Transfusion Medicine 1992. 2, 3 19-322
W O RK S H O P R E P O R T
Self help for platelet serologists: the Australian platelet antibody workshops R.M . Minchinton
Queensland Red Cross Blood Transfuion Senlice. Brirbane. Queensland. Australia
Rereiued 20 May 1992: accepted for publication 6 July 1992
The first three Australian Platelet Antibody Workshops (APAW) were held under the auspices of the Australasian Society of Blood Transfusion (ASBT) in 1989, 1990 and 1991. Participants submitted serum samples to the convenor who coded and distributed samples to laboratories routinely performing platelet serology. Results were summarized and discussed at the annual ASBT meetings. This report documents the evolution of platelet
antibody tests in Australia and demonstrates the marked improvements in the participants’ ability to differentiate platelet-specific antibodies from HLA antibodies and to identify the alloantigenic specificity of platelet antibodies. Five reference laboratories in Australia now hold panels typed for at least three platelet alloantigens.
Australian Platelet Antibody Workshops (APAW) were held under the auspices of the Australasian Society of Blood Transfusion (ASBT) in 1989, 1990 and 1991 with the following aims.
local laboratories, tested for viral markers, packaged, coded and despatched without clinical notes to participating laboratories (Tables I and 2). Technical questionnaires accompanied the samples. Returning data were summarized by the convenor and presented in booklet form for discussion at the annual scientific meeting of the ASBT in the same year. Numbers were too small for meaningful statistical analysis, therefore only raw data and simple percentages were reported.
SUMMARY.
Key wordr: platelet antibody workshop.
1 To establish a communication network for information exchange, education and problem solving. 2 To facilitate a standardized approach to the identification of platelet antibodies and antigens. 3 To consider the further provision of an external quality assurance programme for platelet serology.
1991 workshop
The workshop format was modelled on that successfully used by the ISBT/ICSH Working party on Platelet Serology. Biennial, international, platelet antibody exchanges were first begun by the international group in 1982 (Metcalfe & Waters, 1990). This report reviews the results of the first three annual platelet antibody workshops conducted in Australia and documents the improvements in platelet serology observable through the workshops over the same period.
A requirement of participation in the 1991 wet workshop was the submission of 30 ml of patient serum for use in the exchange. Sixteen samples were despatched to 15 laboratories (Table 2). Handling, analysis and discussion were identical to that in the 1989 and 1990 workshops. Table 1. APAW demographics
METHOD
1989
1990
1991
10 100
10
78
16 93 15 8
~~~
1989, 1990 workshops
Number of sera Return rate (%)
Ten serum samples were drawn from the banks of the Queensland Red Cross Blood Transfusion Service and
Number of participants Number of observers*
Correspondence: Dr Robyn Minchinton. Chief Scientist, Queensland Red Cross Blood Transfusion Service. PO Box 1 0 3 3 Adelaide Street. Brisbane, Queensland 4O00, Australia.
Observers received correspondence and summaries without simples.
3 I9
14
0
19 3
320
Self help f o r platelet serologists
Table 2. Identity of sera issued
Table 4. True- and false-positive and -negative detection rates as a percentage
1989 Negative (AB serum) HLA Platelet specific alloantibody WD* Platelet specific alloantibody UDS Platelet autoantibody HLA +platelet antibody mixture EDTA/quinine dependent Total
1990
4
2
I
I
3
1
1991 2 1 9 2
1
1
0 0 I
2 1
0 I
2
1
10
10
16
Well defined prior to submission.
t Undefined.
