A survey of rubella haemagglutination-inhibition testing in the southern states of Australia*

Bull. Org. mond Sante 11973, 49, 139-142 A survey of rubella haemagglutination-inhibition testing in the southern states of Australia* I. D. GUST,1 A. MATHISON,2 & H. WINSOR2 The accuracy and reproducibility ofrubella haemagglutination-inhibition testsperformed in 17 laboratories in the south of Australia were surveyed and showed that only 8 were providing reliable results. The accurate diagnosis of rubella is of consider- able importance, as infection with this virus during early pregnancy may produce a range of malforma- tions in the fetus. Because of its speed and ease of performance, haemagglutination-inhibition (HI) is the test most widely used for detecting antibodies to rubella virus. Virus laboratories in Australia and other countries have been unable to keep up with the demand for this test and, as a result, it is now performed by many general pathology laboratories. Because of numerous variables in the test and the limited experi- ence ofsome ofthe personnel with haemagglutination techniques, concern has arisen about the reliability and reproducibility of the results being produced. In 1972 the WHO Regional Reference Laboratory for Virus Diseases, Fairfield Hospital, Melbourne, invited all laboratories in the southern states of Australia known to be performing rubella serology to participate in a survey. Every laboratory ap- proached accepted the invitation. Each was then issued with a number of batches of sera to be tested for rubella antibodies by the HI technique routinely used in the laboratory. At the conclusion of the survey, the results were collated and a workshop was held to discuss ways in which performance could be improved. MATERIALS AND METHODS The laboratories that participated in the survey included 2 from South Australia, 1 from Western * From the WHO Regional Reference Laboratory for Virus Diseases, Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield, Victoria 3078, Australia. I Medical Virologist. 2 Virologist. Australia, 1 from Tasmania, and 13 from Victoria. Samples of blood were obtained from a group of 20 people with a range of HI titres. Each serum was tested to ensure that it was negative for hepatitis B antigen by standard techniques, then divided into 1-ml aliquots, ampouled, and stored at -20°C. Six sera were present in sufficient quantities to allow the preparation of duplicate aliquots. Thus, panels of 26 sera were available for distribution to each of the participating laboratories. The virus laboratories at Fairfield Hospital, Mel- bourne, and the Institute of Clinical Pathology and Medical Research, Lidcombe, N.S.W., acted as reference centres, since both have had considerable experience in rubella HI testing. At Lidcombe kaolin was used to remove nonspecific inhibitors of haemagglutination, while at Fairfield the tests were performed in parallel using both kaolin and heparin-manganese(2+) chloride. The precise tech- niques used by the two laboratories have been described elsewhere (1). Although different starting dilutions were used by the reference laboratories, the titres they obtained showed good agreement (see Table 1). After testing by the reference laboratories, the sera were distributed to the participating labora- tories under code; initially 8 were issued in April, 1972, followed by 3 per month over the next 6 months. Each laboratory received sera in a different order. The results and details of the methods used were recorded on standard forms and returned to the coordinating laboratory at Fairfield Hospital. RESULTS The reference laboratories found that 5 sera had no detectable antibody and that the remaining sera 3098 -139- I. D. GUST ET AL. obtained by the two reference centres Code No. Laboratory A Laboratory Bof serum Si S2 S3 S4 S5 S6 S7 S8 S9 S10 Sil S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 < 8 128 256 < 8 1 024 512 256 64 256 1 024 64 4 096 < 8 16 1 024 512 1 024 128 256 256 128 512 256 256 < 8 < 8 < 10 80 160 < 10 640 160 320 20 160 640 80 5120 < 10 20 640 160 640 80 160 160 80 160 160 80 < 10 < 10 had titres from 1: 16 to 1: 4096. The results ob- tained by the participating laboratories are sum- marized in Table 2. Although some variability in titres was expected, it was hoped that most results would fall within a 2-fold dilution of the values obtained by the refer- ence laboratories. Of 442 tests performed, 282 (64 %) fell within these limits, 147 (33%) were reported as having titres at least 4 times lower than expected, and 13 (3%) gave titres that were at least 4-fold higher than expected. The variation in titres obtained by the participa- ting laboratories reflects differences in the sensitivity of their test systems. These differences are less important than the ability to obtain consistent results on replicate tests. This was verified by pre- senting the same six sera to the participating labora- tories on two occasions. Of these 102 pairs, 88 (86.3 %) were reported within a 2-fold dilution of each other and 14 (13.7%) were not. 8 laboratories obtained reproducible titres on all duplicate speci- mens, and 9 reported one or more pairs outside a 2-fold limit. There were several major inconsisten- cies, e.g., one serum with a titre of 1: 128, as deter- mined by the reference laboratories, was presented twice to three laboratories whose reports were as follows: Laboratory A: <1: 10, 1:160 Laboratory B: 1:80, <1:10 Laboratory C: 1:128, <1:8 Two laboratories reported replicate titres of 1: 32, 1: 32 and 1: 80, <1: 8 on a serum in which both reference laboratories found no detectable antibody, and another laboratory reported titres of 1 :128, 1: 128, 1: 32, and 1: 512 for four sera in which the reference laboratories found a titre of 1: 1 024. When the test procedure employed by each labora- tory was analysed, marked differences in technique were found as described below. (a) Removal of nonspecific inhibitors. 