Journal of Immunological Methods, 129 (1990) 111-117 Elsevier
111
JIM 05551
A sensitive and reproducible method for the assay of human islet cell antibodies C.C. Pilcher and R.B. Elliott School of Medicine, University of Auckland, Private Bag~Auckland, New Zealand (Received 26 April 1989, revised received 24 October 1989, accepted 8 January 1990)
The islet cell antibody test is increasingly used to predict insulin-dependent diabetes before it is clinically apparent. Four international workshops on the test have shown wide variations in reproducibility, sensitivity and specificity. This illustrates the need for a more clearly defined method. We report a technique which offers both increased sensitivity and reproducibility. Using this method 94% of newly diagnosed diabetic children have ICA levels > 10 international units, whereas only 1.5% of normal children are positive at his level, and 7% of first degree relatives (aged less then 20 years) of_IDDM patients. Key words: Islet cell antibody; lmmunofluorescent procedure; Insulin-dependent diabetes rnellitus; Prediction; Family study; Reproducibility
Introduction The presence of islet cell antibodies (ICA) has been used to predict the onset of insulin-dependent diabetes meUitus (IDDM) in first degree relatives of IDDM (Gorsuch et al., 1981; Betterle et al., 1984; Srikanta et al., 1985; Chase et al., 1987; Tam et al., 1988; Pilcher and Elliott, 1990) and in 'normal' populations (Maclaren et al., 1985; Bergua et al., 1987; Karjalainen, 1988; Bruining et al., 1989) and to differentiate the disease from non-insulin-dependent diabetes mellitus (Irvine et al., 1979; Gleichmann et al., 1984; Groop et al.,
Correspondence to: C.C. Pileher, Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. Abbreviations: ICA, islet cell antibody; IDDM, insulin-dependent diabetes mellitus; IDW, Immunology and Diabetes Workshop; IU, international unit; JDF, Juvenile Diabetes Foundation; PBS, phosphate-buffered saline.
1986; Kobayashi et al., 1986). Other clinical indications for ICA testing, which have been reviewed by Vardi et al. (1987), include predicting the conversion of gestational diabetics to permanent IDDM (Steel et al., 1980) predicting IDDM in patients with other autoimmune endocrinopathies (Betterle et al., 1987) and monitoring immunosuppressive treatment (Elliott et al., 1981; MandrupPoulsen et al., 1985; Sibley et al., 1985, Bizzaro et al., 1988; Boitard et al., 1988) and the immune response following the transplantation of either islet cells or whole pancreas into diabetic patients (Sutherland et al., 1984). The absence of ICA may also help in the clinical interpretation of atypical forms of IDDM. There is wide variation in the precision, sensitivity and specificity of the test. The results of the four international Immunology and Diabetes Workshops (IDW) on standardization of ICA showed within laboratory variations of up to six doubling dilutions, and up to nine doubling dilu-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
112 tions between laboratories (Bottazzo and Gleichman, 1986; Gleichman and Bottazzo, 1987; Boitard et al., 1988; Bonifacio et al., 1988). Not all of these variations could be accounted for by major methodological differences or corrected by conversion to international units (IU), though some laboratories showed increased concordance both between laboratories and over time when the Juvenile Diabetes Foundation (JDF) reference serum was made available (Bonifacio et al., 1988). Many otherwise specific and precise assays had unacceptably low sensitivity at less than 80 IU. Discordance between laboratories was greatest around the limits of detection. The presence of other antibodies such as antinuclear antibodies, pancreatic acinar cell antibodies or high background fluorescence (usually at high serum concentration), can lead to erroneous ICA results or make tests unreliable. We now sl,ow that these difficulties can be overcome by increasing the sensitivity of the test. Using this method, which incorporates a prolonged incubation step in the presence of a protease inhibitor (Pilcher and Elliott, 1984), it has been possible to obtain a level of sensitivity and reproducibility superior to that of any previously published method.
