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Micromethod for demonstrating increased platelet surface immunoglobulin G Findings in acute chronic and human immunodeficiency virus-1-related immunologic thrombocytopenias.

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American Journal of Hematology 34:275-282 (1990)
Micromethod for Demonstrating Increased Platelet
Surface Immunoglobulin G: Findings in Acute, Chronic,
and Human Immunodeficiency Virus-I -Related
Immunologic Thrombocytopenias
Dahlia Sthoeger, Michael Nardi, Susan Travis, Margaret Karpatkin, and Simon Karpatkin
Departments of Pediatrics and Medicine, New York University Medical Center, New York
A method has been developed for the demonstration of increased platelet surface IgG
that uses 1 ml of blood regardless of the platelet count. Platelets are gel filtered to remove
plasma and contaminating lymphocytes. They are then reacted with fluorescein-conjugated antihuman IgG and analysed by flow cytometry. Percent positive staining cells vary
from 10% to 80% of total cells examined. A platelet antibody index is derived from the
product of percent positive staining cells X mean fluorescence intensity of positive staining cells. All patients studied with chronic idiopathic thrombocytopenia purpura (ITP) or
human immunodeficiencyvirus-1 (HIV-1)-related thrombocytopenia had increased platelet surface IgG. Twelve acute children and 11chronic children had indices averaging 3.5and 8.9-fold greater than 12 normal children, respectively. Five of 12 children with acute
ITP had normal platelet IgG. There was no linear correlation between the platelet antibody
index and platelet count. Platelets of patients with acute, chronic, or HIV-1-related ITP
displayed autofluorescence. In chronic ITP, the percentage of platelets displaying autofluorescence had a significant negative correlation with the platelet count. This technique
will be a valuable diagnostic tool in the pediatric population.
Key words: ITP fluorescence-activated flow cytometry, acute ITP, chronic ATP, HIV-1related ITP, autofluorescence
INTRODUCTION
Idiopathic thrombocytopenic purpura (ITP), also
known as autoimmune thrombocytopenic purpura
(ATP), is an acute or chronic disorder in which IgG is
bound to the platelet membrane in most patients. The
opsonized platelets are removed by macrophages of the
reticuloendothelial system. Demonstration of increased
amounts of IgG on the platelet may be helpful in establishing the diagnosis of ATP [ 11. Numerous techniques
for measurement of platelet-bound IgG have been described [2,3]. Many of these require large volumes of
blood, particularly if the platelet count is very low, and
therefore are not useful in infants and young children.
Here we describe a method for demonstrating increased
platelet surface IgG that requires no more than 1 ml of
blood regardless of the platelet count, does not utilize
radioactive isotopes, and eliminates contaminating lymphocytes, which can give spurious results with low plate0 1990 Wiley-Liss, Inc.
let counts. Gel-filtered platelets are exposed to fluorescein-labeled antihuman IgG and then analyzed by
fluorescence-activated flow cytometry to determine the
number of platelets that fluoresce and the intensity of
their fluorescence.
Received for publication September 22, 1989; accepted February 15,
1990.
Address reprint requests to Margaret Karpatkin, MD, Department of
Pediatrics, NYU Medical Center, 550 First Avenue, New York, NY
10016.
Dahlia Sthoeger is now at Kaplan Hospital, Rehovath, Israel.
Susan Travis is at the University College of Medicine and Dentistry,
New Jersey-Robert Wood Johnson Medical School, Camden, New
Jersey.
276
Technique: Sthoeger et at.
