- Email: [email protected]
Anticardiolipin kits: Techniques of antiphospholipid antibody measurement
Vol. 11, No. 3, 1991
5.
6.
7.
8.
9.
10.
11.
12.
Authors reply. Clin Exp Rheumatol 8:614-615, 1990. Galli M, Comfurius P, Maassen C, et al.: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 335:1544-1547, 1990. Harris EN, Gharavi AE, Boey ML, et al.: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosis. Lancet ii:1211-1214, 1983. Harris EN, Gharavi AE, Wasley GD, et al.: Use of an enzyme-linked immunosorbent assay and inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J Infect Dis 157:23-31, 1988. Hunt JE, McNeil HP, Krilis SA: Anticardiolipin antibodies from autoimmune disease and infection have different binding specificities. FASEB (in press), 1991. Hunt JE, McNeil HP, Crameri R, et al.: A phospholipid I complex is an antigen for anticardiolipin antibodies occurring in autoimmune disease but not with infection (submitted). Keisey PR, Stevenson KJ, Poller L: The diagnosis of lupus anticoagulants by the activated partial thromboplastin time: the central role of phosphatidylserine. Thromb Haemost 52:172-175, 1984. Koppe AL, Walter H, Chopra VP, Bajatzadehm: Investigations on the genetics and population genetics of the 132-glycoprotein I polymorphism. Humangenetik 9:164171, 1970. Matsuurd E, Igarashi Y, Fujimoto M, et al.: Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease.
CLINICAL IMMUNOLOGY
Lancet 336:177-178, 1990. 13. McNeil HP, Krilis SA, Chesterman CN: Purification of antiphospholipid antibodies using a new affinity method. Thromb Res 52:641-648, 1988. 14. McNeil HP, Chesterman CN, Krilis SA: Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br J Haematol 73:506-513, 1989. 15. McNeil HP, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen which includes a lipid-binding plasma glycoprotein. Clin Exp Rheumatol 8:208, 1990. 16. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: 132glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 87:4120-4124, 1990. 17. McNeil HP, Chesterman CN, Krilis SA: Immunology and clinical importance of antiphospholipid antibodies. Adv Immunol 49:(in press), 1991. 18. McNeil HP, Shi W, Chesterman CN, Krilis SA: Elevated plasma 132-glycoprotein I levels in patients with anticardiolipin antibodies (submitted). 19. Moore JE, Mohr CF: Biologically false positive serologic tests for syphilis. JAMA 150:467-473, 1952. 20. Nimpf J, Wurm H, Kostner GM: 132-glycoprotein I (apo H) inhibits the release reaction of human platelets during ADP-induced aggregation. Atherosclerosis 63:109-114, 1987.
Newsletter 41
21. Pangborn MC: A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med 48:484-486, 1941. 22. Pengo V, Thiagarajan P, Shapiro SS, et al.: Immunological specificity and mechanism of action of IgG lupus anticoagulants. Blood 70:69-76, 1987. 23. Polz E, Kostner GM: The binding of 132glycoprotein I to human serum lipoproteins. FEBS Lett 102:183-186, 1979. 24. Ranch J, Meng Q-H, Tannenbaum H: Lupus anticoagulant and anti-platelet properties of human hybridoma autoantibodies. J Immunol 139:2598-2604, 1987. 25. Ranch J, Tannenbaum M, Janoff AS: Distinguishing plasma lupus anticoagulants from anti-factor antibodies using hexagonal (II) phase phospholipids. Thromb Haemost 62:892-896, 1989. 26. Reid KBM, Day AJ: Stucture-function relationships of the complement components. Immunol Today 10:177-180, 1989. 27. Schousboe I: Binding of 132-glycoprotein I to platelets: effect on adenylate cyclase activity. Throm Res 19:225-237, 1980. 28. Schousboe I: 132-glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 66:1086-1091, 1985. 29. Schultze HE, Heide H, Haupt H: Uber ein bisher unbekanntes niedermolekulares 132globulin des human-serums. Naturwissenschafien 48:719, 1961. 30. Staub HL, Khamashta MA, Harris EN, et al.: Lupus anticoagulant and anticardiolipin antibodies: the affinities may differ. Postgrad Med J 65:700, 1989. 31. Tilcock CPS: Lipid polymorphism. Chem Phys Lipids 40:109-125, 1986.
A n t i c a r d i o l i p i n Kits: T e c h n i q u e s o f Antiphospholipid Antibody Measurement Sozos Loizou Rheumatology Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
he first specific solid-phase immunoassay for the measurement of anticardiolipin (aCL) antibodies was the radioimmunoassay (RIA) developed in 1983 by E. N. Harris and colleagues. 5 Since then a large number of immunoassays, mainly ELISAs, have been published for the estimation o f aCL or antiphospholipid antibodies (aPL) in patients' serum or plasma (Tables 1, 2). All
T
ELISA assays for antiphospholipid antibody measurement use purified cardiolipin or a mixture of negatively charged phospholipids as the antigen for coating microtiter plate wells. Antiphospholipid antibodies present in serum or plasma are then allowed to react and bind to the antigen coated onto the plate wells, and the amount of bound antibody is measured by the use of enzyme-conjugated anti-human © 1991 Elsevier Science Publishing Co., Inc.
antibodies o f the appropriate isotype; the amount of antiphospholipid antibody present is measured by the intensity of color produced when a chromogenic substrate is added to the above complex, as shown in the generalized aPL ELISA shown in Figure 1. Antiphospholipid antibody assays are clinically important because of the numerous clinical complications that have 019%1859/91/$0.00 + 2.20
42 C L I N I C A L I M M U N O L O G Y Newsletter
Vol. 11, No. 3, 1991
TABLE 1. PUBLISHED SERIES OF aCL PREVALENCE IN SLE PATIENTS
Reference
Method
No. of Controls
Harris et al., 1983 Koike et al., 1984 Colaco and Male, 1985 Tincani et al., 1985 Meyer et al., 1985 Norberg et al., 1985 Loizou et al., 1985 Manoussakis et al., 1987 Kalunian et al., 1988 Mouritsen et al., 1989
RIA ELISA RIA RIA ELISA ELISA ELISA ELISA ELISA ELISA
30 11 23 40 30 156 135 276 40 100
Positivity Cutoff SD > Mean
No.
% + ve
2.76 a 2.0 3.0 2.0 b 5.0 4.0 5.0 c
65 24 87 51 108 127 65 86 85 86
61.0 41.7 83.9 49.0 40.0 37.0 29.2 20.9 42.4 42.0
SLE Patients
SLE = systemic lupus erythematosus. a Not indicated b Ratio of patient/mean control; positive > 1.6. c 99th percentile.
Commercial aCL or aPL Kits Evaluated and Reference Method Used
been associated with raised levels of these antibodies.I° The importance attached to this antibody test is reflected by the large number of commercial kits that are now available, eight by the end of 1990, and two more that will become available during 1991. This review will examine the discrepancies that have been reported in the prevalence of antiphospholipid antibodies, the means available for standardization of these assays, and finally report on an evaluation of commercial antiphospholipid/anticardiolipin antibody kits. Discrepancies in Prevalence of aCL Antibodies and Currently Available Means of Standardization of aCL/aPL Assays Availability of published methods and commercial kits does not necessarily provide accuracy, specificity, and comparability of results from different centers. Hence, the reported prevalence of anticar-
diolipin in the two groups of patients where levels of these antibodies have been estimated in relatively large numbers of patients, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), have shown marked discrepancies, and the reasons for these will be discussed later on in this review (Tables 1, 2). Because of these discrepancies the need for standardization of aCL assays became evident by 1986, and hence a standardization workshop was organized by Dr. E.N. Harris. 7 As a consequence of the results of this workshop, an International Standardization Committee for Antiphospholipid antibodies was formed, and international reference sera for IgG and IgM aCL antibodies were produced and made available to facilitate uniformity in reporting levels of these antibodies in serum or plasma. These reference sera were designated as the KAPS (Kingston Antiphospholipid Study) reference sera, 4 by means
Reference
Method
No. of Controls
Cutoff SD > Mean
No.
Colaco and Male, 1985 Meyer et al., 1985 Loizou et al., 1985 FoIt et al., 1987 Manoussakis et al, 1987 Keane et al., 1987 Mouritsen et al., 1989
RIA ELISA ELISA ELISA ELISA ELISA ELISA
23 30 135 27 276 40 100
2.0 2.0 5.0 3.0 4.0 4.0 a
16 35 47 57 65 90 20
RA Patients % + ve 50.0 6.0 6.4 33.0 7.7 48.9 15.0
RA = rheumatoidarthritis. • 99th percentile.
