Workshop Report 0 1990 S. Karger AG, Basel 0042-9007/90/0594-0251$2.75/0

Vox Sang 1990;59:251-256

Results of the First International Granulocyte Serology Workshop G . E Lucas", I? A . Carringtonb aBlood Group Reference Laboratory, South Western Regional Blood Transfusion Centre, Bristol, UK; bHaematology Department, Hope Hospital, Salford, UK

Abstract. Eleven laboratories paticipated in the First International Workshop of Granulocyte Serology. Participants were asked to identify any granulocyte-reactive antibodies present in l2human sera distributed as unknown samples. Granulocyte immunofluorescence and granulocyte micro-agglutination were the most commonly used techniques. The incidence of false-positive and false-negative reactions was 15 and 4%, respectively. Most laboratories were able to distinguish between granulocyte-specific and anti-HLA antibodies using platelet absorption. The identification of the specificity of granulocyte-specific antibodies caused the most problems; these were correctly identified on only 57% of occasions.

Table 1.Participating laboratories

Introduction ~

Neutrophil-specific antibodies were first discovered over 30years ago [l] and since that time numerous techniques have been described for their detection [2,3]. However, an organised exchange of sera between laboratories to enable the relevant merits of different techniques to be compared has not been previously attempted. We therefore undertook a Granulocyte Serology Workshop to evaluate the techniques currently used and to identify any problem areas. It is hoped that the results of this workshop will prove useful not only to those who already have experience of granulocyte serology but also to those considering developing these techniques in their own laboratory.

Amsterdam

Central Laboratory of the Netherlands Red Cross

Dr. W. Ouwehand

Colindale

North London Blood Transfusion Centre

Dr. G. Schwartz

G iessen

Giessen University, FDR

Prof. C. MuellerEckhardt

London

St. Bartholomew's Hospital

Prof. A. Waters/ Mr. P. Metcalfe

Minneapolis

American Red Cross

Dr. J. McCullough/ Mrs. C. Sullivan

New York

Montefiore Institute

Prof. Lalezari

Oxford

Blood Group Reference Laboratory

Dr. G. Lucas

Paris

Hospital Necker

Dr. J. Cartrod Dr. J.-L. Celton

Salford

Hope Hospital

Dr. P. Carrington

Tooting

South London Blood Transfusion Centre

Mr. F. Sherwood

Warsaw

Institute of Haematology

Dr. B. Zupanska

Materials and Methods Participants The Workshop was organized for the UK Platelet and Granulocyte Serology Working Group (Chairman: Prof. A.H. Waters) which is affiliated to the British Blood Transfusion Society. The 39 members of the Working Group and 6 overseas laboratories were invited to participate in the Workshop and to supply details of their techniques for granulocyte antibody investigations. A lack of appropriate antisera in sufficient volumes prevented the inclusion of all laboratories wishing to participate and, consequently, participation was restricted. The 12 lab-

The order of the laboratories presented in this table does not correspond to the numeric code used to identify the laboratories in the text or in subsequent tables.

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LucaslCarrington

Table 2. Description of workshop samples

Table 3. Techniques used in the workshop

Sample

Description

A

Plasma pool from male non-transfused AB donors

a. Tests for granulocyte antibodies

B

Unknown sample reactive by LAT but not LCT

C

Anti-NB1 from a case of ANN; detectable by GAT and GIFT full titre 512 and >1,024, respectively

D

Plasma containing multispecific anti-HLA antibodies reactive by GIFT, GCLT, LIFT and 26/33 donors by LCT

E

Serum pool from male non-transfused AB donors, incubated at 56OC for 30 min

F

Serum containing anti-HLA-B12 antibodies detectable by GIFT, GCLT, LIFT and LCT

Granulocyte immunofluorescence test (GIFT) Granulocyte agglutination test (GAT) Mixed leucocyte agglutination test (LAT) Flow cytometry (FACS) Granulocyte chemiluminescence test (GCLT) Granulocytotoxicity test

G

Reconstituted lypholysed serum containing anti-5b antibodies formed during pregnancy

H

Serum containing anti-NA2 antibodies diluted with inert human serum and containing 0.01% sodium azide

