Flow Cytometry

Flow cytometry is a technique of quantitative single cell analysis. The flow

cytometer was developed in the 1970s and rapidly became an essential instrument
for the biologic sciences. purred by the !"# pandemic and a plethora of discoveries
in hematology$ speciali%ed flow cytometers for use in the clinical laboratory were de&
veloped by several manufacturers. The ma'or clinical application of flow cytometry is
diagnosis of hematologic malignancy$ but a wide variety of other applications e(ist$
such as reticulocyte enumeration and cell function analysis. )resently$ more than
*0$000 'ournal articles referencing flow cytometry have been published. This brief
review of the principles and ma'or clinical applications of flow cytometry may be
supplemented by several recent review articles and boo+s.
The technique of analy%ing individual cells in a fluidic channel was first described by
,allace -oulter in the 19.0s$ and applied to automated blood cell counting.
ubsequent developments in the fields of computer science$ laser technology$
monoclonal antibody production$ cytochemistry$ and fluorochrome chemistry led to
the development of the flow cytometer two decades later. /ecause the first
commercial flow cytometers were large$ comple($ e(pensive$ and difficult to operate
and maintain$ they were primarily used in the research laboratory. !owever$ the
enormous value of the flow cytometer in the medical and biologic sciences was
quic+ly appreciated$ and its cost and comple(ity gradually decreased as its analytic
capability increased.0 The present 1state&of&the art2 flow cytometers are capable of
analy%ing up to 13 parameters 4forward scatter$ side scatter$ 11 colors of immuno&
fluorescence5 per cell at rates up to 100$000 cells per second. 6utomation and
robotics is increasingly being applied to flow cytometry to reduce analytic cost and
improve efficiency.
BASIC PRINCIPLES Basic Principles of
Flow Cytometry
)repared single cell or particle suspensions are necessary for flow cytometric
analysis. #arious immunoflurescent dyes or antibodies can be attached to the
antigen or protein of interest. The suspension of cells or particles is aspirated into a
flow cell where$ surrounded by a narrow fluid stream$ they pass one at a time
through a focused laser beam. The light is either scattered or absorbed when it
stri+es a cell. 6bsorbed light of the appropriate wavelength may be re&emitted as
fluorescence if the cell contains a naturally fluorescent substance or one or more
fluorochrome&labeled antibodies are attached to surface or internal cell structures.
7ight scatter is dependent on the internal structure of the cell and its si%e and shape.
Fluorescent substances absorb light of an appropriate wavelength and reemit light of
a different wavelength. Fluorescein isothiocyanate 4F"T-5$ Te(as red$ and
phycoerythrin 4)85 are the most common fluorescent dyes used in the biomedical
sciences. 7ight and9or fluorescence scatter signals are detected by a series of
photodiodes and amplified. :ptical filters are essential to bloc+ unwanted light and
permit light of the desired wavelength to reach the photodetector. The resulting
electrical pulses are digiti%ed$ and the data is stored$ analy%ed$ and displayed
through a computer system.7$ ; The end result is quantitative information about every
cell analy%ed 4Fig. 15. ince large numbers of cells are analy%ed in a short period of
time 4<1$0009sec5$ statistically valid information about cell populations is quic+ly
obtained.
Flow Cytometry And Its Clinical Applications
Flow cytometry measures optical and fluorescence characteristics of single cells (or any other
particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical
properties, such as size represented by forward angle light scatter) and internal
complexity(represented by right-angle scatter) can resolve certain cell populations. Fluorescent
dyes may bind or intercalate with different cellular components such as !" or #!".
"dditionally, antibodies con$ugated to fluorescent dyes can bind specific proteins on cell
membranes or inside cells. %hen labeled cells are passed by a light source, the
fluorescent molecules are excited to a higher energy state. &pon returning to their resting states,
the fluorochromes emit light energy at higher wavelengths. 'he use of multiple fluorochromes,
each with similar excitation wavelengths and different emission wavelengths (or (colors)), allows
several cell properties to be measured simultaneously.