+ 2.20
Six commercial aCL/aPL antibody kits from five commercial companies were evaluated out of the eight commercial kits that were on the market at the time of this evaluation. The general properties and codes of each kit used are given in Table 4. All six kits evaluated used an ELISA method for aPL antibody measurement, and the kits were obtained directly from the manufacturers, who had previously been informed about the aim of the study. Each kit was used exactly in accordance with the manufacturers' instructions. Details of the method used and general characteristics for each kit are given in Table 5. For comparative reasons, the reference method used was the quantitative lgG and IgM aCL antibody ELISA assay (HH) that has been routinely used at Hammersmith Hospital for over six years, 8 and that was also one of the methods used for the First Antiphospholipid Antibody Standardization Workshop. 7 Patient and Normal Control Sera Used in the Evaluation
T A B L E 2. P U B L I S H E D SERIES O F aCL P R E V A L E N C E IN RA P A T I E N T S
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of which results can be reported in International GPL and MPL units for lgG and IgM aCL antibodies, respectively (Table 3). No international reference sera or units are as yet available for reporting lgA aPL antibody levels. For the reasons given above, the need also emerged for the evaluation of commercial anticardiolipin/antiphospholipid antibody kits, and such an evaluation was undertaken as part of the antiphospholipid antibody standardization workshop for the 4th International Antiphospholipid Antibody Symposium in Sirmione (Lake Garda), Italy in April 1990, where these results were first presented.
I © 1991 Elsevier Science Publishing Co., Inc.
Anticardiolipin or antiphospholipid antibodies were estimated by both the reference method and the six commercial kits in 10 normal human sera from healthy laboratory personnel, and in 30 patient sera. The patient sera used were selected on the basis that they covered a very wide range of IgG and IgM aCL antibody levels, from negative to very high positive; the aCL levels in the 30 patient sera were ascertained by the reference method
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Vol. 11, No. 3, 1991
CARDIOLIPIN - antigen dried onto polystyrene elate wells
(HH) in triplicate on three different occasions, and the mean aCL levels in anticardiolipin ELISA units (AEU), for IgG and IgM were taken as the target values for the kit evaluation (1 AEU = - 1 GPL/or 1 MPL, see Table 3). Prior to the evaluation study, all patient, normal control, and KAPS reference sera were subaliquoted into 50 Ixl aliquots and stored frozen at - 2 0 ° C until needed for use, when a set of all the sera was thawed out for use prior to each assay for each kit.
Plates blocked with buffer/FCS.ABS or BSA solution (1 hour) wLh Aoorooriatelv diluted samples and controls added (3 hours)
Assessment of Results
w,Lh Enzyme * con!uaated anti-human antibody added-loG.IoM or leA (90 min) wLh Aoorooriate chromooenic substrate added / Colour reaction stonoea~with aPPropriate solution (stop when top standard OA = 0.8 -0.9)
1 Read ootical absorbance (O~A~at appropriate wavelenoth
* alkaline phosphatase or horse radish peroxidase conjugate ( ) brackets indicate KAPS reccomendations for aCL ELISAs FCS = fetal calf serum: ABS=adult bovine serum; BSA= bovine serum albumin
Figure 1. A generalized flow diagram for anticardiolipin antibody determination by ELISA.
TABLE 3. COMPARISON OF aCL ANTIBODY LEVELS FOR IgG AND IgM KAPS STANDARDS GIVEN BY THE REFERENCE METHOD AND COMMERCIAL APL ANTIBODY KITS KAPS
International
HH
Sample
G/MPL
AEU
GI G2 G3 Normal M1 M2 M3 Normal
107.0 ± 19.6 ± 5.8 ± NG 106.0 ± 21.0 ± 5.4 ± NG
16.4 4.8 3.8 19.3 3.8 3.3
92.9 16.5 4.4 <9.0 92.5 26.0 6.6 <8.0
± 16.4 ± 1.9 ± 0.8 ± 14 ± 4.2 ± 1.5
MI ~/ml
CD G/MPL
WL G/MPL
ST G/MPL
25.8 6.3 3.5 <2.0 44.0 14.7 4.2 <2.0
84.0 14.2 8.9 < 10.0 84.4 23.7 8.3 <9.0
85.2 15.8 9.0 <9.0 63,2 32.4 27.4 <4.0
93.3 14.6 8.1 < 5.0 >100 43.2 16.9 <3.0
HH = Hammersmith Hospital; AEU = anticardiolipin ELISA units; NG = not given; other abbreviations as per Table 4. I
i
© 1991 Elsevier Science Publishing Co., Inc.
Results of aCL or aPL levels in the patient and control sera were assessed using optical absorbance (OA) readings and the KAPS reference sera for comparison of results between the reference method (HH) and all the kits studied (qualitative ST/ GMA, BL/GMA; quantitative MI, CD, WL; semiquantitative ST/G, ST/M, Table 4). Quantitative comparison of results could only be performed between HH and the quantitative kits MI, CD, and WL, which the manufacturers had stated were standardized against the KAPS standards (see Table 3 for IgG and IgM KAPS results). Table 6 shows the Spearman Rank correlations obtained for the 30 patient sera between the OAs of all the assays evaluated. Strong correlations (>0.800) were obtained for all the IgG specific kits. For IgM aCL antibodies, strong correlations (>0.800) were only seen between the HH method and the CD IgM kit. Two of the kits, ST/GMA and BL/GMA, were stated to be qualitative screening kits for a combination of three aPL isotypes IgG, IgM, and IgA (GMA kits); for these two kits, the OA obtained by the reference method (HH) for either IgG or IgM (whichever was the highest) was compared to that obtained by the two combination GMA kits; a strong correlation was obtained with the ST/GMA kit, but the correlation between both HH and ST/GMA and the BL/GMA kit was very Ix)or (Table 6). The detection limits for each kit and the reference method could only be assessed for the quantitative assays, and these were estimated by a graphical method as follows. The mean level of each individual kit or the reference method for each patient serum was plotted together with 0197-1859/91/$0.00 + 2.20
44 CLINICAL
IMMUNOLOGY
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V o l . 1 1, N o . 3, 1991
ii
TABLE 4. PROPERTIES OF COMMERCIAL ANTICARDIOLIPIN ANTIBODY KITS Name of Kit and Company
Code
1. aCL antibody kit, Medical Innovations LTD 2. QACA, Cheshire Diagnostics Ltd. 3. MELISA aCL antibody kit Walker Laboratories Ltd. 4. Asserachrom APA Diagnostica Stago 5. MALAKIT Cardiolipin Biolab, sa, nv 6. SYNELISA aCL kit ELIAS Medizintechnic GmbH 7. SLOR El aCL kit BioHyTech, Israel 8. REAADS aCL kit Biostar Medical Products Inc.
Units
lsotype of aCL
Antigen
MI CD
Units/mL G/MPL
CL CL
lgG or IgM IgG or lgM
WL ST BL
G/MPL G/MPL and SQ QL
CL PL mixture CL
IgG or IgM lgG or IgM G/M/A G/M/A
EL~
QU
CL
lgG or IgM
BHT~ BSa
SQ SQ
CL CL
lgG or IgM IgG or lgM
SQ = semi-quantitative;QL = qualitative;QU = quantitative. • Kits 6. 7, and 8 were not evaluated. I
I
TABLE 5. CHARACTERISTICS OF DIFFERENT ANTIPHOSPHOLIPID ANTIBODY KIT METHODS Kit Code
MI
Proprietary name Instructions Assay procedure Sample dilution
aCL Ab. Good Easy 1:20 on plate 50 Ixl 30 rain Peroxidase 50 ~l 45 m 50 Ixl 15 m Fast Good >2.0
Sample volume increase time Conjugate Volume Increase time Substitute volume Increase time Color development Duplicate agreement Top standard (OA)
CD
WL
ST
BL
QACA Very good Easy 1:100
(MELISA) Good Complex 1:100
Asserachrom Good Easy 1:101
Malakit Complex Easy 1:101
100 I~1 30 min Alkaline phosphate 100 pJ 30 m 100 p.1 " 30 m Good Good <2.0
100 txl 15 min Peroxidase 100 ~1 15 m 200 p.l 10 m Fast Good >3.0
200 Ixl 30 min Peroxidase 200 ill 30 m 200 p,l 6m Fast Good <0.7
100 ~.1 1 hr Peroxidase 100 ixl 1 hr 100 I~1 30 m Slow Variable <1.2
OA = optical absorbance; other abbreviationsas per Table 4.
the m e a n values o f all the methods for each s e r u m in G P L or M P L . For each m e t h o d being investigated, a line can be d r a w n c o n n e c t i n g all values specific for the particular method. If the graph for an individual m e t h o d runs parallel and very near a b o v e or b e l o w the m e a n line, then the kit or m e t h o d can be regarded as g o o d and reproducible. Figure 2 illustrates the procedure for IgG aCL. P e r f o r m a n c e for negative to intermediate positive values ( < 7 5 G P L ) was g o o d for H H , C D , and W L kits, but rather p o o r for the MI kit. At higher a C L levels ( > 7 5 G P L ) , the reference method H H p e r f o r m e d best as very high levels o f lgG a C L c o u l d o n l y be m e a s u r e d by this m e t h o d (highest standard, 270 G P L ) , in contrast to the various kits, C D kit (highest standard, 160 G P L ) , W L kit (highest standard, 100 G P L ) , and MI kit (highest standard, 64 units). T h e results for I g M a C L are s h o w n in Figure 3, w h e r e g o o d a g r e e m e n t is s h o w n for n e g a t i v e to intermediate positive values ( < 5 0 M P L ) with the reference m e t h o d H H (highest standard, 150 M P L ) and the C D kit (highest standard, 120 M P L ) . The two r e m a i n i n g kits, the MI kit (highest standard, 64 units) tended to g i v e lower levels than the mean, and the W L kit (highest standard, 60 M P L ) tended to give higher results than the m e a n values. At highpositive l g M a C L levels ( > 5 0 M P L ) , the H H m e t h o d again p e r f o r m e d better than the kits. Assessment of aCL/aPL Antibody L e v e l s in A c c o r d a n c e w i t h t h e KAPS International Standardization Workshop 6
I
TABLE 6. SPEARMAN RANK CORRELATIONS BETWEEN OPTICAL ABSORBANCES OBTAINED BY THE DIFFERENT METHODS AND KITS ASSESSED IN 30 PATIENT SERA.