Tyrode’s buffer containing 1% bovine serum albumin
(BSA). A second 1 ml aliquot of gel-filtered platelets is
Patient Population
incubated with Tyrode’s buffer plus 1% BSA without
The patient population consisted of 12 children with addition of antibody. The platelets are washed four times
acute ITP (the term is used for the acute childhood dis- in Tyrode’s buffer 1% BSA, and each aliquot is then
order for which the etiology is poorly understood), 11 resuspended in 50 pl of 1% paraformaldehyde. One milwith chronic ATP, and five who were thrombocytopenic liliter of isoton (Coulter Diagnostics, Hielaeh, FL) is
and had antibodies against HIV- 1. The 23 ITP-ATP chil- added, and the platelets are resuspended by gentle tapdren presented with purpura, did not have lymphade- ping and then analyzed by flow cytometry (FACScan;
nopathy or splenomegaly , and had negative antinuclear Becton Dickinson).
antibodies and no risk factors for HIV- 1 . All but one had
undergone bone marrow aspirations, which revealed nor- Fluorescence-Activated Cell Scanning (FACScan)
mal to increased megakaryocytes. The disease was conThe FACScan is equipped with an argon laser operatsidered to be acute if it resolved within 6 months of
ing
at 488 nm. Data acquisition of 10,000 events was
diagnosis (platelet count 150,000/mm3 or more on no
gated
on a cytogram of forward and right-angle scatter to
therapy). Disease lasting longer than 6 months was coninclude
platelets and exclude cellular debris (Fig. 1A).
sidered chronic. In addition, 21 adults with chronic ATP
Under
these
conditions, 98% of gated cells of healthy
were studied.
controls stain with 3B2, a fluorescein-labeled monoclonal antibody for platelet GPIIb [5]. The ungated parCollection of Blood
ticles contain -1% of the total positive staining cells.
Blood specimens were obtained from discarded sam- Under these conditions, less than 10% of lymphocytes
ples routinely employed for relevant clinical tests. are gated as determined from an analysis of lymphocytes
Venous blood was drawn into 0.04 ml of 7.5% potasium separated by Ficoll-Hypaque from thrice-defibrinated
EDTA (Vacutainer Systems, Becton Dickinson, Ruther- blood (less than 5% of these particles stain for GPIIb).
ford, NJ) and centrifuged at lOOg for 10 min to obtain This represents 9% of total “lymphocyte” IgG (Ficollplatelet-rich plasma (PRP). Acetyl salicyclic acid was Hypaque fraction). Prior gel filtration of PRP eliminates
added to the PRP to a final concentration of 0.25 mM to virtually all lymphocyte and red blood cell contaminaprevent clumping.
tion. In severely thrombocytopenic patients (platelet
counts < 30,00O/pl) platelet and red blood cell fragments are often present [6]. Therefore, fewer intact
Separation of Platelets From Plasma
platelets are gated (Fig. 1B). Approximately 80% of the
and Lymphocytes
gated cells of this patient with HIV- 1 thombocytopenia
Depending on the volume of PRP available, between
(platelet count 30,0OO/pl) stain positively for GPIIb. The
0.1 ml and 1.0 ml is applied to a disposable 200 mm
ungated “cellular debris” contains less than 2% of total
column (Kontes, Vineland, NJ) of sepharose 2B in Typositive staining cells.
rode’s buffer, pH 7.4. The column is washed with TyA fluorescence index or platelet antibody index is obrode’s buffer; the first 1 ml of effluent is discarded, and
tained by multiplying the mean intensity of fluorescence
the next 2 ml, which contains the platelets, is collected.
of antibody-treated cells (x axis) by the number of plateTo verify the absence of lymphocytes in the platelet fraclets that fluoresce (y axis) beyond the gate. For each
tion, PRP known to contain HIV-1 was subjected to gel
patient, the value obtained for the nonantibody-incubated
filtration. The platelet fraction was amplified by the
platelets is subtracted from that obtained for the antipolymerase chain reaction using Taq 1 polymerase and
body-treated platelets (Fig. 2). This index, rather than
oligonucleotide primers for the gag gene of HIV-1.
percent positive staining cells or mean intensity, was
Southern blotting of six amplified samples did not reveal
noted to give the largest differences and least overlap
gag, whereas unfiltered samples were positive for gag
between control and patient samples. Control studies
141.
with irrelevant fluorescene-conjugated anti-CD4 antibody revealed no change in fluorescence before and after
Incubation With Fluorescein-Conjugated Antibody addition of antibody to the platelet sample (data not
One milliliter of gel-filtered platelets is centrifuged at shown).
