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Flow cytometry in RBC and platelet disordres

Prof R. Saxena, M.D., Professor and Head of Department, Department of Haematology, 2

New Private Ward, AIIMS, Ansari Nagar, New Delhi. 110029

Flow cytometry is of extensive use in the non-malignant haematological disorders. The chapter will
be discussed in sections covering RBC and Platelet disorders.
RBC disorders:
Flow cytometry has a wide application in the RBC disorders including Haemolytic anaemia such as
reticulocyte counting , PNH, hereditary spherocytosis, autoimmune mediated haemolysis, and F cell
Reticulocyte count
The determination of reticulocyte count is based on the identification of residual RNA and
ribosomes in the RBCs. They are seen on the Romanovsky stained smears as polychromatophils but
accurate counts are performed manually on blood incubated with supravital dyes. The supravital
dyes such as new methylene blue, brilliant cresyl blue precipitate the nucleic acid on incubation with
blood. Manual reticulocyte counts are performed on smears made after this process. The method is
however, labour-intensive, imprecise and tedious.

Fluorescent dyes such as thiazole orange and auramine O bind to the residual RNA in the
reticulocytes and hence, reticulocyte count can be performed using flow cytometry.
fluorescent dyes such as acridine orange, pyronin Y, thioflavin T etc can also be used for automated
reticulocyte counts.
Thiazole orange is the preferred dye as its excitation occurs in the visible light
spectrum (488 nm) while acridine orange, auramine etc require the ultraviolet light for
Flow cytometric reticulocyte counts have been incorporated in many commonly available
automated cell counters.

The use of flow cytometric methods in estimation of reticulocyte count helps not just to determine
the reticulocyte percentage and absolute reticulocyte count but also gives additional parameters
such as reticulocyte maturation index (RMI)
and immature reticulocyte fraction (IRF).
RMI was
found to be affected by iron stores and was later replaced by IRF which is a more accurate
parameter. The fluorescence of reticulocytes after staining with a fluorescent dye is divided into low,
medium and high fluorescence based on the RNA content. The area of medium and high
fluorescence ratios comprises the IRF.
The IRF is correlated with the erythropoietic activity of the
marrow and can differentiate between conditions with reduced erythropoiesis and enhanced
erythropoiesis with a study indicating that IRF 0.23 in patients of anemia was a reflector of
nonresponsive or under-responsive bone marrow.
It is also a significant predictor of recovery from
neutropenia following haematopoietic stem cell transplantation. The serial evaluation of IRF to
calculate the IRF doubling time was shown to precede recovery of absolute neutrophil count by
several days.

However, there are many interfering factors in flow cytometric reticulocyte count. These include
both interference from cellular elements and the RBC inclusions. Since the platelets and reticulocyte
counts are performed in the same channel with the separation between RBCs and platelets done on
Forward angle light scatter or on forward vs side angle side scatter, platelet clumps and large
platelets interfere with reticulocyte counts. In addition, WBC fragments and nucleated RBCs also
interfere with reticulocyte counts. In addition, RBC inclusions containing nucleic acid such as Howell-
Jolly bodies, parasites such as plasmodium, basophilic stippling also interfere with reticulocyte
count. Other RBC inclusions like Pappenheimer bodies, Heinz bodies and haemoglobin H inclusions
also interfere with reticulocyte counts.

Paroxysmal nocturnal haemoglobinuria (PNH)
PNH is an acquired clonal haematopoietic stem cell disorder due to a mutation in the X-linked
phosphatidyl-inositol glycan (PIG-A) gene. This gene synthesises a glycophosphatidyl-inositol anchor
protein (GPI) and there may be a total or partial deficiency of this anchor protein.
Many proteins
on all cells of haematopoietic lineage require this GPI anchor protein. The classical manifestations
include chronic intravascular haemolysis, varying degrees of bone marrow failure and thrombosis
especially at unusual sites such as intra-abdominal circulation. In addition, small PNH clones are also
detectable in aplastic anemia, Myelodysplastic syndromes. Since the first manifestations were
recognised to be as a result of increased complement mediated lysis of erythrocytes, the initial tests
based on activation of complement were the Hams test and sucrose lysis test. Both these are labour
intensive and may be false negative in heavily transfused patients.

