Use of a Solid Phase Red Blood Cell Adherence Method for

Use of a Solid Phase Red Blood Cell Adherence Method for
Pretransfusion Platelet Compatibility Testing
A solid phase red blood cell adherence method has been used
for platelet antibody detection and crossmatching for refractory platelet recipients. Patient sera were first screened for
HLA or platelet-specific antibodies, then crossmatched with
potential apheresis platelet donors. The overall correlation of
platelet crossmatch results with transfusion outcome was 97%
in patients with no evidence of nonimmune platelet destruction.
The solid phase red blood cell adherence method provided a
feasible and effective alternative to HLA matching as a means
of donor selection for refractory platelet recipients. The speed
and simplicity of this method may allow most hospital laboratories to perform platelet antibody screening before routine
platelet transfusions. (Key words: Solid phase; Platelets; Compatibility testing; Crossmatch; Platelet transfusions) Am J
Clin Pathol 1988;90:63-68
IN RECENT YEARS, platelet usage has increased ten
times more than red blood cells (RBCs). Transfusions of
either whole blood or RBCs increased 58%, from 6.3 to
9.9 million units, between 1971 and 1980. During the
same period, platelet usage increased 598%, from 0.4 to
2.8 million units. 12 The growing demand for platelets
has brought about an increased awareness of several
problems associated with platelet transfusion therapy.
Foremost among these is the detection and management
of platelet alloimmunization.
Most platelet transfusions are administered as a pool
of six to ten individual concentrates, selected at random.
As HLA or platelet-specific antibodies develop, posttransfusion platelet increments diminish, because of
immune destruction. Recipients who repeatedly derive
little or no benefit from random-donor platelets are
termed refractory, and alloimmunization is suspected.
Because the antibodies are often directed against HLA
antigens, refractoriness can sometimes be overcome by
providing platelets from HLA-matched donors. A major
disadvantage to this approach is the cost of establishing
Received September 29, 1987; received revised manuscript and accepted for publication November 30, 1987.
Supported by a grant from the Research Foundation of the American Association of Blood Banks.
Dr. Sinor's current address is Immucor, Inc., Norcross, Georgia.
Dr. Plapp's current address is Department of Pathology, St. Luke's
Hospital, Kansas City, Missouri.
Address reprint requests to Ms. Rachel: Community Blood Center
of Greater Kansas City, 4040 Main Street, Kansas City, Missouri
Community Blood Center of Greater Kansas City,
Kansas City, Missouri
and maintaining the necessary panel of several thousand
HLA-typed apheresis donors. 6 " In addition, extensive
experience has determined that 18-35% of HLAmatched platelet transfusions are not beneficial.115 The
reason for these failures is unclear. One possible explanation is the presence of antibodies directed against
platelet-specific antigens. In fact, such antibodies have
been detected in the sera of 22-25% of alloimmunized
patients.7'9 Thus, the assumption that histocompatible
platelets are serologically compatible may not be valid.
Pretransfusion platelet crossmatching is a more direct
means of identifying compatible donors for alloimmunized recipients. Consequently, several techniques that
were initially developed for platelet antibody detection
have been adapted to platelet crossmatching.1-5-81316"19
The major impediment to widespread use of these
methods is that several hours are required to complete
small numbers of tests. This is a significant limitation,
because finding compatible donors for broadly alloimmunized patients may require extensive crossmatching.
Recently, a solid phase RBC adherence (SPRCA)
assay was developed specifically for platelet crossmatching. Platelet preparation was reduced to a single 10minute centrifugation step so that, if necessary, samples
from large numbers of donors could be crossmatched in
a single day. The SPRCA method was initially evaluated
by retrospectively crossmatching platelet samples from
apheresis donors with sera from refractory recipients.
Eighty transfusions given to 20 patients were studied.
