You are on page 1of 9

Vox Sanguinis (2007) 93, 289–297

© 2007 The Author(s)

ORIGINAL PAPER Journal compilation © 2007 Blackwell Publishing Ltd.
DOI: 10.1111/j.1423-0410.2007.00989.x

A modified rapid monoclonal antibody-specific immobilization

Blackwell Publishing Ltd

of platelet antigen assay for the detection of human platelet

antigen (HPA) antibodies: a multicentre evaluation
K. Campbell,1 K. Rishi,2 G. Howkins,2 D. Gilby,1 R. Mushens,3 C. Ghevaert,1 P. Metcalfe,4 W. H. Ouwehand1,5 & G. Lucas3
National Health Service Blood and Transplant, Cambridge Centre, Long Road, Cambridge CB2 2PT, UK
National Health Service Blood and Transplant, Oxford Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
National Health Service Blood and Transplant, Bristol Centre, Southmead Road, Bristol BS10 5ND, UK
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
Department of Haematology, University of Cambridge, Long Road, Cambridge CB2 2PT, UK

Background The monoclonal antibody-specific immobilization of platelet antigens

(MAIPA) assay is the cornerstone technique for the detection and identification of
human platelet antigen (HPA) antibodies. However, the original technique described
by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay.
Moreover, proficiency exercises indicate that there are substantial variations in the
MAIPA protocol, and that these may account for interlaboratory differences in
sensitivity and specificity.
Study Design and Methods A review of current MAIPA assay protocols from six
laboratories together with performance in quality-assessment schemes identified
several key variables potentially affecting the assay results. An optimized protocol
was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA)
assay was evaluated using 61 samples with a range of HPA antibodies typically
encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-
transfusion purpura (n = 8), platelet refractoriness (n = 7) and other platelet immune
conditions (n = 24). The sensitivity of the assay was assessed using three inter-
national standards and the recombinant HPA-1a antibody CamTran007. The results
obtained were compared with the original findings obtained with the local MAIPA
assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies
were evaluated for their effect on assay sensitivity.
Results Complete concordance was found between the original MAIPA results and
those obtained with the new assay when testing a selected panel of clinical samples.
The modified assay had nanogram level sensitivity for the detection of HPA-1a anti-
bodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded
Received: 23 April 2007,
end-points equal to or greater than the mean recorded in international workshops.
revised 3 August 2007,
accepted 13 August 2007, Conclusion The MR-MAIPA assay offers improved turnaround for the detection of
published online 7 August 2007 HPA antibodies without loss of sensitivity.

Human platelet antigen (HPA) antibodies are of clinical
Correspondence : Willem Ouwehand, MD, PhD, Department of significance in fetomaternal alloimmune thrombocytopenia
Haematology, University of Cambridge, Long Road, Cambridge CB2 2PT, UK (FMAIT), post-transfusion purpura (PTP) and platelet refrac-
E-mail: toriness (PR). Rapid and sensitive detection and identification