The enhanced complement activity led to the discovery of two GPI-linked proteins CD55 and CD59
were discovered to be important to control excess complement activity preventing complement
mediated destruction in normal RBCs.
Most flow cytometric analysis has been directed at the
analysis of these two proteins on RBCs and the same markers were also analysed on the
granulocytes. The analysis on RBCs provides a clear distinction between the type I cells with normal
expression, type II cells with a partial deficiency and type III cells with complete deficiency.
CD59 is vital for preventing complement mediated lysis, the types are defined on CD59
The analysis of only RBCs is fraught with problems as any recent increase in haemolysis
and transfusions make the determination of clone size difficult and also inaccurate. This led to
analysis of the granulocytes for expression of GPI linked proteins and since most experience was
available with CD55 and CD59, they were applied to granulocytes also. However, they have been
found to be inferior for analysis of granulocytes.
There are additional GPI-linked proteins suitable
for analysis such as CD24, CD16, CD66b on granulocytes and CD14 on monocytes.
A specific toxin pro-aerolysin was discovered from the bacteria Aeromonas hydrophila which on
activation, specifically binds to GPI anchor protein and causes the formation of pores in the cell
membrane leading to lysis of the cell. PNH cells are resistant to lysis and it was seen that the
percentage lysis after aerolysin exposure paralleled the percentage of CD59 positive cells. It was also
demonstrated that type III cells were resistant and type II cells showed only a partial resistance to
A mutated form of proaerolysin was conjugated with Alexa 488 (FLAER) and this still
retains its ability to bind with GPI-linked structures but does not cause cell lysis. Since FLAER is
directed at the GPI anchor protein, it is highly sensitive for detection of PNH. The inability to analyse
RBCs is only drawback as glycophorin binds weakly to proaerolysin.

The assays for PNH are performed in a lot o0f laboratories but poor concordance was seen on EQA
programs. This led to the recent guidelines on PNH in 2010. These guidelines have differentiated two
types of PNH assays- routine sensitivity and high sensitivity. Routine analysis was defined as
identification of an abnormal population >1% with acquisition of at least 5000 specific events and
high sensitivity analysis with identification of as low as 0.01% PNH cells with acquisition of at least
2,50,000 specific events. The analysis is preferably performed on peripheral blood and EDTA, heparin
and ACD as anticoagulants. RBCs are stable for 7 days while since the scatter properties of
granulocytes is affected, the analysis should be performed in <48 hours. It is also recommended that
granulocytes should be analysed in all cases with monocytes providing confirmatory information.
RBCs can be analysed in all cases but since it is challenging, it should be at least performed in all
cases where a PNH clone is detected on WBC analysis. For granulocyte analysis, in a routine assay,
CD45 vs SSC or CD15 vs SSC gating is sufficient but for a high sensitivity analysis, a granulocyte
specific marker like CD15 should be included for the initial gating. At least two reagents from
FLAER/CD24/CD66b/CD16 should be preferred. CD55/CD59 combination is no longer preferred for
granulocyte analysis. CD16 is not present on eosinophils and should not be used as sole reagent. For
monocytes, routine sensitivity analysis requires initial gating on CD45 vs SSC or CD33 vs SSC and due
to small numbers, high sensitivity analysis may not be possible. FLAER, CD14 are the commonly used
markers while CD48, CD55 and CD157 can be used. CD14 is not present on dendritic cells and should
not be the only marker analysed. For RBC analysis, log FSC vs SSC gating is recommended for routine
analysis and Glycophorin A+ scatter for high sensitivity analysis. CD59 is the recommended reagent
to be used in all cases while CD55 can be added.

Hereditary spherocytosis (HS)
HS occurs due to deficiency in the vertical interactions of the membrane cytoskeleton with the RBC
membrane. The most commonly deficient cytoskeletal protein is ankyrin. This deficiency is a result
of mutations in the ankyrin gene. The other common deficiency is of the spectrin protein. HS is
inherited as an autosomal dominant condition in ~75% cases while is recessive in the residual 25%
It is usually diagnosed on the basis of an increased incubated osmotic fragility test (OFT) and
a normal direct Direct Coombs test (DCT) in the presence of a positive family history.