Kit/Method
HH MI CD WL ST ST/GMA
lgG aCL Antibodies HH MI CD WL
. 0.907 0.948 0.978 0.882 .
.
. . --0.902 -0.948 0.946 0.907 0.948 . . .
IgM aCL Antibodies HH MI CD WL
. ---0.978 .
. . . 0.752 -0.922 0.718 0.778 0.582 0.702 0.654 . . .
--0.704 0.645 .
---0.631
GMA aCL Antibodies ST BL
0.909 a ND ND ND ND
0.164" ND ND ND ND 0.014
ND = not done. • For comparisonof GMA kits to HH, lgG, or IgM OA, whicheverwas the highest for the HH method was used. i
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i i iiii © 1991 Elsevier Science Publishing Co., Inc.
The K A P S reference sera offer a m e a n s for standardizing the way in which results of a C L antibody levels are reported, and enables valid c o m p a r i s o n s b e t w e e n laboratories o f the prevalence and relationship b e t w e e n these antibodies and the numerous associated clinical c o m p l i c a t i o n s . I ° A c c o r d i n g to the r e c o m m e n d a t i o n s of the 2nd International Standardization W o r k s h o p , 13 using the K A P S reference sera ( G I , G2, G3 for IgG and M I , M 2 , M3 for I g M aCL) for reporting results, a serum sample is reported as high positive for l g G a C L if its O A reading is greater iii iiiii
C L I N I C A L I M M U N O L O G Y Newsletter 45
Vol. 11, No. 3, 1991
300 ----
mean of all
J.
mean of HH
250
mean of MI mean of CD mean of WL
200 c.._1 0
150
100
50
l
"
I
0
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2
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8
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10 12 14 16 18 20 22 24 26 28 30
Serum number Figure 2. Comparison of mean IgG aCL levels for HH and the three quantitative kits MI, CD, and WL in 30 patient sera with the mean levels of all four methods in the same sera. The serum number refers to the 30 patient sera that have been arranged consecutively in order of increasing average levels of all four methods. At very high levels >200 units, the result of the HH is shown as the mean as the levels measured were more than twice those of the top standard for each of the kits (serum numbers 27-30).
Figure 3. Comparison of mean IgM aCL levels for HH and the three quantitative kits MI, CD, and WL in 30 patient sera with mean levels of all four methods in the same sera. The serum number refers to the 30 patient sera that have been arranged consecutively in order of increasing average levels of all four methods. For very high levels, results that were <50% of the HH value (serum numbers 26-30) were omitted from the calculation of the mean of all four methods. 120
¢ •¢
100
~.
mean of all mean of HH mean of MI mean of CD mean of WL
80
60
4o
I
0
"
I
2
•
I
4
"
I
6
"
I
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8 10 12 14 16 18 20 22 24 26 28 30
Serum number © 1991 ElsevierScience Publishing Co., Inc.
than the OA reading of the reference serum G1; medium or moderate positive when its OA lies between G1 and G2, low positive if its OA reading is between G2 and G3, and negative if its OA reading is less than G3. For IgM aCL, serum samples are reported in a similar way, where samples are reported as high positive if their OA is greater than that of the KAPS reference serum M 1, medium or moderate positive if the OA reading lies between M1 and M2, low positive when the OA is between M2 and M3, and negative when the OA reading is less than that for M3. For results of the established values for IgG and IgM aCL for the KAPS reference sera in international units GPL or MPL see Table 3. Results for all the commercial aCL kits evaluated and analyzed according to the KAPS recommendations for the 30 patient and 10 normal control sera are shown in Table 7 for IgG and in Table 8 for IgM aCL. At the bottom of these tables are shown the differences in total percentage (%) positivity as found in the serum samples studied, using both the KAPS way of reporting results, and also when results were reported according to each method's or kit's own cutoff criteria for positive-negative results. For IgG aCL antibodies, there was generally good agreement for samples reported as high positive, except for the BL/GMA kit where all aCL-positive samples fell in the high-positive category. Some overlap was also seen between the number of patients that fell in the moderate and low-positive categories, which was expected as different methods vary in their specificity, sensitivity, composition, and concentration of the reagents used. The number of negative patient samples also varied, from two reported by HH to eight reported by the MI kit; this finding was also expected as it depends greatly on the definition of the positive/negative cutoff points of the different methods. For example, the KAPS reference sera cutoff point for both IgG and IgM aCL was set at 2 SD above the mean normal control level, whereas for the HH assay the cutoff point for both isotypes was set at 5 SD above the mean normal control level of the assay. Hence, the HH method gave the lowest number of negatives for 0197-1859/91/$0.00 + 2.20
46 C L I N I C A L I M M U N O L O G Y Newsletter
Vol. 11, No. 3, 1991
TABLE 7. IgG aCL ANTIBODIES COMPARISON OF KAPS LEVELS BY THE DIFFERENT METHODS IN 30 PATIENTS (P) AND 10 NORMAL CONTROLS (N) KAPS Level
High + v e Medium + ve Low + ve Negative % positivity KAPS % positivity KIT or HH Method
HH
MI
P
N
8 12 8 2
0 0 1 9
P
CD N
10 11 1 8
WL
P
N
P
0 0 1 9
7 11 9 3
0 0 0 10
5 16 6 3
ST/G N 0 0 1 9
ST
BL
GMA
GMA
P
N
P
N
P
N
8 13 4 5
0 0 0 10
8 12 5 5
0 0 0 10
25 0 0 5
0 0 0 10
93.3
10
73.3
10
90
0
83.3
10
83.3
0
83.3
0
83.3
0
86.7
0
93.3
10
90
0
86.6
10
83.3
0
76.6
0
83.3
0
Abbreviations as per Table 4.
the patient sera (only two), which can be explained in that a number of patient sera, namely those lying between 2 and 5 SD above the normal mean, which would be negative by the HH method, were positive by the KAPS criteria. This also holds when total percentage positivity was compared by the two different methods of reporting results using the two different cutoff points. As for the HH method this was 86.7%, which is lower than the 93.3% positivity that was found when the KAPS criteria were used. These comparisons were also applied to the results of all the commercial kits, for both the patient and normal control sera for both IgG (Table 7) and IgM aCL (Table 8) antibodies. When the same criteria were applied for IgM aCL antibodies, discrepancies were evident in all results except for those in the high-positive category. These dis-
crepancies were more obvious for the number of sera that were found to be negative by the different methods. Here again for the same reasons given above for IgG aCL, the lowest number of negatives was found using the HH method when the KAPS cutoff was used. The number of positive and negative samples by the different kits varied considerably for all the quantitative and semiquantitative kits, and reached 19 negative patient sera for the WL IgM kit. Here again when the total percentage (%) positivity was examined, only the HH and ST/GMA methods gave a lower total % positivity by their own criteria than by those for KAPS. In all the remaining kits, total % positivity by each kit's cutoff was higher than that for the KAPS, and for one kit (WL), positivity using this kit's cutoff was more than twice that found with the KAPS criteria (Table 8). ii
TABLE 8. IgM aCL ANTIBODIES--COMPARISON OF KAPS LEVELS BY THE DIFFERENT METHODS IN 30 PATIENTS (P) AND 10 NORMAL CONTROLS (N) KAPS
HH
MI
CD
WL
ST/M P
N
ST
BL
GMA
G MA
Level
P
N
P
N
P
N
P
N
P
N
P
8 12 5 5
0 0 2 8
25 0 0 5
High + ve Medium + ve Low + ve Negative % positivity KAPS % positivity KIT or HH Method
5 6 11 8
0 0 0 10
5 3 8 14
0 0 0 10
5 2 9 14
0 0 0 10
5 1 5 19
0 0 3 7
4 2 6 18
0 0 1 9
73.3
0
53.3
0
53.3
0
36.6
30
40
10
83.3
20
83.3
10
66.7
0
70,0
0
63.3
0
80
30
50
0
76.6
0
83.3
10
Abbreviations as per Table 4. i
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I II © 1991 Elsevier Science Publishing Co., Inc.