RESULTS Techniques in use Techniques used by Australian participants to test workshop samples reflect those in use at the same time in other parts of the world (Table 3). The platelet immunofluorescence test (PIFT) (von dem Borne et al., 1979), which is the international standard technique for verification of newly developed platelet antibody assays, remains the most frequently used test in the APAW. Five participants used a flow cytometer for interpretation of the PIFT. The use of enzymelinked immunosorbent assays (ELISA), using whole platelets and polyclonal antihuman globulin reagents, has decreased, while mixed passive haemagglutination (MPHA) type tests, with red cells or latex beads as indicators, are gaining popularity as screening methods. To indicate the presence of HLA antibodies, most laboratories used the lymphocytoxicity test (LCT), alone, or in combination with chloroquine or acid treatment of platelets which strips HLA antigens from Table 3. Changing pattern of techniques used in the APAW
1989 PIFT microscopic PlFT flow cytometer ELlSA MPHA type LCT
Chloroquine/acid treatment Western immunoblot MAIPA
5
3 5 3 6
3 3 0
1990
1991
True positives True negatives False positives False negatives
1989
1990
1991
19 17
90 97
79 75
22 21
3 10
25 21
the platelet surface leaving platelet-specific antigens intact. Western Blotting (WB) is little used except by reference laboratories. The Monoclonal Antibody Immobilisation of Platelet Antigen (MAIPA) assay was used for the first time for Australian workshop samples in 1991, although its use has been steadily increasing in the international workshops since 1988. Some laboratories, in particular those acting as State Reference Laboratories, used a combination of platelet antibody tests. True- and false-positive and -negatice rates An improvement in the ability to detect true positives and true negatives was seen in 1990; however, this returned to 1989 levels in the 1991 workshop (Table 4). Idcntl/ication of cell specificity of antibodies The ability to differentiate between platelet-specific antibodies and HLA antibodies, negative controls and mixtures of platelet-specific and HLA antibodies was charted by plotting the consensus cell specificity (abscissa) versus each laboratory's observed result (ordinate) (Lucas, 1990) (Fig. 1). Ideally, the plot consists of all of the points lying along a straight line through the origin. From 1989, a definite move of the plotted points towards the central axis linecan be seen. In particular, fewer antibodies were classified as positive but of unspecified character. More laboratories were able to identify platelet-specific antibodies, HLA antibodies and platelet/HLA mixtures. The detection of quinine-dependent antibodies also improved. The ability to determine platelet alloantigen specificity Marked improvement has occurred in the successful characterization of the most clinically important antibody. anti-HPA-la (PLA') (Table 5). In 1989, on average. 25% of laboratories correctly identified antiHPA-la. while in 1991, the figure was 44%. The
R.M. Minchinton :O)
um e
wu L
ru
w
nu
UI4
Table 5. Ability of laboratories to determine correct platelet alloantigen specificity (7'0)
..... .... ::::. ......... ..... .... ..... .... .. ..... . . . . .... . . . ....... iiiii: i: :::.......
.....
.....
.... ..... .................. ........ .... .....
I
NIO
YLA
PU
nm~ml
WY IOU
I wuec
I
.... ..
Id
.. .... .... ....
... ..... ........ ........ ..... ..... ......... .............. ................ .............. ......... ..... . . . . . . .... ... ....
Platelet alloantibody
I989
1990
1991
HPA-la HPA-3a HPA-4a HPA-4b HPA-Sb
25 21
-
-
13 33
14 7
7
-
21
Number of laboratories
14 (3)
19 (3)
IS (5)*
44
Figures (in parentheses) are the number or laboratories with panels typed Tor more than three platelet alloantigens.
.....
nso)
321
.....
Consensus
Fig. I . Plot of the consensus cell specificity versus the observed result for each laboratory in (a) 1989 (b) 1990 and (c) I991 Australian Platelet Antibody Workshops.
identification of anti-HPA-Sb (Br") improved from only 7% in 1989 to 21% in 1991. Other antibody specificities are less common, but in 1991, five laboratories in Australia held panels typed for more than three platelet alloantigens.