I

Anti-NAl and multispecific anti-HLA antibodies (43% reactivity by LCT) from the mother of a child with ANN

J

Untreated serum pool from non-transfused male AB donors

K

Anti-NA2 and multispecific anti-HLA antibodies from the mother of a child with ANN

L

Plasma containing multispecific anti-HLA antibodies reactive by GIFT, GCLT, LIFT and 26/33 donors by LCT (also issued as sample D)

ANN GAT GCLT GIFT LAT LCT LIFT

= Allo immune neonatal neutropenia = = = = = =

Granulocyte agglutination test Granulocyte chemiluminescence test Granulocyte immunofluorescence test Leucocyte agglutination test Lymphocytotoxicity test Lymphocyte immunofluorescence test

oratories chosen offered antisera for distribution and provided an established service for the detection of anti-granulocyte antibodies. Eleven of the 12 laboratories completed the workshop (table 1): the order of the laboratories does not correspond to the numeric code used to identify the laboratories in the text or in subsequent tables. Design of the Workshop The workshop comprised two parts. Part I consisted of three diluted murine monoclonal antibodies (ascitic fluids) which were provided together with a description of their specificities for use as reference reagents. Results obtained with these reagents were not specifically requested and are not discussed further. Part I1 consisted of 12human (serum or plasma) samples which were supplied as unknown samples.

Laboratory

I, 11, 111, IV, V, VI, VII, VIII, IX, XI I, 11, 111, V, VI, VII, IX, XI

IV,VII, X I, VI, IX VIII I1

(Gn)

b. Tests for anti-HLA antibodies Lymphocytotoxicity test (LCT) Lymphocyte immunofluorescence test (LIFT) Immune phagocytosis inhibition (IPI)

I, 11,111, IV, v , VI, VII, IX, x I, VIII, IX, XI I1

c. Tests used to distinguish between granulocyte-specific and anti-HLA antibodies Platelet absorption used in conjunction with the above techniques Platelet elution used in conjunction with the above techniques Chloroquine-treated granulocytes in GIFT

I, 11, V, VII, VIII, XI

VIII

IX

d . Other tests Monocyte immunofluorescence test Anti-neutrophil cytoplasmic antigen ELISA

I I

Participants in the workshop were asked to detect any granulocytereactive antibodies present and to identify the specificity(ies), titre and immunoglobulin class of these antibodies. Workshop Samples A description of the workshop samples (part 11) based on information provided by the submitting laboratories is presented in table 2. AB serum and plasma samples consisted of pools from 2 nontransfused male AB donors. Each individual donation was found not to contain granulocyte- or lymphocyte-reactive antibodies as determined by granulocyte immunofluorescence test (GIFT) [4], granulocyte chemiluminescence (GCLT) test [ 5 ] , lymphocyte immunofluorescence test [6] and lymphocytotoxicity [7].The serum pool was

Granulocyte Serology Workshop

253

Table 5. Phenotyped granulocytes used in the 1989 workshop

Table 4. Granulocyte isolation procedures and treatments Laboratory

Test

Anticoagulant

Isolation procedure and treatments ~

gradient

I

I1

111

IV V VI

VII

VIII IX

X XI

GIFT GAT FACS GCT LAT GCLT MeCel FH DFH a b

GIFT GAT FACS GIFT GAT GCT GIFT GAT GIFT LAT GIFT GAT GIFT GAT FACS GIFT GAT LAT GIFT GCLT GIFT GAT FACS LAT GIFT GAT = = = = =

= =

C

= = = = =

d

=

HT F SP

= = =

EDTA EDTA EDTA heparin EDTA heparin EDTA EDTA EDTA EDTA heparin heparin EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA EDTA

MeCel MeCel MeCel dextran dextran dextran plasmagel plasmagel dextran dextran MeCel MeCel dextran dextran dextran dextran dextran dextran -

dextran dextran dextran dextran MeCel MeCel

RBC PFA lysis FH

FH FH FH DFH FH FH FH FH DFH DFH FH FH FH FH FH

+a +a +a +a -

?