290 K. Campbell et al.

of HPA alloantibodies is imperative for good clinical care. domain genes to generate a recombinant immunoglobulin
The monoclonal antibody (mAb)-specific immobilization of G1 (IgG1) antibody [10].
platelet antigens (MAIPA) assay is the cornerstone technique
for HPA antibody detection and identification in many
Standard polyclonal HPA antibody preparations
platelet immunology laboratories [1–3]. However, quality-
assessment exercises have revealed wide interlaboratory The WHO minimum sensitivity reagents for the detection of
variation in the proficiency of HPA antibody detection by anti-HPA-1a (93/710) [11] and anti-HPA-5b (99/666) [8], and
the MAIPA assay [1–3]. Nonetheless, several laboratories the anti-HPA-1a potency standard (03/152) [1] containing a
participating in the platelet immunology quality-assurance 100-international units (IU) polyclonal IgG anti-HPA-1a were
exercises organized by the National Institute for Biological used in the initial validation studies. None of these reagents
Standards and Control (NIBSC) have shown consistently contained HLA class I or other HPA antibodies. All were supplied
good performance over the 10 most recent exercises. The freeze-dried and were reconstituted according to the accom-
MAIPA assay protocols of these laboratories were analysed panying instructions and diluted in phosphate-buffered saline
and it was deduced that minor variations [e.g. sample (PBS) supplemented with 0·2% w/v bovine serum albumin (BSA).
volume, incubation time and temperature, reagents and
glycoprotein (GP) capture mAbs] had significant conse-
Anti-GP mouse mAbs
quences for the sensitivity and sometimes specificity of the
assay. Although a rapid enzyme-linked immunosorbent assay The following mAbs were used in the assay, anti-GPIIb/IIIa:
(ELISA)-based assay for the detection of HPA antibodies is RFGP56, ascites diluted 1 : 1000 (Cymbus, Southampton,
commercially available, it lacks the flexibility of the MAIPA UK; a kind gift from Professor A. Goodall, University of
assay and previous reports have suggested a reduced sensi- Leicester), NBS-PAB-1, culture supernatant (CS) diluted
tivity when compared to the MAIPA assay [4,5]. In a collab- 1 : 10; NBS-PAB-6, CS diluted 1 : 10; NBS-5B12, CS diluted
orative project between the platelet immunology laboratories 1 : 10, NIBSC-85/661, CS, diluted 1 : 3; anti-GPIbα: NBS-
of the National Health Service Blood and Transplant (NHSBT) PAB-5, CS diluted 1 : 10; anti-GPIa/IIa: NBS-P16, CS diluted
and NIBSC, a modified rapid (MR)-MAIPA assay protocol was 1 : 10; and anti-HLA class I: W6/32, CS diluted 1 : 5.
developed. The MR-MAIPA assay was initially validated in
the NHSBT laboratory in Cambridge and a common protocol
Patient samples
was defined. The sensitivity of the assay for the detection of
HPA-1a antibodies was assessed by titration of the recom- Both ethylenediaminetetraacetic acid (EDTA) plasma and
binant HPA-1a antibody CamTran007 [6,7], the polyclonal serum samples from patients and normal controls were used
anti-HPA-1a potency standard 03/152 [1] and two World to validate the MR-MAIPA assay. The 61 blinded samples
Health Organization (WHO) International Reference Rea- were clinical referrals to NHSBT platelet immunology labo-
gents for sensitivity of HPA-1a (93/710) and HPA-5b (99/666) ratories and contained a representative range of HPA and
[8] antibody detection. To determine interoperator variation platelet GP specificities typically found in referrals to refer-
61 blinded samples were tested by four different operators in ence laboratories. The panel was deliberately enriched with
three laboratories. Finally, the 42 samples with antibodies some samples where HPA antibodies had been difficult to
against GPIIb/IIIa were tested with four different capture detect at initial referral. High and low titre HPA antibodies
antibodies (RFGP56, NBS-PAB-1, NBS-PAB-6 and NBS- and examples of single and multiple HPA specificities were
5B12) to determine their effect on the sensitivity of the included, as were panreactive GPIIb/IIIa, GPIbα/IbβIX and
MR-MAIPA assay. GPIa/IIa antibodies. All samples were coded before testing so
Finally, we analysed the results for evidence of coimmuno- that operators were blinded for the antibody specificities. The
precipitation of GPIIb/IIIa with human leucocyte antigen results obtained with the MR-MAIPA assay were analysed
(HLA) class I. and the results were compared with the original data
obtained by the laboratory that had submitted the sample.

Materials and methods

Panel platelets
HPA-1a mAb
Samples were tested for the presence of HPA and HLA class I
The human recombinant HPA-1a mAb CamTran007 with a antibodies against a panel of cryopreserved platelets (Table 1)
Kd of 6 × 108 M/l was used to determine the sensitivity of the from group O, HPA and HLA class I genotyped donors. Each of
assay [9]. This antibody has been obtained from the immune the HPA-1, -2, -3 and -5 alleles were present in homozygosity
B cells of a case of FMAIT by V gene phage display techno- on at least one panel cell. Panel platelets were stored until use
logy [6] and the V genes were recombined with constant at the temperature of liquid nitrogen vapour phase and, after

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
Rapid monoclonal antibody-specific immobilization of platelet antigen assay 291