Analysis of the data indicated that none of 20 crossmatch-incompatible transfusions had produced significant posttransfusion increments. Of the crossmatchcompatible transfusions, 63% (38 of 60) had been successful. 10 These results suggested that the SPRCA
platelet crossmatch method could be effective if used
before apheresis to select platelet donors for alloimmunized patients. We describe the results of a study
designed to evaluate the effectiveness and feasibility of
using the SPRCA method prospectively for pretransfusion platelet antibody detection and crossmatching.
platelet monolayers
serum anti-platelet antibody
antl-lgG-coated Indicator RBCs
FIG. 1. Schematic representation of positive (incompatible) and negative (compatible) reactions using the SPRCA method for platelet antibody detection and crossmatching.
The SPRCA method, as described previously,10 was
used for platelet antibody detection and crossmatching.
Briefly, test platelets were prepared by collecting 7 rnL of
whole blood into ethylenediaminotetraacetate (EDTA)
and centrifuging for 10 minutes at 200 X g to obtain
platelet-rich plasma (PRP). No further isolation or
washing ofplatelets was required, nor was it necessary to
determine platelet counts of PRP obtained from apparently healthy donors. PRP was prepared and used within
48 hours from time of collection. Solid phase platelet
monolayers were formed by adding two drops of PRP to
polystyrene U-bottom microplate wells that had been
pretreated to specifically bind platelets (Capture-P®, Immucor Inc., Norcross, GA 30091). Microplates were
centrifuged for 5 minutes at 35 X g to form solid phase
platelet monolayers. Unbound platelets were removed
by gently washing the wells six to eight times with 0.85%
(w/v) NaCl. One drop of test serum and three drops of a
low ionic strength solution (Capture-P) were added to
the appropriate wells and mixed. Microplates were incubated for 30 minutes at 37 °C, which allowed serum
antibody to bind to platelet monolayers. After incubation, unbound serum components were removed by
washing the wells five times with 0.85% (w/v) NaCl.
Platelet-bound antibodies were detected by the addition
of one drop of a 0.2-0.4% (v/v) suspension of anti-IgGcoated indicator RBCs (Capture-P). Microplates were
then centrifuged at 700 X g for 1 minute, which allowed
indicator RBCs to react with antibodies attached to the
solid phase platelet monolayers. Positive reactions were
thus characterized by the adherence of indicator RBCs
over the surface of the wells, whereas negative reactions
produced discrete pellets of indicator RBCs. These reactions were read visually and are shown schematically in
Figure 1. Results of 96 test wells (one microplate) were
A.J.C.P.-July 1988
available within one hour, including the time required
to prepare platelet monolayers. The SPRCA method
detected antibodies directed against platelet-specific,
HLA, or ABO antigens.
Patients included in the study were those for whom
apheresis platelet transfusions were ordered by the attending physician. Most had proven refractory to random donor platelets, although apheresis platelets were
occasionally requested for patients who were expected to
require long-term platelet support. Diagnoses included
leukemia, aplastic anemia, and various malignancies.
Patients' blood samples were initially screened against
platelet samples obtained from 12 group O whole blood
donors, selected at random with no knowledge of their
HLA or platelet antigens. The antibody screen results
were not considered crossmatches but were used only to
determine the presence and extent of alloimmunization
and to estimate the number of potential donors needed
for crossmatching in order to provide an adequate supply of compatible platelets. For example, patients whose
sera reacted with 9 of the 12 platelet samples in the
antibody screen were expected to be compatible with
approximately 25% of donors. Recipients' sera were
then crossmatched with platelet samples obtained from
ABO-compatible potential donors. Whenever possible,
crossmatch-compatible donors were selected before
apheresis. If no potential donors were compatible, incompatible single donor platelets were provided at the
attending physician's request.
To assess transfusion outcome, pretransfusion and
one-hour posttransfusion platelet counts were requested
from hospitals where transfusions were administered.