+c +c +a -

-

+a +a +a +c +c

other

+

+b

111

-

-

-

-

IV V VI VII VIII IX X XI

+ + +

-

-

-

-

-

+

+

-

+ -

+ +

-

-

-

-

-

-

-

DFH DFH DFH DFH DFH

+d +d

-

HT HT

-

+

-

-

+

-

-

-

DFH DFH

-

+ +

-

NA1, NA2, NB1, NBl (-), NDl, 9a (LAT), FcRIII (-) NA1, NA2, NB1, NB2,5b NA1, NA2, NB1,5b NA1, NA2, NB1, NC1, ND1,9a NA1, NA2, NB1, NB2, NCl, 5a, 5b, 9a, MART NA1, NA2, NB1, NB2, NC1 NA1,5b NA1, NA2, NB1, 5b NA1, NA2, NB1, NB2, LAN Typed panel not available NA1, NA2, NBI, NB2,9a, MART

-

-

-

Granulocyte antigens covered in panel

~

I I1

-

-

Laboratory

-

-

FISP -

Granulocyte immunofluorescence test Granulocyte agglutination test Fluorescence-activated cell sorterlcounter Granulocyte complement fixation test Leucocyte agglutination test Granulocyte chemiluminescence test Methyl cellulose Ficoll Hypaque Double Ficoll Hypaque Lysis in ammonium chloride solution Cryopreserved monocytes and lymphocytes used Lysis in distilled water Lysis in 1% ammonium oxalate at room temperature for 4 min Heat treatment, 52°C for 2 min Frozen cells used occasionally Solid-phase granulocytes used occasionally

divided into two aliquots, one of which was incubated at 56°C for 30 min. All samples in the workshop were either tested and found to be negative for HBsAg antigen and anti-HIV antibody, or were obtained 'high-risk' from donors who were not in any of the currently- recognised groups associated with these infections.

Ro-millilitre aliquots of the workshop samples were packed in dry ice and dispatched to each laboratory. In all cases, the samples were frozen on arrival. Each laboratory was supplied with a response sheet in order to unify the method of reporting the results.

Tests for Granulocyte Antibodies Most laboratories used either the GIFT andor the granulocyte micro-agglutination test (GAT) (10 and 8 laboratories, respectively). Four other techniques were also used: leucocyte agglutination (LAT), flow cytometry (FACS), GCLT and granulocytotoxicity (GCT) (table 3a). All laboratories using the GIFT or FACS used F(ab), fragments of FITC conjugated anti-immunoglobins except laboratory VI (GIFT) and laboratory VII, which used whole antibody preparations. Anti-HLA antibodies were detected using lymphocytotoxicity a n d or lymphocyte immunofluorescence (9 and 4 laboratories, respectively). One laboratory also used immune phagocytosis inhibition [8] (table 3b). Distinction between granulocyte-specific and anti-HLA antibodies was achieved by platelet absorption or by the use of chloroquine-treated granulocytes (table 3c). Other techniques used to screen the samples for granulocyte-reactive antibodies are presented in table 3d. Granulocyte Isolation and Phenotypes The techniques used to isolate granulocytes from whole blood and any subsequent treatments of the isolated granulocytes are summarised in table 4. Seven laboratories used hypo-osmolar solutions to remove red cells from enriched granulocyte preparations. All laboratories used paraformaldehyde (PFA) - treated granulocytes in the GIFT except laboratory VIII, which used granulocytes heat-treated at 52 "C for 2 min. The panels of phenotyped granulocytes which were available to participants are presented in table 5.

Results The conslusions drawn by the 11participant laboratories as to the antibody Of the workshop are presented below (see also table 6).