Table 1 HPA and HLA antigen profile of cryopreserved platelet panel BSA). The resuspended platelets were incubated with 25 μl of
either test or control serum/plasma for 30 min at 37 °C in a
HPA -1a -1b -2a -2b -3a -3b -5a -5b HLA-A HLA-B well of a round bottom microtitre plate (Dynex Technologies,
Worthing, UK). Serum/plasma was removed by two washings
PL No.
with 200 μl TBS/BSA and the pelleted platelets were
1 0 + + 0 0 + + 0 2,11 40,51
resuspended in 50 μl TBS/BSA before the addition of appro-
2 + 0 + 0 + 0 0 + 1,3 8
3 + 0 0 + + 0 + 0 2,29 40,44 priately diluted GP-specific mAb. Following a further
4 + 0 + 0 0 + + 0 2,32 62,27 incubation for 30 min at 37 °C, the platelets were washed
5 0 + + 0 + 0 + 0 2,24 35,44 three times with TBS/BSA, followed by solubilization of the
6 0 + + 0 0 + 0 + 2,23 7,44 platelets with 0·5% Triton-X-100 (BDH Laboratory Supplies,
7 + 0 0 + 0 + + + 2 8,44 Poole, UK) for 15 min at 20 °C. The microtitre plates were
centrifuged at 1400 g for 15 min to precipitate unsolubilized
PL, platelet donor; No., rank number; HPA, human platelet antigen; HLA,
platelet debris. Of the supernatant, 100 μl was transferred to
human leucocyte antigen; 0, antigen absent; +, antigen present.
a flat bottom well microtitre plate (Nunc Maxisorp, Roskilde,
Denmark) that had been previously coated with goat anti-
removal of cryoprotectant by washing with PBS/EDTA/BSA, mouse Ig diluted 1 : 500 (Jackson Immunoresearch Labora-
a suspension 1 × 108 platelets/ml was prepared. Recovered tories, West Grove, PA, USA) and washed and blocked with
panel platelets were stored at 4 °C in PBS/EDTA/BSA for not wash buffer (TBS, Nonidet P40 substitute 0·5% v/v, Tween
more than 3 days. The detection of HPA-15 antibodies was 0·01% v/v, CaCl2 0·01% w/v, BSA 0·1% w/v). The plates were
not evaluated because CD109 is not preserved after cryop- incubated for 30 min at 37 °C to allow effective capture of
reservation of panel platelets [12]. the trimolecular complex of antigen, murine and human
antibodies. The lysate was discarded, and the plate was
washed six times with 125 μl of wash buffer. Bound human
Historical testing
IgG was detected by the addition of 100 μl of a 1 : 5000 dilu-
The 61 blinded samples were investigated at the time of referral tion of horseradish peroxidase-conjugated goat anti-human
for HPA antibodies by the indirect platelet immunofluorescence IgG (Jackson Immunoresearch Laboratories) in wash buffer.
test (PIFT), the local MAIPA assay, and in one sample a whole- After 60-min incubation at 20 °C, the plates were washed a
platelet solid-phase ELISA. The local MAIPA protocols were further six times and the colour was developed by addition
based on the original protocol [13] and subsequent modifi- of 100 μl o-phenylenediamine (OPD) solution (DakoCytoma-
cations [14,15] but none included shortened incubation times tion, Ely, UK; OPD tablets, 2 mg, code no. S2045) and 5 μl of
at higher temperature or a standardized panel of mAbs. 30% w/v hydrogen peroxide (H2O2) in reverse osmosis water.
The enzymatic reaction was stopped by the addition of 100 μl
of 0·5 M H2SO4 to each well. The absorbance was read at
MR-MAIPA assay
490 nm with a reference filter of 630 nm. The major differ-
Panel platelets (100 μl) were centrifuged, the supernatant was ences between the MAIPA assay as described by Kiefel and
decanted and the platelet pellet was resuspended in 50 μl of colleagues [13–15] and the MR-MAIPA are summarized in
TBS/BSA (Tris-buffered saline supplemented with 0·2% w/v Table 2.

Table 2 Comparison of modified original and MR-MAIPA protocol

Step/reagent Modified original MAIPA [13–15] MR-MAIPA

Buffer used in serum and mAb sensitization phases PBS TBS

Platelets/test (×107/l) 2 1
Volume serum used per test (μl) 5–200 25
Solubilization buffer Nonidet P40 Triton-X-100
Centrifugation of lysate 15 000 g for 10 min at 4 °C 1400 g for 15 mins at RT
Incubation of trimolecular complex with capture antibody 90 min at 4 °C 30 min at 37 °C
Incubation with goat anti-human IgG horseradish peroxidase 90 min at 4 °C 60 min at 20 °C
Total assay time ~8 h ~5 h

mAb, monoclonal antibody; PBS, phosphate buffered saline; TBS, Tris-buffered saline; RT, room temperature; trimolecular complex: platelet glycoprotein,
murine monoclonal antibody and human IgG.

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
292 K. Campbell et al.

the nanogram range and that this was independent of the

Study design
capture antibody.
The initial approach was to analyse the details of the Serial dilutions of the anti-HPA-1a potency standard
MAIPA protocols in consistently ‘good performer’ laboratories NIBSC 03/152 in PBS/BSA were tested using the same three
participating in the NIBSC proficiency scheme and to identify capture mAbs. The potency standard was detected at a titre
factors that had a negative or positive effect on sensitivity. of 1 in 512 with NBS-PAB-1 and NBS-5B12, and at a titre of
Based on this analysis, a protocol was derived (the MR-MAIPA 1 in 256 with NBS-PAB-6 (Fig. 2a). Dilutions of the anti-
assay) that was extensively evaluated in a step-wise process. HPA-1a sensitivity standard NIBSC 93/710 showed reactivity
The first step was to use four well-characterized HPA anti- at a titre of 64 with all three capture antibodies (Fig. 2b),
body reference reagents: the human recombinant IgG1 while titres ranging from 1 in 4 to 1 in 64 had been observed
HPA-1a antibody CamTran007 and the three aforementioned in international proficiency exercises [2]. The first stage of
polyclonal anti-HPA standards. The characterization of all the assessment of the MR-MAIPA assay was completed by
four reagents has been extensive and has been reported testing the HPA-5b sensitivity antibody preparation NIBSC
elsewhere [9–11,16]. The validation phase of the study was 99/666 and a titre of 1 in 32 was consistently observed with
preceded by a workshop in which staff from NHSBT platelet capture antibody NBS-P16 (Fig. 2c), which compares well
immunology laboratories were trained to the MR-MAIPA with the titres observed in previous exercises ranging from
standard operating procedure. The 61 blinded samples were undiluted to 1 : 64 (median 1 : 4) [2].
tested twice by a single operator (K. Campbell, Cambridge)
and then by three different operators in NHSBT laboratories
Clinical samples
in Bristol, Cambridge (different operators) and Oxford in
batches of 30, 16 and 15 samples, respectively, using NBS- Clinical samples typically referred to a platelet immunology
PAB-1, NBS-P16 and NBS-PAB-5 and the standard set of laboratory were used in the second phase of the assessment
panel platelets (Table 1). In addition all 42 samples with of the MR-MAIPA. The characteristics of the 61 blinded sam-
GPIIb/IIIa antibodies were tested with four capture mAbs: ples are presented in Table 3, with 49 samples containing
NBS-PAB-1, NBS-PAB-6, NBS-5B12 and RFGP56 by a single HPA antibodies, a unique sample with anti-Moua [17], five
operator in the NHSBT Cambridge laboratory. with panreactive GP antibodies, one that reacted in the PIFT
only, two with HLA antibodies only (no platelet-specific anti-
bodies) and three inert samples. Testing of the 61 blinded
Results samples by a single operator (KC) in the Cambridge laboratory
on two occasions with two different but partially overlapping
Sensitivity of the MR-MAIPA assay
sets of capture mAbs (RFGP56, NBS-P16, CLB-MB45 and W6/
The sensitivity for the detection of HPA-1a antibodies was 32 vs. NBS-PAB-1, NBS-PAB-6, NBS-5B12, NBS-P16, NBS-
assessed with the human recombinant HPA-1a antibody PAB-5 and W6/32) showed complete concordance between
CamTran007 and with the two polyclonal NIBSC reference the two tests with positive results for all but two of the 58
reagents. Antibody CamTran007 was tested over a concen- samples with platelet antibodies and no significant signal
tration range from 10 μg/ml to 0·015 ng/ml using the GPIIb/ with the three inert samples (data not shown). The anti-Moua
IIIa capture antibodies NBS-PAB-1, NBS-PAB-6 and NBS- that was previously only detected in a solid-phase platelet
5B12 (Fig. 1). The results indicate that the sensitivity for the ELISA but not in PIFT or MAIPA, was also non-reactive in the
detection of this intermediate affinity HPA-1a antibody is in modified MAIPA. Similarly, the ‘PIFT only’ sample was still