Flow cytometry for HS includes two tests- Eosin 5-maleimide (EMA) dye binding test and osmotic
EMA binding dye test: This test is available since 2000. The principle is that maleimide moiety of
EMA binds stochiometrically with the band 3 protein. In cases of HS, there is a primary or a
secondary deficiency of band 3 protein and hence the binding of EMA is reduced and the mean
fluorescence intensity (MFI) is reduced than normal. However, the dye stability is an issue and needs
to be stored at -20C otherwise a rapid loss of labelling efficiency is seen. The reported sensitivity
was 92.7% with a specificity of 99.1% for the detection of HS.

In our lab, EMA binding test was shown to be highly sensitive (96.4%) and specific(94.2%)in a series
of 114 anaemia patients including confirmed HS, suspected HS and other haemolytic, microcytic and
macrocytic anaemias. The sensitivity of incubated Osmotic fragility test was ~74% in this study.Some
cases of CDA type II and AIHA can be falsely positive.
A lower EMA binding has also been reported
in other membrane disorders like hereditary elliptocytosis
and South east Asian ovalocytosis
Flow cytometric OFT: This test uses the property of spherocytes to undergo rapid haemolysis when
exposed to osmotic pressure in low concentration saline. The blood sample from a suspected patient
of HS is spiked with de-ionised water and subjected to acquisition in real time to determine the
sequential RBC count. The same test can also be conducted after the sample is incubated for 24
hours. RBC count has to be adjusted prior to analysis so that each tube has the same number of
cells. In this analysis, a time/FSC plot was made and divided into 8 regions. Residual red cells
percentage is calculated by the following formula:

% residual cells=Mean events of the last two regionsX100
Event count of the first regionX1.1/2.0
This analysis differentiated HS from iron deficiency anaemia. However, patients of AIHA were not
included in this analysis.

In an Indian study, using the same technique as above, a sensitivity of 100% and sensitivity of 98%
was achieved. The study included a 20 HS cases, 3 HE cases and 3 patients of distal tubular acidosis
with a band3 defect. A normal control group and a -thalassemia trait but no cases of AIHA were

Autoimmune haemolytic anaemia (AIHA)
AIHA is divided into the warm and cold type. The diagnosis is based on the demonstration of a
positive DCT using polyspecific antisera against IgG and C3d. DAT can be performed by the tube
method/ gel card test which requires the presence of at least 500 and 200 molecules of
autoantibody per cell.
However, cases where haemolysis is clinically strongly suspected and DCT
is negative still constitute 2-6%. A flow cytometric test has been developed which can detect as low
as 30-40 molecules of autoantibody per cell. In a large study which evaluated 380 patients, flow
cytometry for IgG was positive in 6/17 IgG tube-DAT negative patients of AIHA and 23/36 patients of
C3d tube DAT negative patients.
In an Indian study in which flow cytometry was compared to gel
card based DCT, in the 5 cases that were tube DCT negative, gel card was positive in 4 and flow
cytometry in all 5.

Flow cytometry can be an important adjunct to diagnosis of AIHA especially in the cases where it is
strongly suspected clinically but the gel card test is negative.
F cell estimation
Transplacental haemorrhage may occur during pregnancy and can cause haemolytic disease of
newborn. To prevent this, anti-D is administered to the mother and to quantify the amount of anti-D
to be given, the amount of fetomaternal haemorrhage has to be estimated. Two techniques are
available- acid elution/Kleihauer Betke test and flow cytometry for F cell quantification. The CV
reported by the acid elution method is higher than flow cytometry and it is also technically
challenging and laborious. A fluorescent labelled anti-D antibody is used to detect and quantify the D
positive fetal cells and this is the only recommended antibody by the BCSH guidelines.
haemoglobin antibody can also be used for F cell estimation.
F cell estimation is also useful in
patients of sickle cell anaemia on hydroxyurea therapy
and for determining the F cell pattern to
distinguish HPFH and HbS-HPFH from homozygous S and HbS-
Platelet disorders
Platelets can be studied by flow cytometry to evaluate primary platelet disorders like Bernard
Soulier disease (BSS), Glanzmann thrombasthenia (GT), disorders of platelet dysfunction (acute
coronary syndrome, ischemia, cardiopulmonary bypass and disorders of thrombopoiesis by studying
reticulated platelets. It may also help to diagnose heparin-induced thrombocytopenia (HIT). Platelet
flow cytometry can be performed on whole blood or platelet rich plasma or washed platelets. The
latter two techniques can cause platelet activation and since whole blood is more representative of
the in vivo system, requires almost no sample manipulation and uses small volumes, it is the
preferred method.