Unprocessed blood samples may be stored for 1 week
1,OOOg for 6 min at room temperature, the supernatant is
after
collection without change in the antibody index.
discarded, and 50 p,l of 125 ng of fluorescein-conjugated
After
fixation with paraformaldehyde, platelets can be
goat F (ab’), antihuman 1gG (Tago Inc., Burlingame,
stored
for a least 1 month.
CA) is added to the platelet pellet for 30 min at 4” in
MATERIALS AND METHODS
Technique: Micromethod for Platelet Surface IgG
277
DOT PLOT OF LIGHT-SCATTER
RESULTS
Percent Positive Cells
4
The percent positive staining cells was assessed under
instrument conditions in which less than 1% of unstained
normal cells fluoresced (Fig. 2). The mean percent positive staining cells (after addition of fluorescent antibody)
for 12 healthy children aged 9 months to 16 years was 5.1
+. 2.4 (S.D.). The mean percent positive cells for 20
healthy adults was 4.9
2.9 (S.D.). This was not significantly different from the case in healthy children. We
therefore pooled the adult and child controls, obtaining a
mean percent staining of 5.0, with a 2 S.D. upper limit
of 10.4. The percent positive staining cells for chronic
children averaged 35.4 -+ 20.8 S.D. (range 8.0-78.8)
compared to 12.4 +- 8.9 for acute children (range 2.728.4) (Fig. 3). This 2.9-fold difference was statistically
significant ( P = 0.002), possibly due to the presence of
seven ITP children with normal values. HIV-I-ITP children had a value of 28.8 2 12.9. Chronic adults had a
value of 24.3 -+ 17.4, which was not significally different
from the value in chronic children (Fig. 3).
In severely thrombocytopenic patients (< 30,000
plateletslpl) percent positive platelets may represent
minimal values because of the presence of nonplatelet
particles in the "platelet" gated area (see Discussion).
Linear regression analysis of platelet count vs. percent
positive staining cells was performed for acute and
chronic children as well as chronic adults. No correlation
was noted for any of the groups.
r
.< .'
I .
+_
+
u
cn
h
ssc
B
r
7
'
.:
... .
.
.
Platelet Antibody Index
The mean index for 12 healthy children was 217 ? 82
(S.D.), with a range of 54-259. The mean index for 20
healthy adults was 137 & 84, with a range of 23-282.
This was not significantly different from that for the
u
healthy children. The pooled adult and child controls
v3
.
.
.
.
.:
gave a mean index of 166 & 9 1. This provides a + 2
Er
;
.
. . . ... . :.
. .. ,.
. . . . .., . . :._
S.D.
upper limit of 348. The index obtained in each of
J
... .. '.. . . $ ,.: .
four
children
who were thrombocytopenic due to nonim.
.
.
. , .,. . . . .. . , _ . .
munologic diseases was within the normal range
(Table I).
The platelet antibody index in 21 adults and children,
thrombocytopenic for a variety of causes, was compared
with the level of platelet-associated IgG measured by our
standard radioimmune assay [ 11,121. Comparison by regression
analysis was 0.6, with a P value of < 0.01.
Fig. 1. Dot plot of light scatter properties from a health!
Nine patients were negative according to both methods
control subject (A) and a patient with a platelet count o
30,0001p.1 and cellular debris (6). The area in brackets is and 12 were positive.
gated for fluorescence evaluation. x axis depicts sidewards
Seven out of the 12 patients with acute ITP had an
light scatter, y axis depicts forward light scatter.
elevated platelet antibody index and five were normal
(Fig. 3). There is no apparent difference between these
two groups of patients with respect to age, platelet count,
and time elapsing between diagnosis and remission. In
..'I,.
,,
'
I
,
'
.
I
,
~
I.,.. ,
ssc
278
Technique: Sthoeger et al.