N 0 0 1 9
Future Considerations for Antiphospholipid Antibody Measurements and Standardization of Results Although discrepancies regarding the prevalence of aCL antibodies in patient sera are often reported, the clinical associations of moderate to high levels of these antibodies, especially of the IgG class with clinical complications, such as thrombosis and fetal loss, are now widely accepted. 6 The IgM aCL isotype has also been implicated, but the importance of IgA aCL is still not clear. In order that results from different centers can be compared, and aCL/aPL antibody assays can be standardized, a number of fundamental questions have emerged that need to be answered. As with any other clinical laboratory method, methodological differences exist for aCL assays or commercial aCL kits. Besides these, a number of other factors are very important, such as very wide differences in the determination of the cutoff between positive and negative results, use of limited numbers of normal control populations, selection of patients, and lack of complete uniformity in units of measurement, all factors that invariably prevent an accurate comparison of studies. In order that aCL assays are standardized, some further aspects of aCL ELISA assays need to be addressed, such as the binding capacity of different plastics (microtiter plates), which is known to show marked variations; the use and concentrations of different serum proteins such as bovine serum albumin (BSA), fetal calf serum (FCS), and adult bovine serum (ABS) added to the assay buffers for blocking microtiter plates and for sample and reagent dilution; the nonspecific binding of serum proteins to non-antigencoated platesg; the exact way in which the antigen (cardiolipin, another negatively charged phospholipid or a mixture of phospholipids) is bound to the solid phase, and the possibility of false-positive aCL antibody results that might be caused by the presence of other immunoglobulins, such as the false-positive IgM aCL antibody levels seen in IgG positive sera because of the presence of IgM rheumatoid factors, t,2
C L I N I C A L I M M U N O L O G Y Newsletter 47
Vol. 11, No. 3, 1991
Most research centers using "in-house" aCL assays are now aware of the above problems and take great care in eliminating them from their assays. This task has been facilitated by the Antiphospholipid Antibody Workshops that have led to the creation of international units for aCL measurement, and the provision and availability of reference sera (KAPS) for interlaboratory standardization of reported results, Unfortunately, the same criteria do not seem to be applied to these assays by all the companies that are marketing aCL/aPL antibody kits. For understandable commercial reasons, speed and ease of performance seem to be the most important properties of some kits, at the expense of sensitivity and specificity. The cutoff points between positive-negative levels used by commercial aCL kits also vary considerably, and the way these are defined is not always clearly indicated. Low cutoff points could lead to large numbers of patients with otherwise normal or very low aCL levels being treated unnecessarily, especially as aCL levels that are below 5 SD and above the normal mean of the particular aCL assay, may not be clinically important. 6 The future of the clinical significance of aCL antibodies and the most satisfactory methods for their measurement are still important questions that remain largely unanswered. Recent studies have suggested the existence of a cofactor protein for the binding of aCL antibodies in serum or plasma to the cardiolipin antigen on
microtiter plates, and this cofactor has been characterized and shown to be a serum glycoprotein (132-glycoprotein I), 3a2 and to be able to differentiate between the aPL antibodies found in autoimmune disorders and those of infectious diseases. 1] No doubt as a result of this finding new commercial kits will emerge; it is hoped that both these new and the existing commercial aCL kits will pay a lot of attention in proper validation of their methods on the lines indicated above. Only the emergence of satisfactory standardized methods for aCL/aPL antibody measurement, will be able to answer the very important questions regarding the clinical significance and predictive value of these autoantibodies, c~
References 1. Agopian MS, Boctor FN, Peter JB: False positive test result for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum 31:1212-1213, 1988. 2. Cofiner C, Loizou S, Peter JB, Walport MJ: Effect of IgM rheumatoid factor on IgG and IgM anticardiolipin ELISA assays. Clin Exp Rheumatol 8:(202)abstract 5A, 1990. 3. Galli MD, Moussen C, Comfusius P, et al.: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 335:1544-1547, 1990. 4. Harris EN: Solid-phase anti-cardiolipin test revisited. Am J Med 85:599-601, 1988. 5. Hams EN, Gharavi AE, Boey ML, et al.: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythema-
tosus. Lancet ii:1088-1214, 1983. 6. Harris EN, Chart JKH, Asherson RA, et al.: Thrombosis, recurrent fetal loss, and thrombocytopenia: predictive value of the anticardiolipin antibody test. Arch Intern Med 146:2153-2156, 1986. 7. Harris EN, Gharavi AE, Patel S, Hughes GRV: Evaluation of the anticardiolipin antibody test: report of a standardisation workshop held April 4, 1986. Clin Exp lmmunol 68:215-222, 1987. 8. Loizou S, McCrea JD, Rudge AC, et al.: Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 62:738-745, 1985. 9. Loizou S, Cofiner C, Walport MJ: False positive ELISA tests for IgG and IgM anticardiolipin antibodies, due to non-specific binding to blank microtitre plates. Clin Exp Rheumatol 8:(203)abstract 1la, 1990. 10. Love PE, Santoro SA: Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in Non-SLE disorders. Ann Intern Med 112:682-698, 1990. 11. Matsura E, Igarashi Y, Fujimoto M, et al.: Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease (letter). Lancet ii: 177-178, 1990. 12. McNeil HP, Simpson ILI, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen which includes a lipid binding inhibitor of coagulation: 13-2 glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 87:41204124, 1990. 13. Third International Symposium on AntiPhospholipid Antibodies. Clin Exp Immunol 6:189-215, 1988.
• l a b o r a t o r y T u t i l ~ l for Lupus ~ t i ¢ o ~ l o l l m m t s Douglas A. Triplett
• Quantitative lmmqlllol[hloIt'tKelKt Measurements and Standards: Practical Approaches Howard M. Shapiro • AppUcatfons o f F l o w Cytometry to Solid Tumors John S. Coon and Steven D. Bines • Application o f l ~ M l . l l ~ d .,~say~ far Iglow ~ometry Analysis Thomas M. McHugh and Daniel P. Stites
© 1991 ElsevierScience Publishing Co., Inc.
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48 CLINICAL IMMUNOLOGY Newsletter
Vol. 11, No. 3, 1991
In general, the style adheres to the ASM Style Manual for Journals and Books. As a backup, follow The Chicago Manual of
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Reports documenting interesting case presentations are invited for possible publication in the Clinical Immunology Newsletter. Submitted case reports should be written concisely and should contain: a) a brief clinical history summarizing the symptoms and course of the illness; b) a description of immunologic and other laboratory tests performed; and c)t the results of these laboratory tests as related to the clinical observation of patients and the outcome of the disease process and therapy, if appropriate. References Authors are responsible for the accuracy of references, which should include complete publication information. References should be listed in alphabetical order by the first author's last name, numbered consecutively, and cited in the text by these numbers. Some typical examples of references are listed below. Consult this issue for additional examples of Newsletter reference format. 1. Doone FW, Anderson N: Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. New York, Cambridge University Press, 1987. 2. McCulloch EA: Normal stem cells and the clonal hemopathies. In: Cronkite PE (ed): Hematopoietic Stem Cell Physiology, Vol. 184, New York, Alan R. Liss, Inc., pp. 21-38. 3. Forsgren A, Binder G, Callow F, et al.: Quinolones affect thymidine incorporation in the DNA of human lymphocytes. Amimicrob Agents Chemotber 29:506-508, 1986. Letters Letters expressing opinion(s) or offering technical ideas and procedures will be considered for publication (subject to editing) provided they are signed by all authors and do not exceed two typewritten (double-spaced) pages. and Answers Questions about immunologic techniques, laboratory procedures for immunology, and general problems in clinical immunology are welcome. They will be answered by the editorial board or by selected individuals in the field. Questions
Information about Meetings Announcements or reports of meetings and conferences of interest to clinical laboratory immunologists will also be considered for publication. Preparation
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All material submitted for publication in the Newsletter should be typed double-spaced (including references) on standard 81/2 × 11 paper. The original typescript and three copies should be sent to: or
Alan L. Landay, Ph.D. Department of Immunology and Microbiology Rush-Presbyterian-St. Luke's Medical Center 1753 West Congress Parkway Chicago, IL 60612
Henry A. Homhnrger, M.D. Dept. of Laboratory Medicine and Pathology Mayo Clinic 920 Hilton Building Rochester, MN 55905
Editorial Board N. Franklin Adkinson, M.D., Baltimore, MD; Thomas Fleisher, M.D., Bethesda, MD; James Folds, Ph.D., Chapel Hill, NC; David Kemn, M.D., Ann Arbor, MI; Peter Lipsky, M.D., Dallas, TX; S. Breanndan Moore, M.D., Rochester, MN; Robert Nakamura, M.D., La Jolla, CA; Bruce Rabin, M.D., Ph.D., Pi~burgh, PA; Daniel Stites, M.D., San Francisco, CA.
Editors: Alan L. L a n d a y , P h . D , H e n r y A. H o m b u r g e r , M . D .