For platelet analysis, blood samples can be collected in trisodium citrate
but EDTA and heparin are
not suitable. EDTA dissociated GpIIb-IIIa complex and also prevent its binding to fibrinogen
heparin causes platelet activation
. The other suitable anticoagulants are citrate, theophylline,
adenosine, dipyridamole (CTAD), hirudin, corn trypsin inhibitor (CTI) etc.
Certain precautions are
common to any procedure on platelets like gentle drawing of blood, using a large bore needle (21G)
without a tourniquet, ensuring smooth flow, discarding the first 2 mL of blood. Once the sample is
drawn, it should be processed within 15 minutes
and should be stored at room temperature as
cold can cause platelet activation. Platelets have a tendency to aggregate which can be prevented by
diluting whole blood with buffered saline and the use of very gentle mixing techniques.
Glanzmann thrombasthenia (GT)
GT is caused by a complete or partial deficiency or functional impairment of the GpIIb/IIIa complex.
The characteristic platelet aggregation profile is of absent aggregation with all agonists but the
agglutination with ristocetin is preserved. This profile is due to loss of a critical function of GpIIb/IIIa
of undergoing a conformational change upon activation and binding to fibrinogen. Since this is the
final step in platelet aggregation when stimulated by all agonists, aggregation in response to all
agonists is absent.

Flow cytometry is an important tool for confirmation of GT as well as the characterization into the
three subtypes based on the amount of antigen expression on the platelets. The three types are:
type I in which <5% platelets express GpIIb/IIIa complex, type II in which 5-20% platelets express the
GpIIb/IIIa complex and type III in which the levels of GpIIb/IIIa are normal but the protein is
The antibody CD41 is directed against GpIIb and CD61 against GpIIIa. These are the
commonly used antibodies to detect GT but there are other available antibodies directed against
epitopes which are only expressed when GpIIb/IIIa is activated such as PAC-1.
Such antibodies
make the detection of subtle GT with minor dysfunction in the gpIIb/IIIa complex easy. In North and
south India, the most common subtype of GT is type I.

Carrier detection for GT patients is also possible with the use of flow cytometry. In a study from our
center, flow cytometry using CD41 and CD61 was able to detect a carrier state in 85% of the parents
and 55% of the siblings tested. When flow cytometry was compared to western blot analysis, the
sensitivity of flow cytometry was 75% while it was 39% for Western blot.
DNA analysis still remains
the most accurate method for carrier detection in GT.
Flow cytometry also helps detect the acquired GT cases where autoantibodies against the GpIIb/IIIa
complex are formed. Giannini et al successfully used flow cytometry to detect acquired GT in one
case and they were able to demonstrate that the platelets in acquired GT showed normal binding to
some anti-GpIIb/IIIa antibodies (SZ21 & SAP) while reduced binding was seen with P2, SZ22 &
A2A9/6. Also the binding to PAC-1 was absent. Using mixing studies, they were also able to
demonstrate that patients serum inhibited PAC-1 and A2A9/6 binding with the control platelets.

Bernard Soulier disease (BSS)
This syndrome is characterised by macrothrombocytopenia with bleeding manifestations. It is an
autosomal recessive disorder due to the deficiency if GpIb/IX/V complex. The characteristic platelet
aggregation abnormality is the absent agglutination with ristocetin while aggregation to all other
agonists is normal. To differentiate from vWD, patients PRP is mixed with a normal PPP and
correction with ristocentin indicates vWD while correction on mixing with normal platelets indicates

The diagnosis can also be established by flow cytometry by the use of CD42b against GpIb, CD42a
against GpIX and CD42d against GpV.
Since flow cytometry provides a distinction between the
deficient sub type of the GpIb/IX/V complex, it is also useful to identify the candidate gene. In a
study to identify genotype phenotype correlations, it was seen that GpIb, GpIX and GpV expression
was reduced in all BSS cases but GpIb was in particular always <10% of the control value. In the
cases which showed a GP9 or GP1BB mutation, the expression of GpIX was deficient. In cases of
GP1BA mutation, the level of GpIX was reduced to 18-23%. The expression of GpV was the most
variable ranging between 5-33%.
In a study from South India, the expression of GpIb/IX/V was
reduced from 7.7-57%. The most common mutations were missense mutations.