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Lucas/Carrington

Table 6. Conclusions of laboratories Laboratory

I

Sample A NEG

B aNA2

C aNBl

D aHLA multispecific

E NEG

F aHLA B12

*

GS

*

*

*

*

G a5b

UA HLA

H aNA2

I aNAl

J NEG

K aNA2 + HLA

L aHLA multispecific

*

*

HLA

aNDl

*

* *

(?I * *

*

UA

* * *

*

*

*

aNA2 no HLA

UA no HLA

+

*

* *

* * *

NEG

*

*

*

*

GS

HLA

HLA

UA no HLA

*

*

VI

* *

*

*

HLA

*

*

*

*

UA no HLA

*

NEG

UA

*

VII

GS

aNAl

aNAl

*

GS

GS + HLA

GS

GS

GS HLA

UA

GS no HLA

*

VIII

*

*

*

*

*

*

*

*

*

*

*

IX

*

NEG

GS

*

*

HLA

GS

GS

*

*

GS + HLA

*

X

*

UA

UA

*

*

*

UA

GS

XI

*

*

*

a5B or GS + HLA

aNA2 + HLA

I1 111

IV V

GS UA HLA NEG

*

=

*

*

*

* *

GS

*

* *

*

NEG

a5B or GS + HLA

*

* * aNAl no HLA

* aNAl no HLA

+

UA HLA

*

*

+

*

*

UA + HLA GS HLA

* *

+

Granulocyte-specific;

= Unidentified antibody; = Anti-HLA antibody; = =

Negative. Conclusion in accord with description given by the submitting laboratory or (for sample B) in accord with the consensus.

Inert Samples Samples A , E, J from non-transfused AB male donors were unreactive on pre-trial testing and were generally found by the participants to be unreactive. However, laboratory VII obtained positive reactions with all samples and laboratories I and IV reported anti-HLA antibodies in samples J and E, respectively. Samples Containing Granulocyte-Specific Antibodies Only Sample B, submitted as granulocyte-reactive (specificity unknown) but LCT-negative. Anti-NA2 (6 laboratories),

granulocyte-specific (2), anti-NAl (l), unidentified antibody (1) and negative (1). Sample C, submitted as anti-NB1. Anti-NBl(8 laboratories), anti-NAl (l), granulocyte-specific (1) and unidentified antibody (1). Sample G, submitted as an antiJb. Anti-Sb (3 laboratories), unidentified antibody (3), anti-5b or granulocytespecific plus anti-HLA (2), granulocyte-specific (2) and unidentified plus anti-HLA (1). Sample H, submitted as an anti-NA2 only. Anti-NA2 (6 laboratories), granulocyte-specific (3), anti-NA2 plus anti-HLA (1) and granulocyte-specific plus anti-HLA (1).

Granulocyte Serology Workshop

Samples Containing Granulocyte-Specific and Anti-HLA Antibodies Sample I, submitted as an anti-NAl plus anti-HLA. Anti-NAl plus anti-HLA (7 laboratories), anti-NAl only (2), granulocyte-specificplus anti-HLA (1) and unidentified antibody plus anti-HLA (1). Sample K , submitted as anti-NA2 plus anti-HLA. AntiNA2 plus anti-HLA (4 laboratories), granulocyte-specific plus anti-HLA (2), anti-NA2 only (l), granulocyte-specific - no anti-HLA (l), unidentified antibody (l), unidentified antibody plus anti-HLA (1) and anti-ND1 plus anti-HLA (1)* Samples Containing Anti-HLA Antibodies Only Sample D, submitted as a multispecific anti-HLA. AntiHLA only (10 laboratories) and unidentified antibody - no HLA (1). Sample F, submitted as an anti-HLA-B12. Anti-HLABE! (4 laboratories), anti-HLA (3), granulocyte-specific plus anti-HLA (1) and negative (3). Sample L, identical to D. Anti-HLA only (10laboratories) and unidentified antibody - no HLA (1, same laboratory as in D).

Discussion This workshop was not intended to provide a comprehensive survey of all laboratories undertaking granulocyte serology but probably provides the best indication so far of the current status of granulocyte serological testing. The specific aims of the workshop were to determine the ability of laboratories to detect and identify granulocyte-reactive antibodies and to evaluate the methods for distinguishing between granulocyte-specific and anti-HLA antibodies. The results enable a number of conclusions to be made. The GIFT and GAT were the most widely used techniques and, generally, gave consistent answers both between the two techniques and between different laboratories. The LAT was of limited value, and given that the GAT requires little additional effort this latter test should be performed in preference. Some of the conclusions given by laboratories IV,VII, X (table 6,) may reflect the limitations of the LAT Many laboratories also used additional techniques to detect granulocyte-reactive antibodies, i.e. FACS, GCT and GCLT (table 3a). These techniques usually gave results consistent with the GIFT andor GAT. However, the merits of FACS, GCT and GCLT compared to the established GIFT and GAT procedures cannot be determined because the relative importance given to the results of different techniques was not