Information concerning the recipient's clinical status
with regard to fever, active bleeding, sepsis, splenomegaly, or disseminated intravascular coagulation (DIC)
was also requested. The corrected count increment
(CCI) at one hour posttransfusion was calculated as follows2:
_ (post - pretransfusion platelet count) X BSA (m 2 )
number of platelets transfused (X10")
Transfusions were considered successful when the onehour CCI was greater than 10 X 109/L (10 X 10 3 /ML)- 2
Crossmatch results were correlated with transfusion
outcome as follows4: true positive (TP)—incompatible
crossmatch and unsuccessful transfusion; false positive
(FP)—incompatible crossmatch and successful transfusion; true negative (TN)—compatible crossmatch and
successful transfusion; false negative (FN)—compatible
crossmatch and unsuccessful transfusion.
The sensitivity, specificity, predictive value, and efficiency of the SPRCA platelet crossmatch method were
determined by use of standard formulas. 4
Vol. 90 • No. 1
Platelet antibody screen and prospective crossmatch
results are summarized in Table 1. A total of 2,878
crossmatches were performed for 87 patients, of which
only 479 (17%) were compatible. These data were further evaluated by separating patients into one of three
categories based on their initial antibody screen results.
The first category included 35 patients whose sera
were nonreactive when initially screened for platelet antibodies. Most potential donors (96%) were compatible
with these patients. The few incompatible crossmatches
(4%) may have resulted from antibodies directed against
low-frequency antigens present on donor platelets but
not on the 12 randomly selected platelet samples used
for antibody screening. Although these patients were not
considered alloimmunized, they were transfused with
apheresis platelets, at the attending physicians' request.
Category II consisted of 30 patients whose sera had
positive results in the antibody screen against some
platelet samples but not against all 12. These results
indicated that patients were alloimmunized but not so
broadly that compatible donors could not be found.
These patients' sera were compatible with 31% of potential donors. In most instances, crossmatch-compatible platelets were provided for transfusion.
The 22 patients in category III were those whose sera
reacted with all 12 platelet samples in the antibody
screen, indicating broad alloimmunization. A total of
2,144 crossmatches were performed for these patients,
of which only 87 (4%) were compatible. The actual
number of compatible donors per patient ranged from
none to six (data not shown). Patients with three to six
compatible donors were usually transfused with crossmatch-compatible platelets. Transfusions given to patients with less than three compatible donors were sometimes obtained from incompatible donors, at the attending physician's request.
Recipients' samples were retested frequently in order
to detect developing antibodies. As expected, patients
often became more broadly alloimmunized during the
course of therapy, so that previously compatible donors
were subsequently incompatible. Another observation,
which has previously been reported, 314 was the sudden
and complete disappearance of antibody in patients
known to be broadly alloimmunized. Loss of antibody
could not be reliably attributed to incompatible platelet
transfusions or changes in treatment. Antibodies usually
reappeared within several days, so that crossmatches
done during the period of antibody absence were no
longer valid.
During a one-year period of study, 621 apheresis
platelet concentrates were transfused. Unfortunately,
only 67 of these transfusions met our criteria for evalua-
Table 1. Summary of Platelet Antibody Screen Results
and Correlation with Crossmatch Results
Antibody screen
Posl-11* Pos 12f
Number of patients 35
Number of
241(96%) 151(31%)
10(4%) 332(69%) 2,057(96%)
* Patients whose sera reacted with 1-11 of 12 random platelet samples in the antibody
| Patients whose sera reacted with all 12 random platelet samples in the antibody screen.
tion of transfusion outcome and correlation with crossmatch results. A large number of transfusions involved
recipients who received platelets from an individual
donor more than once. For the purposes of this study,
transfusions involving unique donor-recipient pairs
were evaluated only once, because repeat donations
usually resulted from previously successful transfusions.
Thus, inclusion of replicate donor-recipient pairs would
have biased the study in favor of a successful outcome.