Immunophenotyping of Leukemias and Lymphomas
*mmunophenotyping by flow cytometry is an important tool in the diagnosis and staging of
patients with a haematological neoplasm. *t is used in con$unction with classical morphology.*n
the bone marrow, normal blood cells develop from stem cells in a progressive series of
differentiations, branching off to give different lineages of cells (for example, myeloid or lymphoid,
' cell or + cell), each of which has a distinct maturation pathway. "t each stage, the cells carry a
distinctive set of mar,ers classified by the - (cluster of differentiation) nomenclature.'he panel
selected will depend on initial clinical diagnosis. 'he results from the initial screen may indicate
the need for further classification with an extended panel that is more specific for a particular
disease. 'he mar,ers include.
+ cell. -/, -01, -02, -31, -4/, 5appa, 6ambda7
' cell. -3, -8, -4, -/, -9, -:, -4/, -/;7
<yeloid cells. -9, -00b, -08, -04, -0/, -0;, -88, -84, -4/, -/;, -009,
=6"-#, <P>.
Plasma cell. -02, -8:, -4/, -/;, -08:.
Detection of minimal residual disease
<inimal residual disease (<#) was defined as disease beyond the limit of morphological
detection using conventional microscopy. Patients with acute leu,aemia were considered to be in
remission when bone marrow samples contained ?/@ neoplastic cells. Flow cytometric methods
can detect far lower levels of disease, which can be important in the clinical management of
leu,aemia. 'he residual tumour cells are detected using immunofluorescence of surface mar,ers.
Stem cell enumeration
=aemopoietic stem cells in the bone marrow can be identified by their expression of -84.
!ormally, the number of such cells in bone marrow is low and is negligible in peripheral blood.
=owever, if mobilisation of -84 positive cells from the bone marrow is stimulated, stem cells
can be harvested from the peripheral blood as well as the marrow. 'hese cells can be used to
repopulate a depleted bone marrow after, for example, high dose chemotherapy.'o assist in the
identification of a small percentage of -84 positive cells, a double stain of -84 and -4/ is
used. 'he method gives the percentage of -84 Ave cells.
Solid organ transplantation
T cell cross-match
Flow cytometry can be used to crossmatch a recipientBs serum with donor lymphocytes to detect
antibodies that could interfere with engraftment. Prior to organ transplantation, the organ donorBs
lymphocytes are incubated with serum from the potential recipient of the graft. "fter washing,
bound immunoglobulins are detected using an F*'--con$ugated anti-human *gC antibody. 'he '
cells are identified using a PD--8 con$ugate.
Postoperatie monitoring
"fter the organ transplant, analysis of the peripheral blood lymphocytes may help to indicate early
re$ection and bone marrow toxicity during immunosuppressive therapies, and to help in the
differentiation of infections from transplant re$ection. " variety of cell surface mar,ers and
activation antigens can be used depending on the clinical condition and the organ transplanted.
Peripheral blood testing may also be used to monitor the effectiveness of anti-re$ection
immunosuppressive therapy, which may include, for example, antibodies designed to destroy '
cells. 'he number of circulating '-cells is usually determined by measuring the cell surface
mar,er -8.
Detection of autoanti!odies
"utoantibodies to leucocytes, platelets and erythrocytes may be found in a variety of autoimmune
conditions and can cause anaemia, leu,openia, or thrombocytopenia. 'hey are detected by
immunofluorescence in either a direct or an indirect assay. *n the former, anti-human *g
antibodies are used to detect *g on the surface of the patientEs cells. *n the indirect assay, the
reaction of antibodies in the patientEs serum with cells from a normal person is observed. 'he
procedures are similar to those used for a ' cell crossmatch.
"I# infection$
etermination of the numbers of -4 Ave lymphocytes in the peripheral blood is used to monitor
patients with =*F infections.'he percentage of -4 Ave cells can be obtained in a single tube by
staining for -4/G-8G-4. " cytogram of HH versus -4/ is used to identify the lymphocytes
and a cytogram of -4 versus -8 to enumerate the -4Ave ' cells. "n extended panel is used
to obtain a more complete picture of the peripheral blood lymphocytes.
HLA B27 antigen determination
"n,ylosis spondylarthritis is strongly associated with =6" +39 phenotypes. Facing a borderline
symptomatology, the detection of this phenotype can help the diagnosis. -lassically =6"
phenotypes are determined by lymphocytotoxicity assays which are time consuming (/ h) and
costly. &sing an anti-=6" +39 monoclonal antibody, =6" +39 phenotypes can be determined on
all blood it samples by direct fluorescence measured by flow cytometry. >ther =6" antigens such
as =6" +9 cross-react with =6" +39 and are responsible for false positives.!egative
fluorescence cutoff can be determined on a panelof =6" +9 A, =6" +39 A, =6" +39 -G+9 - cells.