Fig. 1 Titration of anti-HPA-1a CamTran007 by

MAIPA with three capture antibodies. Sensitivity
of the MR-MAIPA to detect recombinant human
immunoglobulin G1 (IgG1) anti-HPA1a
CamTran007. Doubling dilutions of the antibody
were tested by MAIPA with an HPA-1a1a
homozygous platelets. The capture mAbs
NBS-PAB-1, NBS-PAB-6 and NBS-5B12 were
used. x-axis, dilution of CamTran007 in μg/ml.
y-axis, absorbance at 490 nm.

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
Rapid monoclonal antibody-specific immobilization of platelet antigen assay 293

Fig. 2 Titration of three NIBSC anti-HPA

standards by MAIPA with three capture
antibodies. Doubling dilutions of three NIBSC
standards with HPA antibodies were tested by
MR-MAIPA. Samples of HPA-1a homozygous
platelets were sensitized with the samples
prepared from the samples. Dilution of the
standard is on the x-axis and absorbance at
490 nm on the y-axis. (a) Anti-HPA-1a potency
standard 03/152 was tested with three GPIIb/IIIIa
capture antibodies, NBS-PAB-1, NBS-PAB-6 and
NBS-5B12; (b) anti-HPA-1a sensitivity standard
93/710 was tested as per (a) and (c); anti-HPA-5b
sensitivity standard 99/666 was tested with the
GPIa/IIa capture antibody P16.

non-reactive in the modified MAIPA. The identified specifi- Cambridge (different operators) and Oxford laboratories,
cities were identical to specificities previously determined by respectively. Again the anti-Moua and the ‘PIFT only’ samples
the indirect immunofluorescence test and the local MAIPA. were non-reactive and the expected results were obtained
HLA class I antibodies were detected in 32 (52%) samples, with all but one of the remaining samples, one weak anti-
and in 28 samples in combination with HPA antibodies. GP- HPA-1a, sample OX-01 that was only positive in the original
reactive antibodies without allelic-restricted reactivity were local MAIPA only when the test serum volume was tripled.
detected in all five (8%) samples known to contain these This sample gave positive results in the modified MAIPA with
antibodies (three against GPIIb/IIIa, one against GPIbα/IbβIX a normal serum volume when tested with two panels of mAbs
and one against GPIIb/IIIa, GPIbα/IbβIX and GPIa/IIa). (above), but was negative in the modified MAIPA when
In a parallel exercise the same 61 blinded samples were retested by the originating laboratory. However, after break-
tested in batches of 30, 16 and 15 by three different operators ing of the code and upon retesting with a double volume of
in the platelet immunology laboratories situated in NBS Bristol, serum a positive signal was obtained.

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
294 K. Campbell et al.