In many instances, transfusions could not be evaluated
because platelet counts either were not ordered by the
attending physician or were not drawn within one hour
after transfusion. Strict adherence to this time limit was
considered necessary in order to minimize the effects of
nonimmune platelet destruction on posttransfusion increments. In general, unsatisfactory platelet increments
at one hour posttransfusion are thought to represent
immune destruction, whereas platelet counts obtained
after this time are more likely to be affected by clinical
factors such as fever, sepsis, bleeding, splenomegaly,
or DIC. 2
The correlation of crossmatch results with transfusion
outcome for 67 transfusions involving 28 patients is expressed in Figure 2. The data include all transfusions for
which crossmatch results were available and a one-hour
CCI could be calculated, regardless of the patient's status
with regard to fever, active bleeding, sepsis, splenomegaly, or DIC. Some transfusions occurred when one or
more of these complications were known to be present.
Although most studies evaluating platelet crossmatch
methods exclude such patients,1'5,8'16"19 we were unable
to obtain the clinical information necessary to allow
such exclusions to be made fairly. We thus chose to
express the total data, with the assumption that many
false negative results may have resulted from nonimmune platelet destruction.
Of the 67 transfusions evaluated, only 32 were given
to patients whose clinical status at the time of transfu-
A.J.C.P.- July 1988
Evaluation of the data derived from both patient populations is shown in Table 4. Chi-square analysis is
highly significant for both groups. The sensitivity, specificity, and predictive values most representative of actual transfusion practice are those of the total patient
population. However, the ability of the SPRCA platelet
crossmatch method to predict serologic compatibility is
best represented by data from the selected patient population, which is presumably unaffected by nonimmune
platelet destruction. Comparisons of this study with
others should be based on these latter figures, because
most platelet crossmatch methods have been evaluated
by use of selected patient populations.
• •
°„ o
O n n nO
mean CCI 4.1
SD 5.1
mean CCI 16.3
SD 9.6
FIG. 2. Correlation of posttransfusion CCI (X109/L) (103/ML) and
crossmatch results for the total patient population. Data represent 67
apheresis platelet transfusions given to 28 patients. Replicate donorrecipient pairs are excluded.
Many hospitals and blood centers are currently using
both HLA matching and platelet crossmatching to select
donors for alloimmunized recipients. The question that
has not been answered is whether or not platelet crossmatching can be used instead of HLA matching. This
study represents an extensive investigation into the feasibility of using platelet crossmatching as the sole crite-
sion was reliably reported and did not include fever,
active bleeding, sepsis, splenomegaly, or DIC. The correlation of crossmatch results with transfusion outcome
for this selected patient population is expressed in Figure
3. As expected, the number of false negative reactions is
greatly reduced, but two false positive results are also
excluded. One of these transfusions involved a reportedly bleeding patient who benefitted from crossmatchincompatible platelets. The other false positive result
occurred when incompatible platelets were given to a
febrile patient.
Tables 2 and 3 contain additional information concerning transfusions evaluated for the total and selected
patient populations, respectively. Initial antibody status
of the recipients, as described in Table 1, is correlated
with the crossmatch results expressed in Figures 2 and 3.
Both tables indicate that most of the transfusions analyzed were given to alloimmunized recipients, that is,
those in categories II and III of Table 1. This distribution
is important, because inclusion of a disproportionate
number of transfusions involving recipients in category
I may have biased the outcome in favor of success.
mean CCI 3.8
SD 3.1
mean CCI 19.2
SD 8.0
FIG. 3. Correlation of posttransfusion CCI (X109/L) (l03/fL) and
crossmatch results for a selected patient population. Data represent 32
apheresis platelet transfusions given to 17 patients. Replicate donorrecipient pairs are excluded.
