to a fluorescent mono-
clonal antibody directed against
immunoglobulin. If im-
munoglobulin has previously
adhered to the surface of these
The Compendium April 1996
platelets, the monoclonal anti-
body will bind and cause the
platelets to fluoresce, indicating
an immunologic cause of the
thrombocytopenia. Patient
serum also can be tested for
the presence of circulating an-
tiplatelet antibody. Normal
platelets are first exposed to
this serum, and if the patient
has free antiplatelet antibody,
binding occurs. The platelets
are then exposed to the mono-
clonal antibody directed against
immunoglobulin, which binds
to the surface antibody and fluo-
resces in the flow cytometer.8,9
Antiplatelet antibodies may
be produced in response to
drugs, infectious agents, or
neoplasia; they may also be id-
iopathic.10 Increased amounts
of antiplatelet antibody have
been found in association with
canine lymphoma, with and
without concurrent thrombocy-
topenia.11 The determination of
the presence and quantity of
antiplatelet antibodies by flow
cytometry therefore has a sig-
nificant impact on the diagno-
sis, prognosis, and therapy of
many patients in veterinary
hospitals.
Flow Cytometry
and Hematology
Flow cytometry and related
laser-based technologies are
rapidly becoming staples in vet-
erinary hematology laborato-
ries. By combining lasers and
peroxidase staining, white
blood cell differentials can be
obtained. Individual cells are
differentiated by size and perox-
idase activity, with neutrophils
having the most activity and
lymphocytes the least. Cy-
tograms are then constructed
and the populations are enu-
merated.12
Flow cytometric reticulocyte
Small Animal
enumeration is becoming the
standard method for the evalu-
ation of anemia in humans. The
standard manual technique in-
volves staining erythrocytes
with new methylene blue, which
precipitates the residual RNA in
reticulocytes. This method is
imprecise, however, because it
relies on a subjective interpreta-
tion of a small number of cells.
Manual counting of canine
reticulocytes has been reported
to have a coefficient of variation
of 8% to 23%.13 Flow cytometry
offers precise and reproducible
enumeration because it uses a
fluorescent nucleic acid stain
and quickly analyzes tens of
thousands of erythrocytes for
the presence of RNA.
The precision of the cytomet-
ric technique also allows a
reticulocyte maturation index to
be calculated and followed as a
patient progresses. This index
is the mean fluorescence of all
erythrocytes analyzed, which
reflects the maturity of the
reticulocyte population rather
than simply calculating a per-
centage. An increase in mean
fluorescence indicates the pres-
ence of more immature reticu-
locytes (having more residual
RNA) and suggests an early
and ongoing need for erythro-
cytes. A decrease in mean fluo-
rescence over time indicates a
more mature reticulocyte popu-
lation and a potentially resolv-
ing or nonregenerative ane-
mia.14
Other Tests
on the Horizon
The sensitivity of the flow cy-
tometer is much greater than
that of the human eye for de-
tecting abnormalities in cells.
Applications now in use in hu-
man medicine have implica-
tions for the veterinary industry
regarding detection of minute
changes in individual cells. Flow
cytometry can, for example,
provide valuable prognostic and
therapeutic information to the
oncologist by detecting a re-
lapse when the neoplastic cells
are in such low numbers that
they cannot be detected by his-
tologic examination. Likewise,
neoplastic cells that remain af-
ter therapy can be detected.
Veterinarians are becoming
increasingly aware of neutrophil
function defects in animals.
Neutrophil activation tests that
use flow cytometry show
promise for the diagnosis of
these disorders. Activation oc-
curs when bacteria are phago-
cytized and destroyed by com-
pounds generated during the
oxidative burst. A fluorescent
dye that passively enters an ac-
tivated neutrophil is also altered
by the burst products. Once al-
tered, the dye cannot exit the
cell and the resulting intracellu-
lar fluorescence identifies the
neutrophil as being activated.15
Hemic parasites can be iden-
tified by flow cytometry even
when they are morphologically
indistinguishable from Howell-
Jolly bodies, basophilic stip-
pling, or Pappenheimer bodies.
A fluorescent dye is altered by
the parasites and incorporated
into their DNA, making them
visible under laser light.16
When specialized flow cy-
tometers are used, viruses can
be detected. Single viruses
have been demonstrated by
light scatter, and virus-infected
cells have been detected using
monoclonal antibodies.17
Bacteria also can be detect-
ed, occasionally speciated, and
evaluated for antibiotic sensitiv-
ity using flow cytometry. By
comparing the light scatter pat-
terns and dye binding patterns
of suspect organisms with
known controls, bacteria can be
quickly identified. Also, the inhi-
bition of growth by the addition
of antibiotics to the media can
be evaluated to determine sen-
sitivity.18
Conclusion
Flow cytometry has become
a valuable tool in the diagnosis
and treatment of human dis-
eases and will certainly become
equally important in the veteri-
nary world in the near future.
As more tests become avail-
able, clinicians will gain addi-
tional information about neo-
plastic, immune-mediated,
parasitic, and hematologic dis-
eases as well as the prognosis
and treatment for these disor-
ders. The future applications of
flow cytometry are limited only
by the availability of monoclon-
al antibodies and the imagina-
tion of the physicians, laborato-
rians, and veterinarians working
on this cutting edge of technol-
ogy.
References
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Small Animal The Compendium April 1996

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