3813
CONTROL
K
W
m
z
3
z
J
J
W
-
0
D
PATIENT + Ab
0
1
FLUORESCENCE
Fig. 2. Fluorescence-activated flow cytometry analysis of
control and patient's platelets. A: Autofluorescence of control platelets. Vertical line represents the marker set at < 1%
autofluorescence. 6: Fluorescence pattern after addition of
fluoresceinatedgoat antihuman IgG antibody to gel-filtered
platelets. C: Autofluorescence of patient's platelets. D: Fluorescence after addition of fluoresceinated goat antihuman
IgG to gel-filtered platelets.
two patients in whom follow-up samples were obtained
after remission, the index had returned to normal.
All of the children with chronic ATP had elevated
indices, as did the children who were HIV-1 positive.
The mean index in the children with chronic ATP, 3,093
+- 2,509 was 2.5-fold higher than in those with acute
ITP, 1,228 +- 1,520 ( P < 0.04 unpaired t test; Fig. 3 ) .
This is due to the presence of five ITP children with
normal values. All five children with HIV-l-related ITP
had elevated indices that averaged 6,910 2 5,017 (Fig.
3 ) . Their mean index was 5.6-fold greater than acute ITP
( P = 0.002) and 2.2-fold greater than chronic ATP,
( P = 0.06).
Nineteen adults with chronic ATP were also studied.
The platelet antibody index was elevated in all of them,
with a mean value was 1,484 -+ 945 (Fig. 4). This was
significantly lower than the value for childhood chronic
ATP ( P < 0.02).
The relationship between the platelet count and the
antibody index was evaluated by linear and by exponential regression analysis in the children with acute ITP,
and in the children and adults with chronic ATP. There
was no correlation in any of these groups.
Percent Autof luorescence
The percent platelets that fluoresced without the addition of antibody (percent autofluorescence) was surprisingly greater in patients than in normal subjects: acute
childhood ITP, 5.6 -+ 0.28 vs. control 0.83 +- 0.28, P
< 0.09; chronic childhood ATP, 8.5 5 8.6 vs. control
0.83 -+ 0.28, P < 0.005; chronic adults ATP, 4.1 ? 4.9
vs. control 0.44 +- 0.28, P < 0.002; HIV-1-ITP, 10.6 ?
6.5 vs. control 0.83 ? 0.28, P < 0.001 (Fig. 5). The
percent autof luorescence correlated inversely with the
platelet count in chronic childhood ATP employing a
log/log plot (r = 0.86, P < 0.001, n = lo). Autofluorescence was not observed in all four nonimmunologic
thrombocytopenic patients (range 0.4-1.3%). We ex-
Technique: Micromethod for Platelet Surface IgG
% Ab Staining
h i e Chronic
Mean Intensity
HIV
Platelet Ab
-
279
Index
lormoi Acute
304
15
9
8
7
120
65
5 9w
100
0
4
0
0
0
8
40
*
Fig. 3. Scattergram of fluorescent antibody-treated platelets of children with immunologic platelet disorders: Percent positive fluorescent platelets, mean intensity of fluorescent platelets, and mean platelet antibody index (percent
Platelet
countlkl
Positive
cells (%)
Platelet
Ab index
1
fluorescent cells x mean intensity of fluorescent cells) for
controls, acute childhood ITP, chronic childhood ATP, and
childhood HIV-1-related ITP.
TABLE 1. Percent Positive Cells and Platelet Antibody Index in
Children With Nonimmunologic
- Thrombocytopenia
.
Acute myeloblastic
leukemia in remission,
on chemotherapy
(12 years, M)
May-Heggelin
anomaly
(2 weeks, F)
Post bone marrow
transplant for acute
lymphocytic leukemia
(10 years, M)
Anorexia nervosa
(15 years, M)
Control children
(n = 12)
2
0
20
Diagnosis (age, sex)
3
0
.
Chronic
Normol
180
Chronic
0
'latelet Ah Index
Normol
1
9
8
74,000
8.6
29 1
40,000
11.5"
435a
7
0
100
5.0
114
115,000
10.0
305
> 150,000
< 10.4
< 381
3
59,
80
30,000
6
m
4
0
60
40
LU
8
0
8
0
.-- -__
_.