Please address editorial correspondence to either Alan L. Landay, Ph.D., Department of Immunology and Microbiology, Rush-Presbyterian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, IL 60612 or Henry Homburger, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic, 920 Hilton Building, Rochester, MN 55905. General Information
ClinicalImmunologyNewsletteris published monthly by Elsevier Science Publishing Co., 655 Avenue of the Americas, New York, NY 10010. See inside front cover for subscription information. This newsletter has been registered with the Copyright Clearance Center, inc. Consent is given for copying articles for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition that the copier pay through the Center the per-page fee stated in the code on each page for copying beyond that parmitted by the U.S. Copyright Law. If no code appears on an article, the author has not given broad consent to copy, and permission to copy must be obtained directly from the author. This consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works. Address orders, changes of address, and claims for missing issues to Journal Fulfillment Department, Elsevier Science Publishing Co., Inc.. 655 Avenue of the Americas, New York, NY 10010. Claims for missing issues can be honored only up to three months for domestic addresses and six months for foreign addresses. Duplicate copies will not be sent to replace ones undelivered due to failure to notify Elsevier of change of address.
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Authors reply. Clin Exp Rheumatol 8:614-615, 1990. Galli M, Comfurius P, Maassen C, et al.: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 335:1544-1547, 1990. Harris EN, Gharavi AE, Boey ML, et al.: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosis. Lancet ii:1211-1214, 1983. Harris EN, Gharavi AE, Wasley GD, et al.: Use of an enzyme-linked immunosorbent assay and inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J Infect Dis 157:23-31, 1988. Hunt JE, McNeil HP, Krilis SA: Anticardiolipin antibodies from autoimmune disease and infection have different binding specificities. FASEB (in press), 1991. Hunt JE, McNeil HP, Crameri R, et al.: A phospholipid I complex is an antigen for anticardiolipin antibodies occurring in autoimmune disease but not with infection (submitted). Keisey PR, Stevenson KJ, Poller L: The diagnosis of lupus anticoagulants by the activated partial thromboplastin time: the central role of phosphatidylserine. Thromb Haemost 52:172-175, 1984. Koppe AL, Walter H, Chopra VP, Bajatzadehm: Investigations on the genetics and population genetics of the 132-glycoprotein I polymorphism. Humangenetik 9:164171, 1970. Matsuurd E, Igarashi Y, Fujimoto M, et al.: Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease.
CLINICAL IMMUNOLOGY
Lancet 336:177-178, 1990. 13. McNeil HP, Krilis SA, Chesterman CN: Purification of antiphospholipid antibodies using a new affinity method. Thromb Res 52:641-648, 1988. 14. McNeil HP, Chesterman CN, Krilis SA: Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br J Haematol 73:506-513, 1989. 15. McNeil HP, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen which includes a lipid-binding plasma glycoprotein. Clin Exp Rheumatol 8:208, 1990. 16. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: 132glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 87:4120-4124, 1990. 17. McNeil HP, Chesterman CN, Krilis SA: Immunology and clinical importance of antiphospholipid antibodies. Adv Immunol 49:(in press), 1991. 18. McNeil HP, Shi W, Chesterman CN, Krilis SA: Elevated plasma 132-glycoprotein I levels in patients with anticardiolipin antibodies (submitted). 19. Moore JE, Mohr CF: Biologically false positive serologic tests for syphilis. JAMA 150:467-473, 1952. 20. Nimpf J, Wurm H, Kostner GM: 132-glycoprotein I (apo H) inhibits the release reaction of human platelets during ADP-induced aggregation. Atherosclerosis 63:109-114, 1987.
Newsletter 41
21. Pangborn MC: A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med 48:484-486, 1941. 22. Pengo V, Thiagarajan P, Shapiro SS, et al.: Immunological specificity and mechanism of action of IgG lupus anticoagulants. Blood 70:69-76, 1987. 23. Polz E, Kostner GM: The binding of 132glycoprotein I to human serum lipoproteins. FEBS Lett 102:183-186, 1979. 24. Ranch J, Meng Q-H, Tannenbaum H: Lupus anticoagulant and anti-platelet properties of human hybridoma autoantibodies. J Immunol 139:2598-2604, 1987. 25. Ranch J, Tannenbaum M, Janoff AS: Distinguishing plasma lupus anticoagulants from anti-factor antibodies using hexagonal (II) phase phospholipids. Thromb Haemost 62:892-896, 1989. 26. Reid KBM, Day AJ: Stucture-function relationships of the complement components. Immunol Today 10:177-180, 1989. 27. Schousboe I: Binding of 132-glycoprotein I to platelets: effect on adenylate cyclase activity. Throm Res 19:225-237, 1980. 28. Schousboe I: 132-glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 66:1086-1091, 1985. 29. Schultze HE, Heide H, Haupt H: Uber ein bisher unbekanntes niedermolekulares 132globulin des human-serums. Naturwissenschafien 48:719, 1961. 30. Staub HL, Khamashta MA, Harris EN, et al.: Lupus anticoagulant and anticardiolipin antibodies: the affinities may differ. Postgrad Med J 65:700, 1989. 31. Tilcock CPS: Lipid polymorphism. Chem Phys Lipids 40:109-125, 1986.
A n t i c a r d i o l i p i n Kits: T e c h n i q u e s o f Antiphospholipid Antibody Measurement Sozos Loizou Rheumatology Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
he first specific solid-phase immunoassay for the measurement of anticardiolipin (aCL) antibodies was the radioimmunoassay (RIA) developed in 1983 by E. N. Harris and colleagues. 5 Since then a large number of immunoassays, mainly ELISAs, have been published for the estimation o f aCL or antiphospholipid antibodies (aPL) in patients' serum or plasma (Tables 1, 2). All
T
ELISA assays for antiphospholipid antibody measurement use purified cardiolipin or a mixture of negatively charged phospholipids as the antigen for coating microtiter plate wells. Antiphospholipid antibodies present in serum or plasma are then allowed to react and bind to the antigen coated onto the plate wells, and the amount of bound antibody is measured by the use of enzyme-conjugated anti-human © 1991 Elsevier Science Publishing Co., Inc.
antibodies o f the appropriate isotype; the amount of antiphospholipid antibody present is measured by the intensity of color produced when a chromogenic substrate is added to the above complex, as shown in the generalized aPL ELISA shown in Figure 1. Antiphospholipid antibody assays are clinically important because of the numerous clinical complications that have 019%1859/91/$0.00 + 2.20
42 C L I N I C A L I M M U N O L O G Y Newsletter
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TABLE 1. PUBLISHED SERIES OF aCL PREVALENCE IN SLE PATIENTS
Reference
Method
No. of Controls
Harris et al., 1983 Koike et al., 1984 Colaco and Male, 1985 Tincani et al., 1985 Meyer et al., 1985 Norberg et al., 1985 Loizou et al., 1985 Manoussakis et al., 1987 Kalunian et al., 1988 Mouritsen et al., 1989
RIA ELISA RIA RIA ELISA ELISA ELISA ELISA ELISA ELISA
30 11 23 40 30 156 135 276 40 100
Positivity Cutoff SD > Mean
No.
% + ve
2.76 a 2.0 3.0 2.0 b 5.0 4.0 5.0 c
65 24 87 51 108 127 65 86 85 86
61.0 41.7 83.9 49.0 40.0 37.0 29.2 20.9 42.4 42.0
SLE Patients
SLE = systemic lupus erythematosus. a Not indicated b Ratio of patient/mean control; positive > 1.6. c 99th percentile.
Commercial aCL or aPL Kits Evaluated and Reference Method Used
been associated with raised levels of these antibodies.I° The importance attached to this antibody test is reflected by the large number of commercial kits that are now available, eight by the end of 1990, and two more that will become available during 1991. This review will examine the discrepancies that have been reported in the prevalence of antiphospholipid antibodies, the means available for standardization of these assays, and finally report on an evaluation of commercial antiphospholipid/anticardiolipin antibody kits. Discrepancies in Prevalence of aCL Antibodies and Currently Available Means of Standardization of aCL/aPL Assays Availability of published methods and commercial kits does not necessarily provide accuracy, specificity, and comparability of results from different centers. Hence, the reported prevalence of anticar-
diolipin in the two groups of patients where levels of these antibodies have been estimated in relatively large numbers of patients, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), have shown marked discrepancies, and the reasons for these will be discussed later on in this review (Tables 1, 2). Because of these discrepancies the need for standardization of aCL assays became evident by 1986, and hence a standardization workshop was organized by Dr. E.N. Harris. 7 As a consequence of the results of this workshop, an International Standardization Committee for Antiphospholipid antibodies was formed, and international reference sera for IgG and IgM aCL antibodies were produced and made available to facilitate uniformity in reporting levels of these antibodies in serum or plasma. These reference sera were designated as the KAPS (Kingston Antiphospholipid Study) reference sera, 4 by means
Reference
Method
No. of Controls
Cutoff SD > Mean
No.
Colaco and Male, 1985 Meyer et al., 1985 Loizou et al., 1985 FoIt et al., 1987 Manoussakis et al, 1987 Keane et al., 1987 Mouritsen et al., 1989
RIA ELISA ELISA ELISA ELISA ELISA ELISA
23 30 135 27 276 40 100
2.0 2.0 5.0 3.0 4.0 4.0 a
16 35 47 57 65 90 20
RA Patients % + ve 50.0 6.0 6.4 33.0 7.7 48.9 15.0
RA = rheumatoidarthritis. • 99th percentile.