Diagnosis of platelet type vWD and vWD type 2B:
Flow cytometry has been used to detect ristocetin induced binding of vWF to platelets. Type 2B
shows a reduced binding of vWF to formalin fixed platelets but increased binding is seen with the
autologous platelets. In a study on 12 patients of type 2B vWD, there was a good correlation
between flow cytometry and vWF:Ag, VWF:RCo, VWF:CB & RIPA.
The same assay has been applied
to the diagnosis of platelet type vWD. Using a combination of normal plasma with patient platelets
and a combination of patient plasma with normal plasma, it is possible to distinguish type 2B vWD
from platelet type vWD.

Measurement of platelet activation
The activation of platelets induces the release of granule contents and also causes conformational
changes in some glycoproteins like GpIIb/IIIa. There are specific monoclonal antibodies direct against
these which can be used in ELISA as well as in flow cytometric assays. The PAC-1 antibody specifically
recognises the activated form of GpIIb/IIIa complex while an increase in CD62P/ P-selectin
expression on release from alpha granules upon activation can be measured by flow cytometry.

The study of platelet activation is relevant to many cardiac disorders as well as other systemic
disorders like uremia, diabetes mellitus, multiorgan failure, myeloproliferative disorders and
Alzheimers disease.

Anti-platelet antibodies
The analysis of anti-platelet antibodies was used to diagnose Immune thrombocytopenia but since
platelets express about 1000-2000 molecules of Fc receptor for IgG and hence a large number of
non-specifically bound antibodies may overlap with the platelet specific antibodies. The most recent
type III assays which detect platelet glycoprotein specific autoantibodies like the monoclonal
antibody-specific immobilizationof platelet antigen (MAIPA) and its modified PAIgG characterization
assay (PAICA) have been developed.
Flow cytometric assays to detect platelet specific
autoantibodies have been developed. The method is based on the detection of autoantibodies
which were reacted with microbeads coated with CD41a, CD42b, CD61, CD41b.
Also, flow
cytometric assays similar PAIgG
have also been developed. The reported sensitivity is 80-
but the sensitivity is low ~30%.
However, the detection of anti-platelet antibodies is not
available in most clinical laboratories.
Reticulated platelets/Immature platelet fraction
Reticulated platelets are the young platelets that contain a higher amount of mRNA. They can be
measured using thiazole orange as this dye permeates cell membranes and binds to nucleic acids. It
provides an easy measure of normal or increased megakaryocytes in thrombocytopenic patients. In
patients of thrombocytopenia with reduced marrow megakaryocytes, the reticulated platelets are
lowered when compared to normal controls.
This measurement is available in automated cell
counters now (Sysmex XE-2100).
The detection of IPF has been found to be closely associated with
engraftment post haematopoietic stem cell transplantation.
Reference method for platelet counts
The reference method for platelet count for a long time was manual counts by phase contrast
microscopy but in 2000, International Council for Standardization in Haematology (ICSH) replaced
this by the flow cytometric PLT/RBC ratio as the new reference method.

Platelet microparticles
The detection of platelet micropartiocles and platelet-leucocyte conjugates was made possible with
the use of flow cytometry. Microparticles are 0.02 to 0.1 m in size and are formed as result of
vesiculation and apoptosis of cells. The outer membrane of the cells undergoes flip-flop mechanism
exposing phosphatidyserine on the surface. Multiple platelet antibodies have been used including
CD42a, CD42b, CD41, CD61, CD62p. Platelet derived microparticles have surface receptors for factor
VIII & factor Va and prothrombinase complex is assembled on this surface. Since their identification,
platelet microparticles have been demonstrated in a wide variety of clinical conditions including ITP
patients post splenectomy and myeloproliferative disorders.
The use of flow cytometry in platelets can help easily confirm the diagnosis if the facilities of
genetic testing are not available. It can also help to detect a carrier state in the siblings and
parents of the propositus with the bleeding disorder like Glanzmann thrombasthenia.
The application of flow cytometry is also extended for the diagnosis of the many primary
immunodeficiency disorders.