+
3
2
1
"The normal values for children under 6 months have not been established.
plored the possibility that 1% paraformaldehyde fixation
or IgG bound to the membrane could increase autofluorescence. Stained normal platelets, with or without
paraformaldehyde fixation, demonstrated no autofluorescence (data not shown). Neither did PLAl-positive
platelets incubated with polyclonal anti-PLA1 antibody,
washed, and then analyzed with FACScan. No increase
Fig. 4. Scattergram of fluorescent antibody-treated platelets of adults with chronic autoimmune thrombocytopenic
purpura: Percent positive fluorescent platelets, mean intensity of fluorescent platelets, and mean platelet antibody index.
in percent autofluorescence occurred, under conditions
where the second antibody-stained sample fluoresced intensely (data not shown). The intensity of autofluores-
Technique: Sthoeger et al.
280
36
F
-
Normal
dormol
Child
Adult
Acute
Child
-
:hronic
Child
:hronic
Adult
HIV
Child
0
0
0
8
b
c
’“i
4
0
0
b
0
0
0
8
B
0
0
0
0
8
-4--
Fig. 5. Scattergram of percent positive autofluorescent
platelets of nonfluorescent antibody-treated platelets of
children and adults with immunologic platelet disorders:
Acute ITP, chronic childhood ATP, chronic adult ATP, HIV1-related ITP of childhood.
cence was no greater in any of the patients with ITP
compared to controls.
DISCUSSION
We have developed a method for demonstrating increased IgG on the platelet surface using no more than 1
ml of whole blood, with a platelet count as low as
lO,OOO/pl. Studies of acute childhood ITP have been
hampered by the need to draw unacceptably large volumes of blood from small children in order to obtain a
sufficient number of platelets. Results of the test correlate well (r = 0.6, P < 0.01) but not perfectly with those
obtained in the same patients using an established radioimmunoassay that utilizes much larger volumes of blood
[ 11,121. The lack of a perfect correlation may be because
the radioimmunoassay measures released IgG [ 13,141 as
well as surface IgG, whereas fluorescence flow cytometry measures cell surface IgG.
Other groups employing washed platelets have developed similar methods demonstrating the usefulness of
flow cytometry for the measurement of antiplatelet antibody [7-lo]. Our method differs from the others in the
use of gel-filtered platelets, which eliminates lymphocytes and lymphocyte fragments and the use of a platelet
antibody index that evaluates both percent positive platelets as well as fluorescence intensity of the positive platelets. This antibody index was found to be more useful
than percent positive platelets alone in providing the
largest differences and least overlap between control and
patient samples. Our study provides new information on
the utilization of flow cytometry to evaluate and compare
platelet antibody of acute, chronic, and HIV-1 immunologic thrombocytopenic children as well as chronic ATP
adults and on the presence of autofluorescence in unstained platelet samples of acute, chronic, or HIV-1 ITP
patients.
The platelet antibody index was compared in children
with acute ITP and chronic ATP. The mean value was
higher in those with chronic ATP, probably due to the
presence of normal values in five of the ITP children (see
below). This is in contrast to two other pediatric studies
that have reported the level of platelet-associated IgG to
be higher in acute ITP at time of presentation than in
chronic ATP [15,16]. It has been suggested that this
finding may have been an artefact due to the fact that
platelet counts in acute ITP at onset are frequently lower
than in chronic, as was the case in one of these studies
[15]. Because the subjects were young children, a
smaller volume of blood than usual was drawn, providing too few platelets, perhaps resulting in a spuriously
low total platelet count. In addition, it is likely that a
proportionately higher number of lymphocytes would
have been present in the PRP that was tested. The IgG on
these lymphocytes may have resulted in an artefactually
high reading of IgG in the specimen. In the studies reported herein, there was no lymphocyte contamination
and no relationship between the patients’ antibody index
and the platelet counts, indicating that no such artefact
was present, Ware et al. [16] have suggested that the
higher level they noted in acute ITP was present only in
those children tested at the onset of disease, before therapy. However two of our five acute ITP patients with
normal levels of platelet IgG were tested before therapy.