+ 2.20
Six commercial aCL/aPL antibody kits from five commercial companies were evaluated out of the eight commercial kits that were on the market at the time of this evaluation. The general properties and codes of each kit used are given in Table 4. All six kits evaluated used an ELISA method for aPL antibody measurement, and the kits were obtained directly from the manufacturers, who had previously been informed about the aim of the study. Each kit was used exactly in accordance with the manufacturers' instructions. Details of the method used and general characteristics for each kit are given in Table 5. For comparative reasons, the reference method used was the quantitative lgG and IgM aCL antibody ELISA assay (HH) that has been routinely used at Hammersmith Hospital for over six years, 8 and that was also one of the methods used for the First Antiphospholipid Antibody Standardization Workshop. 7 Patient and Normal Control Sera Used in the Evaluation
T A B L E 2. P U B L I S H E D SERIES O F aCL P R E V A L E N C E IN RA P A T I E N T S
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of which results can be reported in International GPL and MPL units for lgG and IgM aCL antibodies, respectively (Table 3). No international reference sera or units are as yet available for reporting lgA aPL antibody levels. For the reasons given above, the need also emerged for the evaluation of commercial anticardiolipin/antiphospholipid antibody kits, and such an evaluation was undertaken as part of the antiphospholipid antibody standardization workshop for the 4th International Antiphospholipid Antibody Symposium in Sirmione (Lake Garda), Italy in April 1990, where these results were first presented.
I © 1991 Elsevier Science Publishing Co., Inc.
Anticardiolipin or antiphospholipid antibodies were estimated by both the reference method and the six commercial kits in 10 normal human sera from healthy laboratory personnel, and in 30 patient sera. The patient sera used were selected on the basis that they covered a very wide range of IgG and IgM aCL antibody levels, from negative to very high positive; the aCL levels in the 30 patient sera were ascertained by the reference method
C L I N I C A L I M M U N O L O G Y Newsletter 43
Vol. 11, No. 3, 1991
CARDIOLIPIN - antigen dried onto polystyrene elate wells
(HH) in triplicate on three different occasions, and the mean aCL levels in anticardiolipin ELISA units (AEU), for IgG and IgM were taken as the target values for the kit evaluation (1 AEU = - 1 GPL/or 1 MPL, see Table 3). Prior to the evaluation study, all patient, normal control, and KAPS reference sera were subaliquoted into 50 Ixl aliquots and stored frozen at - 2 0 ° C until needed for use, when a set of all the sera was thawed out for use prior to each assay for each kit.
Plates blocked with buffer/FCS.ABS or BSA solution (1 hour) wLh Aoorooriatelv diluted samples and controls added (3 hours)
Assessment of Results
w,Lh Enzyme * con!uaated anti-human antibody added-loG.IoM or leA (90 min) wLh Aoorooriate chromooenic substrate added / Colour reaction stonoea~with aPPropriate solution (stop when top standard OA = 0.8 -0.9)
1 Read ootical absorbance (O~A~at appropriate wavelenoth
* alkaline phosphatase or horse radish peroxidase conjugate ( ) brackets indicate KAPS reccomendations for aCL ELISAs FCS = fetal calf serum: ABS=adult bovine serum; BSA= bovine serum albumin
Figure 1. A generalized flow diagram for anticardiolipin antibody determination by ELISA.
TABLE 3. COMPARISON OF aCL ANTIBODY LEVELS FOR IgG AND IgM KAPS STANDARDS GIVEN BY THE REFERENCE METHOD AND COMMERCIAL APL ANTIBODY KITS KAPS
International
HH
Sample
G/MPL
AEU
GI G2 G3 Normal M1 M2 M3 Normal
107.0 ± 19.6 ± 5.8 ± NG 106.0 ± 21.0 ± 5.4 ± NG
16.4 4.8 3.8 19.3 3.8 3.3
92.9 16.5 4.4 <9.0 92.5 26.0 6.6 <8.0
± 16.4 ± 1.9 ± 0.8 ± 14 ± 4.2 ± 1.5
MI ~/ml
CD G/MPL
WL G/MPL
ST G/MPL
25.8 6.3 3.5 <2.0 44.0 14.7 4.2 <2.0
84.0 14.2 8.9 < 10.0 84.4 23.7 8.3 <9.0
85.2 15.8 9.0 <9.0 63,2 32.4 27.4 <4.0
93.3 14.6 8.1 < 5.0 >100 43.2 16.9 <3.0
HH = Hammersmith Hospital; AEU = anticardiolipin ELISA units; NG = not given; other abbreviations as per Table 4. I
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© 1991 Elsevier Science Publishing Co., Inc.
Results of aCL or aPL levels in the patient and control sera were assessed using optical absorbance (OA) readings and the KAPS reference sera for comparison of results between the reference method (HH) and all the kits studied (qualitative ST/ GMA, BL/GMA; quantitative MI, CD, WL; semiquantitative ST/G, ST/M, Table 4). Quantitative comparison of results could only be performed between HH and the quantitative kits MI, CD, and WL, which the manufacturers had stated were standardized against the KAPS standards (see Table 3 for IgG and IgM KAPS results). Table 6 shows the Spearman Rank correlations obtained for the 30 patient sera between the OAs of all the assays evaluated. Strong correlations (>0.800) were obtained for all the IgG specific kits. For IgM aCL antibodies, strong correlations (>0.800) were only seen between the HH method and the CD IgM kit. Two of the kits, ST/GMA and BL/GMA, were stated to be qualitative screening kits for a combination of three aPL isotypes IgG, IgM, and IgA (GMA kits); for these two kits, the OA obtained by the reference method (HH) for either IgG or IgM (whichever was the highest) was compared to that obtained by the two combination GMA kits; a strong correlation was obtained with the ST/GMA kit, but the correlation between both HH and ST/GMA and the BL/GMA kit was very Ix)or (Table 6). The detection limits for each kit and the reference method could only be assessed for the quantitative assays, and these were estimated by a graphical method as follows. The mean level of each individual kit or the reference method for each patient serum was plotted together with 0197-1859/91/$0.00 + 2.20
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TABLE 4. PROPERTIES OF COMMERCIAL ANTICARDIOLIPIN ANTIBODY KITS Name of Kit and Company
Code
1. aCL antibody kit, Medical Innovations LTD 2. QACA, Cheshire Diagnostics Ltd. 3. MELISA aCL antibody kit Walker Laboratories Ltd. 4. Asserachrom APA Diagnostica Stago 5. MALAKIT Cardiolipin Biolab, sa, nv 6. SYNELISA aCL kit ELIAS Medizintechnic GmbH 7. SLOR El aCL kit BioHyTech, Israel 8. REAADS aCL kit Biostar Medical Products Inc.
Units
lsotype of aCL
Antigen
MI CD
Units/mL G/MPL
CL CL
lgG or IgM IgG or lgM
WL ST BL
G/MPL G/MPL and SQ QL
CL PL mixture CL
IgG or IgM lgG or IgM G/M/A G/M/A
EL~
QU
CL
lgG or IgM
BHT~ BSa
SQ SQ
CL CL
lgG or IgM IgG or lgM
SQ = semi-quantitative;QL = qualitative;QU = quantitative. • Kits 6. 7, and 8 were not evaluated. I
I
TABLE 5. CHARACTERISTICS OF DIFFERENT ANTIPHOSPHOLIPID ANTIBODY KIT METHODS Kit Code
MI
Proprietary name Instructions Assay procedure Sample dilution
aCL Ab. Good Easy 1:20 on plate 50 Ixl 30 rain Peroxidase 50 ~l 45 m 50 Ixl 15 m Fast Good >2.0
Sample volume increase time Conjugate Volume Increase time Substitute volume Increase time Color development Duplicate agreement Top standard (OA)
CD
WL
ST
BL
QACA Very good Easy 1:100
(MELISA) Good Complex 1:100
Asserachrom Good Easy 1:101
Malakit Complex Easy 1:101
100 I~1 30 min Alkaline phosphate 100 pJ 30 m 100 p.1 " 30 m Good Good <2.0
100 txl 15 min Peroxidase 100 ~1 15 m 200 p.l 10 m Fast Good >3.0
200 Ixl 30 min Peroxidase 200 ill 30 m 200 p,l 6m Fast Good <0.7
100 ~.1 1 hr Peroxidase 100 ixl 1 hr 100 I~1 30 m Slow Variable <1.2
OA = optical absorbance; other abbreviationsas per Table 4.