Table 3 Characteristics of the 61 blinded samples PAB-6 and NBS-5B12 and two with RFGP56. The results of
another unusual sample (OX-13) are included in Table 4. This
Anti-HPA/GP FMAIT PTP PR Other Total sample had been selected on basis that discrepant results had
been reported in two quality assurance exercises [Sample 4
1a 10 5 6 21
(01/448) in workshop 2001-A and Sample 3 (03/138) in
1a+5b 2 2
workshop 2003-A]. In both exercises the detection of this
1a+2b+3a 1 1
HPA-1a antibody varied between laboratories with clear
1b 2 2 1 5
1b+2b 1 1 positive results in 17 but negative results in the remaining
1b+3b 1 1 nine laboratories. The failure to detect this antibody in some
1b+3b+5b+15b 1 1 laboratories may be multifactorial, but results from this
3a 1 2 3 study showed that robust positive signals were obtained with
3a+5a 1 1 all four capture antibodies (Table 4).
3b 1 1 Overall, the absorbance signal produced by NBS-PAB-1
5a 1 1 was slightly higher, but not significantly different from the
5b 6 4 10 NBS-PAB-6 signal (2·61 ± 1·25 vs. 2·44 ± 1·30, P = 0·068,
5b+2b 1 1
t-test for paired samples; average ± 1 × SD on panel cell 1 with
Moua 1 1
24 samples containing anti-HPA-1a).
GPIIb/IIIa 3 3
There is a potential risk with MAIPA assays that incomplete
GPIb/IX 1 1
GPIIb/IIIa, Ibα/IbβIX, Ia/IIa 1 1 solubilization of the platelet membrane may lead to coimmu-
PIFT only 1 1 noprecipitation of platelet-specific GP complexes with HLA
HLA only 2 2 class I. This is thought to be one of the important causes of false-
Inert 3 3 positive results for HPA antibodies with panel cells that carry
Total 22 8 6 25 61 the cognate HLA antigen. The results were analysed for pos-
sible evidence of coimmunoprecipitation (Fig. 3). All samples
HPA, human platelet antigen; GP, glycoprotein; FMAIT, fetomaternal
were tested for reactivity against HLA class I using mAb W6/
alloimmune thrombocytopenia; PTP, post-transfusion purpura; PR,
32. In 18 of the 24 samples with anti-HPA-1a, reactivity on
platelet refractoriness.
one or more panel cells with HLA class I was observed. In 14
of the 18 samples the cognate HLA class I antigen was present
The effect of capture mAb on assay sensitivity was inves- on the one or more of the HPA-1b1b cells. Of the 14 samples
tigated in detail with the 42 samples with anti-GPIIb/IIIa with HPA-1a antibodies, five produced positive reactions
reactivity. These samples were tested in the MR-MAIPA assay with the captured GPIIb/IIIa of the HPA-1b1b genotype
with four panel cells (cells 1, 2, 3 and 6) and capture clones (Fig. 3; average ± 1 SD, 0·288 ± 0·071, range 0·194–0·385).
RFGP56, NBS-PAB6, NBS-5B12 in addition to PAB-1. The
correlation of signal intensities between capture antibodies
was generally good, but interestingly, in five samples
(Table 4) false-negative results were obtained with one of the The detection of HPA antibodies is of clinical relevance for
four capture antibodies, one each with NBS-PAB-1, NBS- the management of patients with FMAIT, PTP and PR. The

Table 4 Selected samples with GPIIb/IIIa antibodies that produced false-negative results in the MR-MAIPA with one of the four capture mAbs

Capture mAb

Sample code Condition NBS-PAB-1 NBS-PAB-6 NBS-5B12 RFGP56 Specificity anti-HPA

OX-01 PTP 0·118 0·087 0·254 0·107 1a+5b

OX-06 ITP 0·015 0·380 0·160 0·701 1a+5b
CAM-16 PR 0·141 0·187 0·140 0·074 1b
OX-09 PR 0·179 0·216 0·549 0·052 1b
CAM-04 NAIT 0·169 0·140 0·097 0·121 3a
OX-13 Blood donor 0·914 0·865 0·981 0·609 1a

HPA, human platelet antigen; sample OX-13 is positive with all four capture antibodies but was included because it produced a clear positive–negative split
in previous NIBSC proficiency schemes. False-negative results are in bold and italic.
PTP, post-transfusion purpura; ITP, immune thrombocytopenic purpura; FMAIT, fetomaternal alloimmune thrombocytopenia.

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
Rapid monoclonal antibody-specific immobilization of platelet antigen assay 295

Fig. 3 Evidence for coimmunprecipitation of

HLA class I with GPIIb/IIIa. Five of the 14 samples
with HPA-1a antibodies and concurrent HLA class
I antibodies also produced a significant reading
with the GPIIb/IIIa derived from the HPA-1b1b
platelets. The results by MR-MAIPA with capture
mAbs NBS-PAB-1 and W6/32 on the HPA-1a1a
(first set of two columns) and HPA-1b1b panel
platelets (second set of two columns). Absorbance
at 490 nm is presented on the y-axis. Sample
BR-04 is an FMAIT case and the four others are
transfusion purpura cases.