DISCUSSION Platelet antibody workshops have now been conducted in Australia for 3 years. The APAW emphasizes education and communication and is a means by
which laboratories gain confidence in their diagnostic ability in this rapidly evolving field. Since 1989, regular participants in the APAW have shown improvement in their ability to detect platelet-reactive antibodies, to differentiate platelet-specific antibodies from HLA antibodies and mixtures and to characterize the platelet alloantigenic specificity of a platelet antibody. The improvement seen in the detection of true positives and negatives from I989 to 1990 is probably a reflection of the greater familiarity with the workshop format. Platelet antibody techniques used for the 1989 and 1990 workshops did not alter greatly and this improvement also reflects a greater familiarity with the technology in each laboratory. In contrast, many new techniques were introduced by participants for the 1991 workshop and their novelty and technical complexity probably contributed to the decrease in correctly reported true positives and negatives to 1989 levels. In 1991, the improvement in the ability to identify correctly the alloantigenic specificity of the workshop samples was largely a result of increased serum exchange between platelet antibody reference laboratories in each Australian state. Furthermore, the participation of some Australian laboratories in the International Platelet Antibody Workshop, and the establishment of valuable overseas contacts, has encouraged overseas exchange of typing sera to our advantage. As a result, five Australian laboratories now have platelet panels typed for at least three alloantigens. This enables them to use the PIFT and the MAIPA to characterize defined or new platelet antibody specificities. The ASBT Australian Platelet Antibody Workshops have allowed the documentation of the evolution of platelet serology in Australia since 1989. The strong collaborative emphasis of these workshops and
322 Self help /or platelet serologisfs the resulting national serum exchanges will ensure that Australia will be able to contribute to the international search for and characterization of, new human platelet alloantigens. ACKNOWLEDGEMENTS The author would like to thank Mrs Sandy Woodward for careful typing of the manuscript and Mrs Debra Hislop for expert technical assistance. All participants in the Australian workshop are gratefully acknowledged for their contributions and continued support.
REFERENCES Lucas, G.F. (1990) A survey of platelet serology in UK laboratories. Clinical Laboratory Haematology, 12, 185200. Metcalfe P. & Waters, A.H. (1990) Report on the fourth ISBT/ISCH platelet serology workshop (London 1988). Vox Sanguinis, 58, 170-175. von dem Borne A.E.G. Kr, Verheugt F.W.A., Oosterhof F., von Reisz E.. Brute1 de la Riviere A. & Engelfriet C.P. ( 1 979) A simple immunofluorescence test for the detection of platelet antibodies. British Journal of Haematology, 39, 195-207.
W O RK S H O P R E P O R T
Self help for platelet serologists: the Australian platelet antibody workshops R.M . Minchinton
Queensland Red Cross Blood Transfuion Senlice. Brirbane. Queensland. Australia
Rereiued 20 May 1992: accepted for publication 6 July 1992
The first three Australian Platelet Antibody Workshops (APAW) were held under the auspices of the Australasian Society of Blood Transfusion (ASBT) in 1989, 1990 and 1991. Participants submitted serum samples to the convenor who coded and distributed samples to laboratories routinely performing platelet serology. Results were summarized and discussed at the annual ASBT meetings. This report documents the evolution of platelet
antibody tests in Australia and demonstrates the marked improvements in the participants’ ability to differentiate platelet-specific antibodies from HLA antibodies and to identify the alloantigenic specificity of platelet antibodies. Five reference laboratories in Australia now hold panels typed for at least three platelet alloantigens.
Australian Platelet Antibody Workshops (APAW) were held under the auspices of the Australasian Society of Blood Transfusion (ASBT) in 1989, 1990 and 1991 with the following aims.
local laboratories, tested for viral markers, packaged, coded and despatched without clinical notes to participating laboratories (Tables I and 2). Technical questionnaires accompanied the samples. Returning data were summarized by the convenor and presented in booklet form for discussion at the annual scientific meeting of the ASBT in the same year. Numbers were too small for meaningful statistical analysis, therefore only raw data and simple percentages were reported.
SUMMARY.
Key wordr: platelet antibody workshop.
1 To establish a communication network for information exchange, education and problem solving. 2 To facilitate a standardized approach to the identification of platelet antibodies and antigens. 3 To consider the further provision of an external quality assurance programme for platelet serology.
1991 workshop
The workshop format was modelled on that successfully used by the ISBT/ICSH Working party on Platelet Serology. Biennial, international, platelet antibody exchanges were first begun by the international group in 1982 (Metcalfe & Waters, 1990). This report reviews the results of the first three annual platelet antibody workshops conducted in Australia and documents the improvements in platelet serology observable through the workshops over the same period.