the m e a n values o f all the methods for each s e r u m in G P L or M P L . For each m e t h o d being investigated, a line can be d r a w n c o n n e c t i n g all values specific for the particular method. If the graph for an individual m e t h o d runs parallel and very near a b o v e or b e l o w the m e a n line, then the kit or m e t h o d can be regarded as g o o d and reproducible. Figure 2 illustrates the procedure for IgG aCL. P e r f o r m a n c e for negative to intermediate positive values ( < 7 5 G P L ) was g o o d for H H , C D , and W L kits, but rather p o o r for the MI kit. At higher a C L levels ( > 7 5 G P L ) , the reference method H H p e r f o r m e d best as very high levels o f lgG a C L c o u l d o n l y be m e a s u r e d by this m e t h o d (highest standard, 270 G P L ) , in contrast to the various kits, C D kit (highest standard, 160 G P L ) , W L kit (highest standard, 100 G P L ) , and MI kit (highest standard, 64 units). T h e results for I g M a C L are s h o w n in Figure 3, w h e r e g o o d a g r e e m e n t is s h o w n for n e g a t i v e to intermediate positive values ( < 5 0 M P L ) with the reference m e t h o d H H (highest standard, 150 M P L ) and the C D kit (highest standard, 120 M P L ) . The two r e m a i n i n g kits, the MI kit (highest standard, 64 units) tended to g i v e lower levels than the mean, and the W L kit (highest standard, 60 M P L ) tended to give higher results than the m e a n values. At highpositive l g M a C L levels ( > 5 0 M P L ) , the H H m e t h o d again p e r f o r m e d better than the kits. Assessment of aCL/aPL Antibody L e v e l s in A c c o r d a n c e w i t h t h e KAPS International Standardization Workshop 6
I
TABLE 6. SPEARMAN RANK CORRELATIONS BETWEEN OPTICAL ABSORBANCES OBTAINED BY THE DIFFERENT METHODS AND KITS ASSESSED IN 30 PATIENT SERA.
Kit/Method
HH MI CD WL ST ST/GMA
lgG aCL Antibodies HH MI CD WL
. 0.907 0.948 0.978 0.882 .
.
. . --0.902 -0.948 0.946 0.907 0.948 . . .
IgM aCL Antibodies HH MI CD WL
. ---0.978 .
. . . 0.752 -0.922 0.718 0.778 0.582 0.702 0.654 . . .
--0.704 0.645 .
---0.631
GMA aCL Antibodies ST BL
0.909 a ND ND ND ND
0.164" ND ND ND ND 0.014
ND = not done. • For comparisonof GMA kits to HH, lgG, or IgM OA, whicheverwas the highest for the HH method was used. i
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The K A P S reference sera offer a m e a n s for standardizing the way in which results of a C L antibody levels are reported, and enables valid c o m p a r i s o n s b e t w e e n laboratories o f the prevalence and relationship b e t w e e n these antibodies and the numerous associated clinical c o m p l i c a t i o n s . I ° A c c o r d i n g to the r e c o m m e n d a t i o n s of the 2nd International Standardization W o r k s h o p , 13 using the K A P S reference sera ( G I , G2, G3 for IgG and M I , M 2 , M3 for I g M aCL) for reporting results, a serum sample is reported as high positive for l g G a C L if its O A reading is greater iii iiiii
C L I N I C A L I M M U N O L O G Y Newsletter 45
Vol. 11, No. 3, 1991
300 ----
mean of all
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mean of MI mean of CD mean of WL
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10 12 14 16 18 20 22 24 26 28 30
Serum number Figure 2. Comparison of mean IgG aCL levels for HH and the three quantitative kits MI, CD, and WL in 30 patient sera with the mean levels of all four methods in the same sera. The serum number refers to the 30 patient sera that have been arranged consecutively in order of increasing average levels of all four methods. At very high levels >200 units, the result of the HH is shown as the mean as the levels measured were more than twice those of the top standard for each of the kits (serum numbers 27-30).
Figure 3. Comparison of mean IgM aCL levels for HH and the three quantitative kits MI, CD, and WL in 30 patient sera with mean levels of all four methods in the same sera. The serum number refers to the 30 patient sera that have been arranged consecutively in order of increasing average levels of all four methods. For very high levels, results that were <50% of the HH value (serum numbers 26-30) were omitted from the calculation of the mean of all four methods. 120
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than the OA reading of the reference serum G1; medium or moderate positive when its OA lies between G1 and G2, low positive if its OA reading is between G2 and G3, and negative if its OA reading is less than G3. For IgM aCL, serum samples are reported in a similar way, where samples are reported as high positive if their OA is greater than that of the KAPS reference serum M 1, medium or moderate positive if the OA reading lies between M1 and M2, low positive when the OA is between M2 and M3, and negative when the OA reading is less than that for M3. For results of the established values for IgG and IgM aCL for the KAPS reference sera in international units GPL or MPL see Table 3. Results for all the commercial aCL kits evaluated and analyzed according to the KAPS recommendations for the 30 patient and 10 normal control sera are shown in Table 7 for IgG and in Table 8 for IgM aCL. At the bottom of these tables are shown the differences in total percentage (%) positivity as found in the serum samples studied, using both the KAPS way of reporting results, and also when results were reported according to each method's or kit's own cutoff criteria for positive-negative results. For IgG aCL antibodies, there was generally good agreement for samples reported as high positive, except for the BL/GMA kit where all aCL-positive samples fell in the high-positive category. Some overlap was also seen between the number of patients that fell in the moderate and low-positive categories, which was expected as different methods vary in their specificity, sensitivity, composition, and concentration of the reagents used. The number of negative patient samples also varied, from two reported by HH to eight reported by the MI kit; this finding was also expected as it depends greatly on the definition of the positive/negative cutoff points of the different methods. For example, the KAPS reference sera cutoff point for both IgG and IgM aCL was set at 2 SD above the mean normal control level, whereas for the HH assay the cutoff point for both isotypes was set at 5 SD above the mean normal control level of the assay. Hence, the HH method gave the lowest number of negatives for 0197-1859/91/$0.00 + 2.20
46 C L I N I C A L I M M U N O L O G Y Newsletter
Vol. 11, No. 3, 1991
TABLE 7. IgG aCL ANTIBODIES COMPARISON OF KAPS LEVELS BY THE DIFFERENT METHODS IN 30 PATIENTS (P) AND 10 NORMAL CONTROLS (N) KAPS Level
High + v e Medium + ve Low + ve Negative % positivity KAPS % positivity KIT or HH Method
HH
MI
P
N
8 12 8 2
0 0 1 9
P
CD N
10 11 1 8
WL
P
N
P
0 0 1 9
7 11 9 3
0 0 0 10
5 16 6 3
ST/G N 0 0 1 9
ST
BL
GMA
GMA
P
N
P
N
P
N
8 13 4 5
0 0 0 10
8 12 5 5
0 0 0 10
25 0 0 5
0 0 0 10
93.3
10
73.3
10
90
0
83.3
10
83.3
0
83.3
0
83.3
0
86.7
0
93.3
10
90
0
86.6
10
83.3
0
76.6
0
83.3
0
Abbreviations as per Table 4.