definition of the molecular basis of HPA antigens and the antibodies, CamTran007 and 19-7 (Kd 6·0 and 3·97 × 10–8 M/l,
introduction of DNA-based genotyping assays has greatly respectively [9]), is substantially lower than most monoclonal
improved the accuracy of typing of donors and patients RhD antibodies, with some having affinities in the lower
[18–20]. In contrast, the detection and identification of HPA nanomolar range [22]. Second, GPIIb/IIIa is known to
antibodies remains cumbersome and proficiency of HPA undergo a dramatic shape change upon platelet activation
antibody detection varies considerably between laboratories with a major repositioning of the plexn–semaphorin–integrin
that participate in quality-assurance exercises [2,3]. There are (PSI) domain that carries the HPA-1 antigens [23,24]. It is
a number of reasons for poor performance in HPA antibody possible that after membrane solubilization, GPIIb/IIIa may
detection. First, sensitivity for antibody detection is tech- undergo conformational change that may lead to the displace-
nique dependent [2,3]. Second, within a single technique, ment of HPA antibodies. If so, then one could speculate that
interoperator variation may occur and even in this study in certain capture antibodies may, depending on their epitope
which all operators were trained to a standard procedure, and affinity, constrain this gymnastic tour de force of
discrepancies were observed between two laboratories with GPIIb/IIIa.
one example of anti-HPA-1a. Finally, the composition of the These results indicate that the sensitivity of the MR-MAIPA
platelet panel cells and particularly the need for homozygo- may be greater than current MAIPA assays but that question
sity for all HPA alleles is important. cannot be unequivocally answered by this study. However, there
Half the laboratories participating in the biannual Inter- is evidence that the MR-MAIPA is at least as sensitive as the
national Society of Blood Transfusion platelet immunology original assay. First, the end-point titres of the recombinant
exercise use the MAIPA assay for antibody detection and HPA-1a antibody and of the three polyclonal NIBSC refer-
identification [3]. This technique originally described by ence reagents were similar or superior to those obtained in
Kiefel and colleagues [13–15] has the advantage that the both the NIBSC proficiency schemes [2] and the International
HPAs are in the native configuration during the sensitization Society of Blood Transfusion platelet immunology workshops
phase. Therefore, the assay has remained the cornerstone [25] (Figs 1 and 2). Second, there was complete concordance
technique in many platelet immunology laboratories in spite between the original results and the MR-MAIPA results for
of the availability of a commercial ELISA for the detection of the panel of 61 blinded samples that had been deliberately
HPA antibodies. Since the original description of the assay, enriched for samples with HPA antibodies that had been dif-
modifications of the MAIPA principle have been reported and ficult to detect at initial referral. Third, the HPA-3a antibodies
variables have been identified that may influence sensitivity in sample CAM-04 from a clinically significant case of
[14,15]. FMAIT were reactive in the MR-MAIPA but not in our previ-
Using the recombinant HPA-1a antibody CamTran007 ously used MAIPA assays. In addition, it has been shown that
that has a Kd of 6 × 10–8 M/l, it was demonstrated that the the sensitivity of the MR-MAIPA is maintained when the
sensitivity of the MR-MAIPA is in the upper nanogram range assay is transferred to other operators in the same or other
(Fig. 1). This level of sensitivity compares poorly with the laboratories provided this process is supported by a standard
sensitivities of the antiglobulin and autoanalyser techniques operating procedure and training.
for anti-D detection that are in the lower nanogram range Our study confirmed the observation made by several
[21]. There are several possible reasons for the lower sensitiv- groups that the type of capture mAb influences the sensitivity
ity of the MAIPA assay. First, the affinity of HPA antibodies of the MAIPA (Table 4). It demonstrates that no single capture
plays a critical role. The affinity of two recombinant HPA-1a antibody is ideal. The observed differences between them

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
296 K. Campbell et al.