5 labora- tories tested all sera twice using the kaolin and heparin-manganese chloride treatments in parallel, 6 used kaolin alone, and 6 used heparin-manganese chloride alone. The relative proportion of heparin to manganese chloride varied considerably. (b) Type of red blood cells used. 2 laboratories used red blood cells obtained from 1-day-old chicks; the remaining 15 used pigeon cells, which were obtained commercially by 11. (c) Source of antigen. The coordinating labora- tory prepared its own antigen by a method based on that of Liebhaber et al. (2); nine used antigen obtained from the Commonwealth Serum Labora- tories, Melbourne; and the remainder, which included the other reference laboratory, used commercial antigens obtained from a variety of sources. All laboratories used the microtitre system, with basic volumes of 0.025 ml or 0.050 ml. Table 1. Titres for all test sera _ 140 RUBELLA HAEMAGGLUTINATION-INHIBITION TESTS Table 2. Results obtained by 17 laboratories on test sera Reference No. of laboratories obtaining the H.I. titres shown below a2 Total No. oftests performedaoratOry (< 10) (10) (20) (40) (80) (160) (320) (640) (1 280) (2560) (5120) on sera of each < 8 8 16 32 64 128 256 512 1 024 2048 4096 titre < 8 75 4 2 3 1 85 16 2 1 6 4 2 1 1 17 64 2 2 7 12 7 3 1 34 128 2 1 10 20 12 6 51 256 2 4 30 50 25 6 1 1 119 512 1 2 10 15 14 9 51 1 024 2 5 12 26 15 6 2 68 4 096 2 1 8 3 3 17 a Some laboratories prepared their tests with sera initially diluted 1: 8, others started from 1: 10. The titres in parentheses are considered equivalent to those without parentheses. (d) Removal of nonspecific agglutinins. The cell concentration used for absorption varied from 0.25% to 50%; and that used in the test itself varied from 0.02% to 0.4%. (e) Incubation. The time and temperature of the antigen-antibody reaction ranged from 60 min at 37°C to 60 min at 4°C. Some laboratories used a mixture of temperatures. In all cases the red cell reaction was maintained at 4°C, but the reaction time varied from 30 min to overnight. After the plates had been removed from the refrigerator, they were left at room temperature for 30 min to 2 h before reading. (f) Starting dilutions. Starting dilutions of serum were 1: 8 (8 laboratories), 1 : 10 (6 laboratories), or 1: 20 (3 laboratories). DISCUSSION A detailed analysis of the results showed that 8 of the 17 laboratories involved were producing satis- factory and reproducible results, while the remainder were not. In some cases the serum from a seronega- tive person was reported as containing antibodies or vice versa and, in addition, some laboratories would have reported diagnostic rises in titre on pairs of sera that had stationary levels. Conversely, the failure of several laboratories to detect high titres per se could result in failure to detect true diagnostic rises. At the conclusion of the survey a meeting was held to discuss these findings. It appeared that the main causes of discrepant results were incomplete removal of nonspecific inhibitors of haemagglutina- tion, and incomplete absorption of agglutinins from the sera. The ability to detect four-fold rises in titre may vary with different antigen preparations, whilst the composition of the diluent used may affect anti- body titres and certainly has a marked effect on antigen titres. The proceedings of this meeting are to be published in full elsewhere (1). It was decided that an attempt would be made to standardize the technique used, and that sera of known titres should be available to each laboratory for regular inclusion in batches of tests. As a result of these recommendations, the virus laboratory at Fairfield Hospital has prepared and titrated a second batch of sera, which it is currently issuing every month to each participating laboratory. It is hoped that this voluntary quality control scheme will improve the reliability of the results obtained in different centres. ACKNOWLEDGEMENTS The authors acknowledge the help of Mr A. A. Murphy of the Institute of Clinical Pathology and Medical Research, Lidcombe, N.S.W., and of the directors and staff of the 17 participating laboratories. 3 141 142 1. D. GUST ET AL. RtSUMI ENQUETE SUR LA PRATIQUE DE L'EPREUVE D'INHIBITION DE L'HIMAGGLUTINATION POUR LE DIAGNOSTIC DE LA RUBIOLE DANS LES ETATS DU SUD DE L'AUSTRALIE En 1972, le Laboratoire regional OMS de reference pour les maladies a virus, a Melbourne (Australie), a fait une enquete sur la fiabilite et la reproductibilite des resul- tats des epreuves d'inhibition de l'hemagglutination (IH) pratiquees dans 17 laboratoires des Etats du sud de l'Australie. Chacun de ces laboratoires a requ pour titrage un jeu de 26 serums, dont 6 doubles, en 7 mois. Les resultats des tests et les details sur la technique IH employee par chaque laboratoire ont WtE analys6s au Laboratoire regional OMS de r6fdrence et a l'Institut de Pathologie clinique et de Recherche medicale, a Lid- combe, agissant comme centres de ref6rence. Sur les 442 dpreuves IH pratiquees, 282 (64%) don- naient des titres concordant, avec une tolerance de 1 dilution, avec ceux des centres de refdrence, 147 (33 %) des titres infdrieurs de 4 fois au moins, et 13 (3%) des titres superieurs de 4 fois au moins aux titres escomptes. On a releve un certain nombre de discordances graves et 8 laboratoires seulement sur 17 ont fourni des rdsultats satisfaisants et reproductibles. Il est apparu que les insuffisances constatees etaient dues a 1'emploi de techniques inadequates, et on a decid6 de tenter de mettre au point une epreuve IH standardis6e pour le titrage des anticorps antirubeoliques. REFERENCES 1. GUST, I. D., ed. A symposium on aspects of rubella serology. Aust. j. med. technol., 4, special issue, November 1973. 2. LIEBHABER, H. ET AL. Proc. Soc. Exp. Biol. Med., 230, 12 (1969).