Vol. 90 • No. 1
Table 2. Correlation of Recipient Antibody Status with
Crossmatch Results for Transfusions Shown in Figure 2
Table 3. Correlation of Recipient Antibody Status with
Crossmatch Results for Transfusions Shown in Figure 3
Compatible transfusions
Incompatible transfusions
* As described in Table 1.
ridn of donor selection for alloimmunized platelet recipients. The results indicate that use of the SPRCA
method for both platelet antibody screening and crossmatching before apheresis is an effective approach. Antibody screen results provide useful information concerning the presence and extent of alloimmunization
and the likelihood of finding compatible donors. The
sensitivity, specificity, and predictive values obtained
for the SPRCA platelet crossmatch method indicate that
the ability of this method to select compatible platelet
donors is better than that generally reported for HLA
matching. 115
Refractoriness in the first group of patients listed in
Table 1 may have resulted from either nonimmune
platelet destruction or undetectable levels of alloimmunization. Most prospective donors (96%) were compatible. The high degree of correlation between antibody
screen and crossmatch results indicates that a panel of
12 randomly selected platelet samples provides adequate pretransfusion platelet antibody detection.
Prospective platelet crossmatching appeared to be
most beneficial for patients who were alloimmunized
but not broadly. The 30 patients in category II were
incompatible with slightly more than two-thirds of potential donors. Pretransfusion testing thus provided an
adequate supply of compatible platelet donors, while
preventing many apheresis procedures that would have
resulted in ineffective transfusions.
Data expressed in Table 1 also suggest that crossmatching for patients who are broadly alloimmunized
may not be productive. Only 4% of all donors were
compatible with these patients' sera. However, the speed
and simplicity of the SPRCA method allowed 100
crossmatches to be completed within eight hours by a
single technologist. It thus seems reasonable to initially
crossmatch 100-200 potential donors. Identification of
even a few compatible donors may be worthwhile if
their use is restricted to periods of extreme need. If no
compatible donors are found, additional crossmatching
cannot be justified.
In most instances, potential donors were friends and
family members of the intended recipient. Some pa-
Compatible transfusions
Incompatible transfusions
* As described in Table 1.
tients who were broadly alloimmunized recruited
hundreds of potential donors in an attempt to obtain
compatible platelets for transfusion. This was most
often accomplished by organizing mass phlebotomies at
the recipient's place of business or during meetings of
interested church or social groups. Platelet samples were
crossmatched with serum from the intended recipient
within 48 hours, to eliminate the need for further isolation of test platelets from autologous plasma. The logistics of this system were complex at best. The most practical solution to this problem would be the establishment of a large group of committed apheresis donors,
whose platelets could be stored and crossmatched
against all patients' sera.
In our study, 25% of patients for whom apheresis
platelets were requested were broadly alloimmunized
when initially screened for platelet antibodies (category
III). These 22 patients required 2,144 crossmatches, of
which only 87 (4%) were compatible. Subsequent plate-
Table 4. Evaluation and Statistical Analysis of the
Correlation Between Crossmatch Results and
Transfusion Outcome for Both Total and
Selected Patient Populations
Total Patient
Crossmatch correlation
Number of:
True positive results
False positive results
True negative results
False negative results
Crossmatch evaluation
Predictive values
Incompatible crossmatch
Compatible crossmatch
Selected Patient
let transfusions were often incompatible and ineffective.
The disappointing outcome for this group of patients
might have been improved if samples had been referred
for platelet antibody screening at an earlier time, when
they were presumably less broadly alloimmunized. Unfortunately, pretransfusion detection of platelet alloimmunization has been impeded by the lack of a method
suitable for routine use in hospital transfusion service
Consequently, most platelet transfusions are administered with no prior antibody screening or compatibility
tests. Platelet alloimmunization is usually detected clinically, rather than serologically, when a recipient repeatedly fails to benefit from random platelets. During the
time required to demonstrate refractoriness, large numbers of platelet concentrates are wasted, and the recipient is exposed, without benefit, to platelets from a large
number of donors.