A requirement of participation in the 1991 wet workshop was the submission of 30 ml of patient serum for use in the exchange. Sixteen samples were despatched to 15 laboratories (Table 2). Handling, analysis and discussion were identical to that in the 1989 and 1990 workshops. Table 1. APAW demographics
METHOD
1989
1990
1991
10 100
10
78
16 93 15 8
~~~
1989, 1990 workshops
Number of sera Return rate (%)
Ten serum samples were drawn from the banks of the Queensland Red Cross Blood Transfusion Service and
Number of participants Number of observers*
Correspondence: Dr Robyn Minchinton. Chief Scientist, Queensland Red Cross Blood Transfusion Service. PO Box 1 0 3 3 Adelaide Street. Brisbane, Queensland 4O00, Australia.
Observers received correspondence and summaries without simples.
3 I9
14
0
19 3
320
Self help f o r platelet serologists
Table 2. Identity of sera issued
Table 4. True- and false-positive and -negative detection rates as a percentage
1989 Negative (AB serum) HLA Platelet specific alloantibody WD* Platelet specific alloantibody UDS Platelet autoantibody HLA +platelet antibody mixture EDTA/quinine dependent Total
1990
4
2
I
I
3
1
1991 2 1 9 2
1
1
0 0 I
2 1
0 I
2
1
10
10
16
Well defined prior to submission.
t Undefined.
RESULTS Techniques in use Techniques used by Australian participants to test workshop samples reflect those in use at the same time in other parts of the world (Table 3). The platelet immunofluorescence test (PIFT) (von dem Borne et al., 1979), which is the international standard technique for verification of newly developed platelet antibody assays, remains the most frequently used test in the APAW. Five participants used a flow cytometer for interpretation of the PIFT. The use of enzymelinked immunosorbent assays (ELISA), using whole platelets and polyclonal antihuman globulin reagents, has decreased, while mixed passive haemagglutination (MPHA) type tests, with red cells or latex beads as indicators, are gaining popularity as screening methods. To indicate the presence of HLA antibodies, most laboratories used the lymphocytoxicity test (LCT), alone, or in combination with chloroquine or acid treatment of platelets which strips HLA antigens from Table 3. Changing pattern of techniques used in the APAW
1989 PIFT microscopic PlFT flow cytometer ELlSA MPHA type LCT
Chloroquine/acid treatment Western immunoblot MAIPA
5
3 5 3 6
3 3 0
1990
1991
True positives True negatives False positives False negatives
1989
1990
1991
19 17
90 97
79 75
22 21
3 10
25 21
the platelet surface leaving platelet-specific antigens intact. Western Blotting (WB) is little used except by reference laboratories. The Monoclonal Antibody Immobilisation of Platelet Antigen (MAIPA) assay was used for the first time for Australian workshop samples in 1991, although its use has been steadily increasing in the international workshops since 1988. Some laboratories, in particular those acting as State Reference Laboratories, used a combination of platelet antibody tests. True- and false-positive and -negatice rates An improvement in the ability to detect true positives and true negatives was seen in 1990; however, this returned to 1989 levels in the 1991 workshop (Table 4). Idcntl/ication of cell specificity of antibodies The ability to differentiate between platelet-specific antibodies and HLA antibodies, negative controls and mixtures of platelet-specific and HLA antibodies was charted by plotting the consensus cell specificity (abscissa) versus each laboratory's observed result (ordinate) (Lucas, 1990) (Fig. 1). Ideally, the plot consists of all of the points lying along a straight line through the origin. From 1989, a definite move of the plotted points towards the central axis linecan be seen. In particular, fewer antibodies were classified as positive but of unspecified character. More laboratories were able to identify platelet-specific antibodies, HLA antibodies and platelet/HLA mixtures. The detection of quinine-dependent antibodies also improved. The ability to determine platelet alloantigen specificity Marked improvement has occurred in the successful characterization of the most clinically important antibody. anti-HPA-la (PLA') (Table 5). In 1989, on average. 25% of laboratories correctly identified antiHPA-la. while in 1991, the figure was 44%. The
R.M. Minchinton :O)
um e
wu L
ru
w
nu
UI4
Table 5. Ability of laboratories to determine correct platelet alloantigen specificity (7'0)
..... .... ::::. ......... ..... .... ..... .... .. ..... . . . . .... . . . ....... iiiii: i: :::.......