All of the children with chronic ATP had increased antiplatelet antibody indices, as did the adults.
In five of 12 patients with acute ITP, the index was
within the normal range. No difference between these
five patients and the others could be determined with
respect to platelet count, age, sex, or duration of disease.
Cheung et a1 [ 171 also noted normal levels of platelet IgG
in four of 26 acute ITP patients, with elevated values in
all 22 pediatric chronic ATP patients studied. One can
speculate that the clinical entity of acute ITP of childhood has a number of etiologies that do not all result in
the presence of increased IgG on the platelet surface.
These may be the children who do not develop chronic
ATP. An alternative explanation could be that some patients have IgM exclusively bound to their platelet membrane, which would not have been detected with the
second antibody employed in this test. In the two patients
who had raised indices and were available for follow-up
in remission, the index had returned to the normal range.
When patients with ITP or chronic ATP were com-
Technique: Micromethod for Platelet Surface IgG
pared to controls, there were a significantly larger number of platelets displaying autofluorescence; i.e., some
platelets fluoresced without exposure to antibody. In addition, there was a significant inverse relationship between platelet count and percent autofluorescence in
chronic childhood ATP. The reason for this is not clear.
It does not appear to be due to antibody binding to the
surface of the platelets as judged from experiments with
anti PLAl antibody, or is it due to the fixative employed.
Of particular interest was the observation of the heterogeneous distribution of antibody in the patients studied, in that only a fraction of the patient’s platelets examined had antibody detectable with this method. This
varied from 10% to 80% positive staining cells in children as well as adults, with a mean of approximately
25% positive staining cells for all four groups studied.
Similar results have been reported by others [8,10]. It is
conceivable that some of the negative-staining cells represent IgM or complement bound to platelets since their
presence was not examined in this study. However, Ault
[lo], who examined the interrelationship between platelet IgG, IgM, and C3 by flow cytometry, found strong
correlations between the presence of IgG and IgM on the
platelet surface, and no cases of C3 alone on the platelet
surface, ruling out this possibility. Another possibility
could be heterogeneity of antigen density or incomplete
saturation by circulating antiplatelet antibody. A third
possibility could be the presence of nonplatelets in the
gated area. This appears to be the case in patients with
severe thrombocytopenia, as was reported by Ault [lo],
who noted nonplatelet fragments within the gated platelet
region. We have confirmed this observation in three severely thrombocytopenic ATP patients with platelet
counts of 6,0OO/p1, lO,OOO/pl, and 25,OOO/ul. Utilizing
our monoclonal antibody against platelet membrane
GPIIb, these three patients had 33%, 12%, and 25%
GPIIb staining particles, respectively, in the gated area.
However, we cannot rule out the possibility that the antiGPIIb monoclonal antibody employed to detect “true”
platelets was not blocked by IgG bound to the GPIIbGPIIIa epitope [ 5 ] .
This method will be of value in studying thombocytopenia in sick premature infants. Low platelet counts
occur in up to 20% of this group of patients. Some studies have suggested that increased platelet destruction
may pay a role [ 18-201. In one of these studies, elevated
PAIgG was demonstrated in some patients [15]. The
level of platelet surface IgG in healthy newborn infants
has not been established. The method described here will
allow the determination of a normal range in nonthrombocytopenic infants of different gestational ages, allowing a more meaningful evaluation of thrombocytopenic
infants.
In conclusion, a method has been developed for dern-
281
onstrating increased PAIgG that has the following advantages. 1) It requires a small volume of blood. 2) It
measures surface platelet IgG. The platelets are not manipulated by washing and multiple centrifugations, with
consequent activation and release of granule IgG on their
surface. 3) There is no lymphocyte contamination that
could spuriously affect low platelet counts. 4)It does not
require radioisotopes. 5) The test is less labor intensive
than most other procedures, requiring 2.5 hr of working
time compared to 7 hr for the radioimmunoassay or
ELISA.
ACKNOWLEDGMENTS
This study was supported by a grant from the National
Heart, Lung and Blood Institute (HL-13336-18).
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