255

indicated. The use of hypo-osmolar solutions to lyse erythrocytes contaminating the granulocyte preparations did not appear to impair assay performance. Similarly,while PFA treatment of granulocytes for use in the GIFT was undertaken by 8 out of 9 laboratories, the substitution of heat treatment for PFA treatment did not appear to influence the results of laboratory VIII. The incidence of false-positive results, i. e. antibodies detected in inert sera, was 5/33 (15%); three of these reactions were attributable to one laboratory. This finding is remarkable given that background fluorescence in the GIFT is a recognised problem (some laboratories recorded ‘l+’reactions with ‘in-house’ negative control sera) and is lower than the incidence of false-positive results found in some platelet serology workshops [9,10]. The incidence of false-negative results, i. e. antibodies not detected in sera containing granulocyte-specific, anti-5b or anti-HLA antibodies was also low 4/99 (4%). The specificity of the granulocyte-specific and anti-5b antibodies was correctly assigned on only 38/66 (57%) occasions (table 6). A considerable proportion of the errors were attributed to results obtained with serum G which contained anti-5b antibodies. The antigens of the 5 system [ll] are present on granulocytes, platelets, lymphocytes and other cells. Consequently, in the absence of cells typed for antigens of the 5 system, it is difficult to distinguish between sera containing anti-5d5b antibodies and sera containing granulocyte-specific plus anti-HLA antibodies. If the results for sample G and for laboratories VII, IX, X are excluded, the proportion of correct identifications increases to 34/40 (85Y0).The ability to detect antibodies is a reflection of technical performance, whereas the ability to correctly identify granulocyte-specific antibodies also reflects the size and scope of the available granulocyte panel (table 4). Laboratories I1 and V identified all samples correctly with the exception of sample F (anti-HLA-Bl2). The overall trend suggests that those laboratories providing a centralised reference service for their country have a greater ability to identify the specificity of granulocyte-reactiveantibodies. Anti-HLA antibodies were rarely mistaken for granulocyte - specific antibodies (2133 occasions), although the detection of weakly reactive HLA-specific antibodies (sample F) proved more difficult. Sera containing both granulocyte-specific and anti-HLA antibodies (samples I and K) were investigated by 6 laboratories using a platelet absorption technique and by 1laboratory using chloroquine-treated granulocytes in conjunction with the GIFT (table 3c). In most cases platelet absorption enabled the laboratories to make correct conclusions about the antibody composition of the sera. However, it was noted that absorption of sera

256

with platelets from random donors occasionally failed to remove all anti-HLA antibodies. A possible solution to this problem is to absorb the test sera with platelets or lymphocytes isolated from the blood of the granulocyte donor(s) to ensure the presence of the appropriate HLA antigens. No conclusion can be drawn regarding the benefit of chloroquine treatment from this study. The main problem identified in this workshop was not the occurrence of false-negative/positive results or confusion caused by anti-HLA antibodies, but rather the correct identification of granulocyte-specific antibodies. This problem may have increasing clinical relevance with respect to febrile and pulmonary transfusion reactions. Febrile reactions are best managed with leucocyte-depleted blood products, but there may be occasions when the identification of specific antibodies (often anti5b) [Carrington, unpublished observations] and the use of products lacking that antigen may be clinically valuable. In pulmonary transfusion reactions, there is a stronger case for detecting granulocyte-specific antibodies. These reactions are due to the passive transfer of granulocyte-specific antibodies in the donor plasma [12-141 and may be apparent after transfusion of as little as 25 ml plasma [15]. At present, the frequency of pulmonary transfusion reactions is probably underestimated as the condition may often be ascribed to other causes, e. g. fluid overload or shock lung associated with trauma. These reactions could be prevented by screening donors for granulocyte-specific antibodies. Such screening would pose severe logistical problems but may become necessary with increasing recognition of the clinical problem and the demand for safer blood products. In conclusion, the overall standard of granulocyte antibody testing in most laboratories in this workshop was very high. The main problem area was the correct identification of granulocyte-specific and anti-5b antibodies. Increased co-operation between between laboratories at national and international level either through future workshops and/or exchange of resources should lead to improved antibody identification and an increased awareness of the clinical importance of granulocyte-specific antibodies.