could be explained by epitope overlap between capture anti- laboratories. In addition, the sensitivity of the MR-MAIPA
body and the HPA antibody, or variation in the ability of assay for the detection of HPA-1a antibodies is in the higher
capture antibodies to constrain the GPIIb/IIIa conformation nanogram range and appears better than the standard MAIPA
after membrane solubilization. The three different capture assay. Improved assay sensitivity has clinical relevance as it
antibodies did not affect the sensitivity of the MR-MAIPA for has been shown that HPA-1a negative, HLA DRB3*0101 positive
the detection of recombinant anti-HPA-1a CamTran007 (Fig. 1). mothers with thrombocytopenic neonates may have a negative
This observation is compatible with the notion that monoclonal HPA-1a antibody screen during pregnancy with antibodies only
or polyclonal HPA-1a antibodies of good affinity are detected becoming detectable in the postpartum period [29]. In addi-
irrespective of the capture antibody, as long as there is no tion, severe FMAIT cases with cerebral bleeds occur in women
epitope overlap. Interestingly, the signal produced with the with low-titre HPA-1a antibodies. This study demonstrates
anti-HPA-1a potency standard 03/152 was less good with that the detection of some antibodies (possibly low titre) by
clone NBS-PAB-6 when compared with NBS-PAB-1 (Fig. 2a). MAIPA assay can be significantly influenced by the choice
Recent studies from our laboratory have shown that NBS- of capture antibody. The use of recombinant HPA antigens [30]
PAB-1 and NBS-PAB-6 target overlapping epitopes [26], and alternative immunoassay platforms like Luminex® beads
suggesting that it may be a difference in affinity, which may be a possible avenue to further increase the sensitivity
explains the poorer performance of NBS-PAB-6. of HPA-1a antibody detection. Experiments are currently
Two samples (Table 4) that produced equivocal results proceeding in our laboratories to investigate this [31].
between our laboratories or in quality exercises were partic-
ularly informative. Sample OX-13 with anti-HPA-1a showed
a positive–negative split between laboratories participating
in the NIBSC proficiency schemes. This sample produced We thank the staff of the NHSBT platelet immunology labora-
robust positive results in the MR-MAIPA with all four capture tories at Bristol, Cambridge and Oxford for their expertise in
mAbs. The HPA-3a antibodies in sample CAM-04 was asso- platelet immunology. We gratefully acknowledge Stephen
ciated with widespread petechiae and a platelet count of Garner, Dr Nick Watkins, Graham Smith, Dave Allen, Rizwan
12 × 109/l in a term neonate after an uneventful delivery. Yusuf and Gerry Campbell for their assistance and valuable
This antibody was not consistently detected by the MAIPA comments during the manuscript preparation.
protocols that were previously operational in our laboratories
but a good signal was observed in the MR-MAIPA with three
out of four mAbs.
The issue of coimmunoprecipitation remains a concern 1 Allen D, Rigsby P, Bessos H, Berry J, Wilson D, Ouwehand WH,
when using the MAIPA assay for HPA antibody detection. Urbaniak S, Metcalfe P: Collaborative study to establish the first
Incomplete solubilization may cause immunoprecipitation of international standard for quantitation of anti-HPA-1a. Vox
the HLA class I molecule by anti-integrin antibodies. In 14 of Sang 2005; 89:100–104
the 24 samples with anti-HPA-1a and anti-HLA, HLA anti- 2 Metcalfe P, Allen D, Chapman J, Ouwehand WH: Interlaboratory
variation in the detection of clinically significant alloantibodies
bodies were against HLA class I antigens present on the HPA-
against human platelet alloantigens. Br J Haematol 1997;
1b1b panel cell. Nine of the 14 samples were non-reactive on
the HPA-1b1b panel cell with GPIIb/IIIa capture antibodies.
3 Bessos H, Wilson DW, Metcalfe P, Allen D, Urbaniak SJ: Report
The remaining five samples produced some signal (Fig. 4) but on the 12th International Society of Blood Transfusion platelet
four of these were from PTP patients and the reactivity with immunology workshop. Vox Sang 2005; 89:105–113
the HPA-1b1b platelets is most likely caused by autoanti- 4 Lucas GF, Rogers SE: Evaluation of an enzyme-linked immuno-
bodies against GPIIb/IIIa [27,28]. The single FMAIT sample sorbent assay kit [GTI PakPlus(R)] for the detection of antibodies
(BR-04) with HPA-1a antibodies and positive results with the against human platelet antigens. Transfus Med 1999; 9:385–386
HPA-1b1b platelets (OD 0·385) contained strong HLA class I 5 Lubenko A, Savage J: Antigen capture ELISA for platelet anti-
antibodies that may have caused the weak positive reaction body detection: choice of conjugate influences assay result.
on the antithetical HPA-1b1b panel cell. There were insuffi- Transfus Med 2000; 10:213–218
6 Griffin HM, Ouwehand WH: A human monoclonal antibody
cient samples containing HPA-2 or HPA-5 reactive antibodies
specific for the leucine-33 (P1A1, HPA-1a) form of platelet
to answer the question of possible coimmunoprecipitation of
glycoprotein IIIa from a V gene phage display library. Blood
HLA class I with GPIbα/IbβIX and GPIa/IIa, respectively.
1995; 86:4430–4436
In conclusion, this study describes the extensive validation 7 Watkins NA, Schaffner-Reckinger E, Allen DL, Howkins GJ,
of a modified and rapid MAIPA assay protocol using a large Brons NHC, Smith GA, Metcalfe P, Murphy MF, Kieffer N,
number of samples with HPA antibodies and several capture Ouwehand WH: HPA-1a phenotype–genotype discrepancy reveals
mAbs. The reduction of the duration of the assay from 8 to a naturally occurring Arg93Gln substitution in the platelet β3 integrin
5 h without loss of sensitivity is of key importance to diagnostic that disrupts the HPA-1a epitope. Blood 2002; 99:1833–1839

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297
Rapid monoclonal antibody-specific immobilization of platelet antigen assay 297