"ll phenotypes of cell samples tested with a level of fluorescence higher than the cutoff must be
controlled by classical lymphocytotoxicity assay.
Foeto-maternal haemorrhage
Foeto-maternal bleeding can sensitise a #hesus blood group -ve mother to Ave blood cells
from the foetus. *n a subseIuent pregnancy, haemolytic disease of the new born child can be
caused by the destruction of #hesus Ave blood cells of the foetus by maternal anti-
antibodies. Prophylactic anti- given to the #hesus -ve mother shortly after delivery of a
#hesus Ave child significantly reduces the incidence of anti- sensitisation in the mother and
has led to the virtual elimination of the disease from mothers so treated. Hince the dose of anti-
given is related to the size of the foeto-maternal haemorrhage, Iuantitation of foetal-maternal
haemorrhage is therefore important.Juantitation is achieved by labelling the erythrocytes in a
sample of maternal blood with F*'--con$ugated, non-agglutinating anti- antibodies. " population
of as few as 1.0@ foetal cells is sufficient to sensitise the parent so at least /11,111 cells should
be analysed to obtain a statistically significant estimation
Immunodeficiency diseases
iseases resulting from primary immunodeficiencies, which are usually found in infants and
young children, can be a result of defects in '-cells, +-cells, granulocytes or monocytes. *n many
of these diseases surface or cytoplasmic proteins are missing or have impaired function. 'hey
are characterized by immunophenotyping, the selection of antibodies being based on the clinical
presentation.
Paro%ysmal nocturnal haemoglo!inuria
Paroxysmal nocturnal haemoglobinuria (P!=) is an acIuired disease characterised by the
development of an abnormal clone of precursor cells in the bone marrow. 'he white cells and red
cells produced are dysfunctional and are susceptible to lysis. "nalysis of this clonal abnormality
by flow cytometry, in general, accomplished by analysis of -// and -/2 on red cells
&eticulocyte analysis
#eticulocytes can be distinguished from erythrocytes by their high content of #!". 'here are
several stains that can be used for #!", one of them is thiazole orange
Applications in !lood transfusion
Contaminating leucocytes
'he presence of contaminating leu,ocytes in transfused products of blood may cause a number
of adverse effects. 'hese include.
K nonhaemolytic febrile reactions
K graft-versus-host disease
K cytomegalovirus transmission
K pulmonary oedema
K alloimmunization to =6" class * antigens
Flow cytometry offers a sensitive measure of the number of remaining leucocytes in leucofiltered
products, such as plasma and red blood cells.
Platelet counting and function
Flow cytometry can be used to count platelets and also to measure their surface proteins. 'he
latter change during activation of the platelets and can be used to measure their activation state.
Platelets are easily activated and blood has to be ta,en and handled with care7 for this reason,
whole blood methods are generally preferred.
Platelet analysis by flow cytometry can have application in
K *dentification of inherited disorders
K <onitoring of anti-platelet therapy
K <onitoring clinical course of disease
K <onitoring platelet production in thrombocytopenia
K *dentification of patients at ris, of thrombosis
K "ccurate platelet counting in thrombocytopenia
K iagnosis of heparin induced thrombocytopenia
D'A Content Analysis

'he measurement of cellular !" content by flow cytometry uses fluorescent dyes, such as
propidium iodide, that intercalate into the !" helical structure. 'he fluorescent signal is directly
proportional to the amount of !" in the nucleus and can identify gross gains or losses in !".
"bnormal !" content, also ,nown as (!" content aneuploidy), can be determined in a tumor
cell population. !" aneuploidy generally is associated with malignancy7 however, certain benign
conditions may appear aneuploid. !" aneuploidy correlates with a worse prognosis in many
types of cancer but is associated with improved survival in rhabdomyosarcoma, neuroblastoma,
multiple myeloma, and childhood acute lymphoblastic leu,emia ("66). *n multiple myeloma, "66,
and myelodysplastic syndromes, hypodiploid tumors cells portend a poor prognosis. *n contrast,
hyperdiploid cells in "66 have a better prognosis.
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