LucasKarrington

ance in the pre-workshop testing of the samples. This study was supported by the Central Blood Laboratories Authority, Elstree.

References 1 Lalezari P, Nussbaum M, Gelman S, et al: Neonatal neutropenia due to maternal isoimmunization. Blood 1960;15:236-244. 2 McCullough J: Granulocyte antigen systems and antibodies and their clinical significance. Hum Pathol 1983;14:228-234. 3 Clay ME, Kline WE: Detection of granulocyte antigens and antibodies: Current perspectives and approaches; in Garraty G (ed): Transfusion Therapy. Arlington, American Association of Blood Banks, 1985, pp 183-266. 4 Verheught FWA, von dem Borne AEGKr, DCcary F, et al: The detection of granulocyte alloantibodies with an indirect immunofluorescence test. Br J Haematol 1977;36:533-544. 5 Hadley AG, Holburn AM: The detection of anti-granulocyte antibodies by chemiluminescence. Clin Lab Haematol1984;6:351-361. 6 DCcary F, Vermeulen A, Engelfriet CP: A look at H L A antisera in the indirect immunofluorescence technique (IIFT); in Amos DB, van Rood JJ (ed): Histocompatibility Testing. Copenhagen, Munksgaard, 1975, pp380-390. 7 Terasaki PI, McClelland JD: Microdroplet assay of human cytotoxins. Nature 1964;204:998-1000. 8 Neppert J, Pohl E, Mueller-Eckhardt C: Inhibition of immune phagocytosis by human sera with HLA A, B, C and DR but not with DQ or EM type reactivity. Vox Sang 1986;51:122-126. 9 DCcary F: Report of the second Canadian workshop on platelet serology; in DBcary F, Rock G (ed): Current Studies in Hematology and Blood Transfusion, Basel, Karger, 1988, vol 54: The Platelet Membrane in Transfusion, pp 1-12. 10 Lucas GF, Holburn AM, Saunders PWG, et al: A survey of antiplatelet antibody detection: Report of the UK platelet and granulocyte serology working group, 1985. Clin Lab Haematol1987;9:147155. 11 van Leeuwen A, Eeernise JG, van Rood JJ: A new leucocyte group with two alleles: Leucocyte group five. Vox Sang 1964;9:431446. 12 Yomtovian R, Kline W, Press C, et al: Severe pulmonary hypersensitivity associated with passive transfusion of a neutrophil-specific antibody. Lancet 1984;i:244-245. 13 Popovsky MA, Moore SB: Diagnostic and pathogenetic considerations in transfusion-related acute lung injury. Transfusion 1985;25:573-577. 14 Nordhagen R, Conradi M, Dromtrop SM: Pulmonary reaction associated with transfusion of plasma containing anti-5b. Vox Sang 1986;51:102-107. 15 Brittingham TE: Immunologic studies on leukocytes. Vox Sang 1957;2:242-248.

Acknowledgements We are grateful to the following for supplying additional antisera: Dr. M. Bhavnani (Wigan), Dr. J. Cartron (Necker Hospital, Paris), Mrs. C. Sullivan (American Red Cross, Minneapolis), Mr. J. Davis (Oxford RTC), Dr. W. Ouwehand (Central Laboratories of the Netherlands Red Cross, Amsterdam) and Mr. E Sherwood (South London RTC). Thanks also to Mr. J. Davis for screening the samples for antiHLA antibodies by LCT and to Mr. N. W. Amphlett (BGRL.) for assist-

Received: February 22,1990 Accepted: April 2, 1990

G. E Lucas, PhD Blood Group Reference Laboratory South Western Regional Blood Transfusion Centre Southmead Road Bristol BSlO 5ND (UK)