8 Metcalfe P, Ouwehand WH, Sands D, Barrowcliffe TW: Collabora- 20 Metcalfe P, Cavanagh G, Hurd C, Ouwehand WH: HPA genotyp-
tive studies to establish the first WHO Reference Reagent for ing by PCR-SSP: report of 4 exercises. Vox Sang 1999; 77:40–43
detection of human antibody against human platelet antigen- 21 Anstee D, Klein H: Mollison’s Blood Transfusion in Clinical
5b. Vox Sang 2003; 84:237–240 Medicine, 11th edn. Oxford, UK, Blackwell Publishing, 2005
9 Santoso S, Kroll H, Andrei-Selmer CL, Socher I, Rankin A, 22 Bye JM, Carter C, Cui Y, Gorick BD, Songsivilai S, Winter G,
Kretzschmar E, Watkins NA, Ouwehand WH: A naturally occur- Hughes-Jones NC, Marks JD: Germline variable region gene seg-
ring LeuVal mutation in β3-integrin impairs the HPA-1a epitope: ment derivation of human monoclonal anti-Rh(D) antibodies.
the third allele of HPA-1. Transfusion 2006; 46:790–799 Evidence for affinity maturation by somatic hypermutation and
10 Armour KL, Clark MR, Hadley AG, Williamson LM: Recombinant repertoire shift. J Clin Invest 1992; 90:2481–2490
human IgG molecules lacking Fcγ receptor I binding and mono- 23 Xiao T, Takagi J, Coller BS, Wang JH, Springer TA: Structural
cyte triggering activities. Eur J Immunol 1999; 29:2613–2624 basis for allostery in integrins and binding to fibrinogen-
11 WHO: WHO Expert Committee on Biological Standardisation, mimetic therapeutics. Nature 2004; 432:59–67
48th report. WHO Technical Report Series 1999; 889:17 24 Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL,
12 Berry JE, Murphy CM, Smith GA, Ranasinghe E, Finberg R, Joachimiak A, Goodman SL, Arnaout MA: Crystal structure of
Walton J, Brown J, Navarrete C, Metcalfe P, Ouwehand WH: the extracellular segment of integrin α Vβ3. Science 2001;
Detection of Gov system antibodies by MAIPA reveals an 294:339–345
immunogenicity similar to the HPA-5 alloantigens. Br J Haematol 25 Metcalfe P: Platelet antigens and antibody detection. Vox Sang
2000; 110:735–742 2004; 87:82–86
13 Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C: Mono- 26 Jennings NS, Harmer IJ, Campbell K, Stafford P, Smith GA,
clonal antibody – specific immobilization of platelet antigens Metcalfe P, Benton MA, Marsh JC, Ouwehand WH: Molecular
(MAIPA): a new tool for the identification of platelet-reactive characterization of the variable domains of an αIIbβ3-specific
antibodies. Blood 1987; 70:1722–1726 immunoglobulin M κ platelet cold agglutinin in a follicular
14 Kiefel V: The MAIPA assay and its applications in immunohae- lymphoma patient with treatment refractory autoimmune
matology. Transfus Med 1992; 2:181–188 thrombocytopenia: idiotypic overlap between αIIbβ3 integrin
15 Kiefel V, Scheld S, Freitag E, Kroll H, Mueller-Eckhardt C: [Two antibodies. Transfusion 2007; 47:499–510
modifications of MAIPA assays for the demonstration of platelet 27 Watkins NA, Smethurst PA, Allen D, Smith GA, Ouwehand WH:
antibodies]. Beitr Infusionsther Transfusionsmed 1994; 32:237– Platelet αIIbβ3 recombinant autoantibodies from the B-cell
239 repertoire of a post-transfusion purpura patient. Br J Haematol
16 Watkins NA, Armour KL, Smethurst PA, Metcalfe P, Scott ML, 2002; 116:677–685
Hughes DL, Smith GA, Williamson LM, Clark MR, Ouwehand 28 Taaning E, Tonnesen F: Pan-reactive platelet antibodies in post-
WH: Rapid phenotyping of HPA-1a using either diabody-based transfusion purpura. Vox Sang 1999; 76:120–123
hemagglutination or recombinant IgG1-based assays. Transfusion 29 Williamson LM, Hackett G, Rennie J, Palmer CR, Maciver C,
1999; 39:781–789 Hadfield R, Hughes D, Jobson S, Ouwehand WH: The natural
17 Masters R, Taaning E: Three cases of platelet alloimmunisation history of fetomaternal alloimmunization to the platelet-specific
associated with the presence of a novel platelet-specific anti- antigen HPA-1a (PlA1, Zwa) as determined by antenatal screen-
body. Vox Sang 1998; 75:242–246 ing. Blood 1998; 92:2280–2287
18 Bugert P, McBride S, Smith G, Dugrillon A, Klüter H, Ouwehand 30 Li CQ, Garner SF, Davies J, Smethurst PA, Wardell MR,
WH, Metcalfe P: Microarray-based genotyping for blood groups: Ouwehand WH: Threonine-145/methionine-145 variants of
comparison of gene array and 5′-nuclease assay techniques with baculovirus produced recombinant ligand binding domain of
human platelet antigen as a model. Transfusion 2005; 45:654– GPIbα express HPA-2 epitopes and show equal binding of von
659 Willebrand factor. Blood 2000; 95:205–211
19 Hurd CM, Cavanagh G, Schuh A, Ouwehand WH, Metcalfe P: 31 Stafford P, Ghevaert C, Campbell K, Smith G, Williamson L,
Genotyping for platelet-specific antigens: techniques for the Watkins NA, Ouwehand WH: Recombinant mini-β3 integrin
detection of single nucleotide polymorphisms. Vox Sang 2002; fragments for the detection of HPA-1 antibodies. Blood 2005,
83:1–12 106:Abstract 218

© 2007 The Author(s)

Journal compilation © 2007 Blackwell Publishing Ltd., Vox Sanguinis (2007) 93, 289–297