Our experience during this study indicates that the
SPRCA method is comparable in terms of speed and
simplicity to those currently in use for RBC compatibility testing and could thus be used in hospital laboratories before most platelet transfusions to detect platelet
alloimmunization. Patients with negative antibody
screens could still be transfused with pools of randomly
selected platelet concentrates. Alloimmunized patients
would be identified earlier and could then be supplied
with platelets obtained by apheresis from crossmatchcompatible donors. The delay encountered would certainly be less than the time currently required for clinical documentation of refractoriness. We thus conclude
that pretransfusion platelet compatibility testing may
now be feasible.
Acknowledgment. The authors thank B. J. Schulenburg for review17.
ing the manuscript.
1. Brand A, van Leeuwen A, Eernisse JG, van Rood JJ: Platelet
transfusion therapy. Optimal donor selection with a combination of lymphocytotoxicity and plateletfluorescencetests.
Blood 1978;51:781-788.
2. Daly PA, SchifTer CA, Aisner J, Wiernik PH: Platelet transfusion
A.J.C.P.-July 1988
therapy. One-hour posttransfusion increments are valuable in
predicting the need for HLA-matched preparations. JAMA
Dutcher JP, SchifTer CA, Aisrier J, Wiernik PH: Long-term follow-up of patients with leukemia receiving platelet transfusions: identification of a large group of patients who do not
become alloimmunized. Blood 1981;58:1007-1011.
Galen RS, Gambino SR: Beyond normality: the predictive value
and efficiency of medical diagnoses. New York: John Wiley
and Sons, 1975:10-14.
Kickler TS, Braine HG, Ness PM, Koester A, Bias W: A radiolabeled antiglobulin test for crossmatching platelet transfusions.
Blood 1983;61:238-242.
Lohrmann HP, Bull MI, Decter JA, Yankee RA, Graw RG: Platelet transfusions from HL-A compatible unrelated donors to
alloimmunized patients. Ann Intern Med 1974;80:9-14.
Murphy MF, Waters AH: Immunological aspects of platelet
transfusions. Br J Haematol 1985;60:409-414.
Myers TJ, Kim BK, Steiner M, Baldini MG: Selection of donor
platelets for alloimmunized patients using a platelet-associated
IgG assay. Blood 1981;58:444-450.
Pegels JG, Bruynes ECE, Engelfreit CP, von dem Borne AEGKr:
Serological studies in patients on platelet- and granulocytesubstitution therapy. Br J Haematol 1982;52:59-68.
Rachel JM, Sinor LT, Tawfik OW, et al: A solid-phase red cell
adherence test for platelet cross-matching. Med Lab Sci
SchifTer CA, Keller C, Dutcher JP, Aisner J, Hogge D, Wiernik
PH: Potential HLA-matched platelet donor availability for alloimmunized patients. Transfusion 1983;23:286-289.
Surgenor DM, Schnitzer SS: The nation's blood resource: a summary report. NIH Publication #85-2028. National Institutes of
Health, March 1985.
Tamerius JD, Curd JG, Tani P, McMillan R: An enzyme-linked
immunosorbent assay for platelet compatibility testing. Blood
Tejada F, Bias WB, Santos GW, Zieve PD: Immunologic response
of patients with acute leukemia to platelet transfusions. Blood
Tosato G, Applebaum FR, Deisseroth AB: HLA-matched platelet
transfusion therapy of severe aplastic anemia. Blood
van5lder Velden KJ, Sintnicolaas K, Lowenberg B: The value of a
Cr platelet lysis assay as crossmatch test in patients with leukaemia on platelet transfusion therapy. Br J Haematol
Ware R, Reisner EG, Rosse WF: The use of radiolabeled and
fluorescein-labeled antiglobulins in assays to predict platelet
transfusion outcome. Blood 1984;63:1245-1248.
Wu KK, Hpak JC, Koepke J A, Thompson JS: Selection of compatible platelet donors: a prospective evaluation of three crossmatching techniques. Transfusion 1977;17:638-643.
Yam P, Petz LD, Scott EP, Santos S: Platelet crossmatch tests
using radiolabeled staphylococcal protein A or peroxidase
anti-peroxidase in alloimmunized patients. Br J Haematol