.....
.....
.... ..... .................. ........ .... .....
I
NIO
YLA
PU
nm~ml
WY IOU
I wuec
I
.... ..
Id
.. .... .... ....
... ..... ........ ........ ..... ..... ......... .............. ................ .............. ......... ..... . . . . . . .... ... ....
Platelet alloantibody
I989
1990
1991
HPA-la HPA-3a HPA-4a HPA-4b HPA-Sb
25 21
-
-
13 33
14 7
7
-
21
Number of laboratories
14 (3)
19 (3)
IS (5)*
44
Figures (in parentheses) are the number or laboratories with panels typed Tor more than three platelet alloantigens.
.....
nso)
321
.....
Consensus
Fig. I . Plot of the consensus cell specificity versus the observed result for each laboratory in (a) 1989 (b) 1990 and (c) I991 Australian Platelet Antibody Workshops.
identification of anti-HPA-Sb (Br") improved from only 7% in 1989 to 21% in 1991. Other antibody specificities are less common, but in 1991, five laboratories in Australia held panels typed for more than three platelet alloantigens.
DISCUSSION Platelet antibody workshops have now been conducted in Australia for 3 years. The APAW emphasizes education and communication and is a means by
which laboratories gain confidence in their diagnostic ability in this rapidly evolving field. Since 1989, regular participants in the APAW have shown improvement in their ability to detect platelet-reactive antibodies, to differentiate platelet-specific antibodies from HLA antibodies and mixtures and to characterize the platelet alloantigenic specificity of a platelet antibody. The improvement seen in the detection of true positives and negatives from I989 to 1990 is probably a reflection of the greater familiarity with the workshop format. Platelet antibody techniques used for the 1989 and 1990 workshops did not alter greatly and this improvement also reflects a greater familiarity with the technology in each laboratory. In contrast, many new techniques were introduced by participants for the 1991 workshop and their novelty and technical complexity probably contributed to the decrease in correctly reported true positives and negatives to 1989 levels. In 1991, the improvement in the ability to identify correctly the alloantigenic specificity of the workshop samples was largely a result of increased serum exchange between platelet antibody reference laboratories in each Australian state. Furthermore, the participation of some Australian laboratories in the International Platelet Antibody Workshop, and the establishment of valuable overseas contacts, has encouraged overseas exchange of typing sera to our advantage. As a result, five Australian laboratories now have platelet panels typed for at least three alloantigens. This enables them to use the PIFT and the MAIPA to characterize defined or new platelet antibody specificities. The ASBT Australian Platelet Antibody Workshops have allowed the documentation of the evolution of platelet serology in Australia since 1989. The strong collaborative emphasis of these workshops and
322 Self help /or platelet serologisfs the resulting national serum exchanges will ensure that Australia will be able to contribute to the international search for and characterization of, new human platelet alloantigens. ACKNOWLEDGEMENTS The author would like to thank Mrs Sandy Woodward for careful typing of the manuscript and Mrs Debra Hislop for expert technical assistance. All participants in the Australian workshop are gratefully acknowledged for their contributions and continued support.
REFERENCES Lucas, G.F. (1990) A survey of platelet serology in UK laboratories. Clinical Laboratory Haematology, 12, 185200. Metcalfe P. & Waters, A.H. (1990) Report on the fourth ISBT/ISCH platelet serology workshop (London 1988). Vox Sanguinis, 58, 170-175. von dem Borne A.E.G. Kr, Verheugt F.W.A., Oosterhof F., von Reisz E.. Brute1 de la Riviere A. & Engelfriet C.P. ( 1 979) A simple immunofluorescence test for the detection of platelet antibodies. British Journal of Haematology, 39, 195-207.