the patient sera (only two), which can be explained in that a number of patient sera, namely those lying between 2 and 5 SD above the normal mean, which would be negative by the HH method, were positive by the KAPS criteria. This also holds when total percentage positivity was compared by the two different methods of reporting results using the two different cutoff points. As for the HH method this was 86.7%, which is lower than the 93.3% positivity that was found when the KAPS criteria were used. These comparisons were also applied to the results of all the commercial kits, for both the patient and normal control sera for both IgG (Table 7) and IgM aCL (Table 8) antibodies. When the same criteria were applied for IgM aCL antibodies, discrepancies were evident in all results except for those in the high-positive category. These dis-
crepancies were more obvious for the number of sera that were found to be negative by the different methods. Here again for the same reasons given above for IgG aCL, the lowest number of negatives was found using the HH method when the KAPS cutoff was used. The number of positive and negative samples by the different kits varied considerably for all the quantitative and semiquantitative kits, and reached 19 negative patient sera for the WL IgM kit. Here again when the total percentage (%) positivity was examined, only the HH and ST/GMA methods gave a lower total % positivity by their own criteria than by those for KAPS. In all the remaining kits, total % positivity by each kit's cutoff was higher than that for the KAPS, and for one kit (WL), positivity using this kit's cutoff was more than twice that found with the KAPS criteria (Table 8). ii
TABLE 8. IgM aCL ANTIBODIES--COMPARISON OF KAPS LEVELS BY THE DIFFERENT METHODS IN 30 PATIENTS (P) AND 10 NORMAL CONTROLS (N) KAPS
HH
MI
CD
WL
ST/M P
N
ST
BL
GMA
G MA
Level
P
N
P
N
P
N
P
N
P
N
P
8 12 5 5
0 0 2 8
25 0 0 5
High + ve Medium + ve Low + ve Negative % positivity KAPS % positivity KIT or HH Method
5 6 11 8
0 0 0 10
5 3 8 14
0 0 0 10
5 2 9 14
0 0 0 10
5 1 5 19
0 0 3 7
4 2 6 18
0 0 1 9
73.3
0
53.3
0
53.3
0
36.6
30
40
10
83.3
20
83.3
10
66.7
0
70,0
0
63.3
0
80
30
50
0
76.6
0
83.3
10
Abbreviations as per Table 4. i
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N 0 0 1 9
Future Considerations for Antiphospholipid Antibody Measurements and Standardization of Results Although discrepancies regarding the prevalence of aCL antibodies in patient sera are often reported, the clinical associations of moderate to high levels of these antibodies, especially of the IgG class with clinical complications, such as thrombosis and fetal loss, are now widely accepted. 6 The IgM aCL isotype has also been implicated, but the importance of IgA aCL is still not clear. In order that results from different centers can be compared, and aCL/aPL antibody assays can be standardized, a number of fundamental questions have emerged that need to be answered. As with any other clinical laboratory method, methodological differences exist for aCL assays or commercial aCL kits. Besides these, a number of other factors are very important, such as very wide differences in the determination of the cutoff between positive and negative results, use of limited numbers of normal control populations, selection of patients, and lack of complete uniformity in units of measurement, all factors that invariably prevent an accurate comparison of studies. In order that aCL assays are standardized, some further aspects of aCL ELISA assays need to be addressed, such as the binding capacity of different plastics (microtiter plates), which is known to show marked variations; the use and concentrations of different serum proteins such as bovine serum albumin (BSA), fetal calf serum (FCS), and adult bovine serum (ABS) added to the assay buffers for blocking microtiter plates and for sample and reagent dilution; the nonspecific binding of serum proteins to non-antigencoated platesg; the exact way in which the antigen (cardiolipin, another negatively charged phospholipid or a mixture of phospholipids) is bound to the solid phase, and the possibility of false-positive aCL antibody results that might be caused by the presence of other immunoglobulins, such as the false-positive IgM aCL antibody levels seen in IgG positive sera because of the presence of IgM rheumatoid factors, t,2
C L I N I C A L I M M U N O L O G Y Newsletter 47
Vol. 11, No. 3, 1991
Most research centers using "in-house" aCL assays are now aware of the above problems and take great care in eliminating them from their assays. This task has been facilitated by the Antiphospholipid Antibody Workshops that have led to the creation of international units for aCL measurement, and the provision and availability of reference sera (KAPS) for interlaboratory standardization of reported results, Unfortunately, the same criteria do not seem to be applied to these assays by all the companies that are marketing aCL/aPL antibody kits. For understandable commercial reasons, speed and ease of performance seem to be the most important properties of some kits, at the expense of sensitivity and specificity. The cutoff points between positive-negative levels used by commercial aCL kits also vary considerably, and the way these are defined is not always clearly indicated. Low cutoff points could lead to large numbers of patients with otherwise normal or very low aCL levels being treated unnecessarily, especially as aCL levels that are below 5 SD and above the normal mean of the particular aCL assay, may not be clinically important. 6 The future of the clinical significance of aCL antibodies and the most satisfactory methods for their measurement are still important questions that remain largely unanswered. Recent studies have suggested the existence of a cofactor protein for the binding of aCL antibodies in serum or plasma to the cardiolipin antigen on
microtiter plates, and this cofactor has been characterized and shown to be a serum glycoprotein (132-glycoprotein I), 3a2 and to be able to differentiate between the aPL antibodies found in autoimmune disorders and those of infectious diseases. 1] No doubt as a result of this finding new commercial kits will emerge; it is hoped that both these new and the existing commercial aCL kits will pay a lot of attention in proper validation of their methods on the lines indicated above. Only the emergence of satisfactory standardized methods for aCL/aPL antibody measurement, will be able to answer the very important questions regarding the clinical significance and predictive value of these autoantibodies, c~
References 1. Agopian MS, Boctor FN, Peter JB: False positive test result for IgM anticardiolipin antibody due to IgM rheumatoid factor. Arthritis Rheum 31:1212-1213, 1988. 2. Cofiner C, Loizou S, Peter JB, Walport MJ: Effect of IgM rheumatoid factor on IgG and IgM anticardiolipin ELISA assays. Clin Exp Rheumatol 8:(202)abstract 5A, 1990. 3. Galli MD, Moussen C, Comfusius P, et al.: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 335:1544-1547, 1990. 4. Harris EN: Solid-phase anti-cardiolipin test revisited. Am J Med 85:599-601, 1988. 5. Hams EN, Gharavi AE, Boey ML, et al.: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythema-
tosus. Lancet ii:1088-1214, 1983. 6. Harris EN, Chart JKH, Asherson RA, et al.: Thrombosis, recurrent fetal loss, and thrombocytopenia: predictive value of the anticardiolipin antibody test. Arch Intern Med 146:2153-2156, 1986. 7. Harris EN, Gharavi AE, Patel S, Hughes GRV: Evaluation of the anticardiolipin antibody test: report of a standardisation workshop held April 4, 1986. Clin Exp lmmunol 68:215-222, 1987. 8. Loizou S, McCrea JD, Rudge AC, et al.: Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 62:738-745, 1985. 9. Loizou S, Cofiner C, Walport MJ: False positive ELISA tests for IgG and IgM anticardiolipin antibodies, due to non-specific binding to blank microtitre plates. Clin Exp Rheumatol 8:(203)abstract 1la, 1990. 10. Love PE, Santoro SA: Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in Non-SLE disorders. Ann Intern Med 112:682-698, 1990. 11. Matsura E, Igarashi Y, Fujimoto M, et al.: Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease (letter). Lancet ii: 177-178, 1990. 12. McNeil HP, Simpson ILI, Chesterman CN, Krilis SA: Antiphospholipid antibodies are directed against a complex antigen which includes a lipid binding inhibitor of coagulation: 13-2 glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 87:41204124, 1990. 13. Third International Symposium on AntiPhospholipid Antibodies. Clin Exp Immunol 6:189-215, 1988.
• l a b o r a t o r y T u t i l ~ l for Lupus ~ t i ¢ o ~ l o l l m m t s Douglas A. Triplett
• Quantitative lmmqlllol[hloIt'tKelKt Measurements and Standards: Practical Approaches Howard M. Shapiro • AppUcatfons o f F l o w Cytometry to Solid Tumors John S. Coon and Steven D. Bines • Application o f l ~ M l . l l ~ d .,~say~ far Iglow ~ometry Analysis Thomas M. McHugh and Daniel P. Stites
© 1991 ElsevierScience Publishing Co., Inc.
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48 CLINICAL IMMUNOLOGY Newsletter
Vol. 11, No. 3, 1991
In general, the style adheres to the ASM Style Manual for Journals and Books. As a backup, follow The Chicago Manual of
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Reports documenting interesting case presentations are invited for possible publication in the Clinical Immunology Newsletter. Submitted case reports should be written concisely and should contain: a) a brief clinical history summarizing the symptoms and course of the illness; b) a description of immunologic and other laboratory tests performed; and c)t the results of these laboratory tests as related to the clinical observation of patients and the outcome of the disease process and therapy, if appropriate. References Authors are responsible for the accuracy of references, which should include complete publication information. References should be listed in alphabetical order by the first author's last name, numbered consecutively, and cited in the text by these numbers. Some typical examples of references are listed below. Consult this issue for additional examples of Newsletter reference format. 1. Doone FW, Anderson N: Electron Microscopy in Diagnostic Virology. A Practical Guide and Atlas. New York, Cambridge University Press, 1987. 2. McCulloch EA: Normal stem cells and the clonal hemopathies. In: Cronkite PE (ed): Hematopoietic Stem Cell Physiology, Vol. 184, New York, Alan R. Liss, Inc., pp. 21-38. 3. Forsgren A, Binder G, Callow F, et al.: Quinolones affect thymidine incorporation in the DNA of human lymphocytes. Amimicrob Agents Chemotber 29:506-508, 1986. Letters Letters expressing opinion(s) or offering technical ideas and procedures will be considered for publication (subject to editing) provided they are signed by all authors and do not exceed two typewritten (double-spaced) pages. and Answers Questions about immunologic techniques, laboratory procedures for immunology, and general problems in clinical immunology are welcome. They will be answered by the editorial board or by selected individuals in the field. Questions
Information about Meetings Announcements or reports of meetings and conferences of interest to clinical laboratory immunologists will also be considered for publication. Preparation
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Alan L. Landay, Ph.D. Department of Immunology and Microbiology Rush-Presbyterian-St. Luke's Medical Center 1753 West Congress Parkway Chicago, IL 60612
Henry A. Homhnrger, M.D. Dept. of Laboratory Medicine and Pathology Mayo Clinic 920 Hilton Building Rochester, MN 55905
Editorial Board N. Franklin Adkinson, M.D., Baltimore, MD; Thomas Fleisher, M.D., Bethesda, MD; James Folds, Ph.D., Chapel Hill, NC; David Kemn, M.D., Ann Arbor, MI; Peter Lipsky, M.D., Dallas, TX; S. Breanndan Moore, M.D., Rochester, MN; Robert Nakamura, M.D., La Jolla, CA; Bruce Rabin, M.D., Ph.D., Pi~burgh, PA; Daniel Stites, M.D., San Francisco, CA.
Editors: Alan L. L a n d a y , P h . D , H e n r y A. H o m b u r g e r , M . D .
Please address editorial correspondence to either Alan L. Landay, Ph.D., Department of Immunology and Microbiology, Rush-Presbyterian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, IL 60612 or Henry Homburger, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic, 920 Hilton Building, Rochester, MN 55905. General Information
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