Experimental procedure (1) Pancreata from young group O kidney donors were collected premortem into a beaker in an ice bath. Fatty tissue was first removed before 5 mm cubes of pancreas were cut and then frozen in liquid nitrogen as quickly as possible before being stored in prechilled containers at - 7 0 ° C for up to 3 years. The whole procedure took less than 1 h. Some pancreata were not suitable as morphological changes had occurred (e.g., acinar degeneration), or the islets failed to bind antibody avidly. (2) Glass microscope slides were treated with silicone (see appendix) leaving uncoated 'wells' to permit ten sections per slide without sample mixing, and to speed up the handling time. No adhesive compounds were applied to the slides. (3) The base of the pancreatic block was mounted in OCT Compound (Tissue Tek, Miles
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Scientific), using dry ice to prevent thawing of the tissue. 4 # m sections were cut at - 2 0 ° C, placed on the unsiliconised portions of the slide and fan-dried at room temperature (RT) for approximately 1 h before immediate use or storage at - 7 0 ° C in an histology slide box for up to 1 month. On removal from - 7 0 o C, the slides were fan-dried to remove moisture. (4) Serum samples were initially diluted 1/32 (see Fig. 1) in phosphate-buffered saline (PBS) (NaC1 0.137 M; N a 2 H P O 4 0.008 M; KH2PO 4 0.0015 M; KC1 0.0027 M; pH 7.3) containing an aprotinin concentration of 2500 k l U / m l (Trasylol, Bayer) to inhibit the proteolytic activity of the exocrine pancreas. The 1 / 3 2 dilution (equivalent to 10 IU) was initially used for screening, and those demonstrating antibodies at the screening level were tritrated to end point. (5) 25 #1 of diluted serum were pipetted onto the pancreatic section and incubated in a small, airtight, horizontal chamber saturated with water vapour at room temperature for 24 h. The samples were then rinsed with PBS and the sections soaked in fresh PBS for 10 rain with occasional gentle manual agitation. (6) Without allowing the sections to dry, excess moisture was removed by giving the slide a sharp flip, then 20/~1 of rhodamine-labelled anti-human IgG (Atlantic Antibodies) diluted in PBS were applied to cover each entire pancreatic section. An optimum dilution of 1/500 was established for our conditions. The slides were incubated as above for 30 min at RT, washed as above, then mounted
113 TABLE I CHARACTERISTICS OF THE STUDY GROUPS Category Newly diagnosed IDDM children First degree relatives of IDDM patients Normal school children
Number
Age
100 335 276 605
5 months-14 years 2 weeks-9 years 10-19 years 20-75 years
467
dried. The siliconised slides were fixed by heating to 200 ° C for 2 h. 'Wells' for sections were created using a desiliconising solution of 15 N N a O H in glycerol at a ratio of 1 : 1. This was applied within circles scribed with a diamond pen. After 24 h the slides were rinsed and dried ready for use. Brief washing with detergent permitted re-usage of these slides many times.
5-15 years Results
with a coverslip and Dabco mounting medium (Johnson et al., 1982) to minimise fading. (7) The sections were observed on an Olympus epifluorescence microscope using a 100 W mercury vapour lamp, x 2 0 UV objective and rhodamine filters. Islet cytoplasmic fluorescence intensity was scored over an arbitrary scale of 5, and end-point titres assigned only if a negative was preceded by a weak positive. Where an antibody was only weakly detectable at the screening dilution, the result was confirmed by repeating the test together with a lower dilution. The weak reaction should then be preceded by a stronger reaction. (8) Conversion of end-point titres to IU is essential for interlaboratory comparisons. T o accomplish this, the J D F standard (80 U) was serially double diluted in a normal human serum found to have less than 0.3 IU and no other interfering factors. Each of these was then titrated to end point and the relationship between the J D F standard diluted in this way and the titre was shown to be linear (Fig. 1). Thereafter, the J D F standard (or an in-house standard calibrated against the J D F standard) was subsequently titrated to end point together with only two predilutions (1/8, 1/64) which normally plot as a straight line, and allows the conversion of titre to international units in this range. (9) Sera from newly diagnosed I D D M children, first degree relatives of insulin-dependent subjects, and normal children with characteristics shown in Table I were measured for ICA.
Reproducibility A plot of the end point titres of the J D F reference serum (80 IU) over 12 weeks demonstrates the between run variation (Fig. 2), and the threshold of sensitivity (0.32 IU + 1 dilution, i.e., 0.16-0.63). Participants of stage 2 of the IDW standardization of ICA received coded samples which included the standard serum serially doubly diluted and sent in duplicate. A second distribution of the same samples (recoded) allowed within run, between run and between laboratory analyses. Fig. 3, from the report of the second workshop by Bonifacio et al. (1988) (reproduced with permission from the Journal of Immunological Methods) demonstrates the reproducibility of the method. G o o d reproducibility was achieved by some laboratories, but with significantly lower sensitivity than the reported method. Laboratories I,J,M,N,P incorporated a prolonged incubation step, but reproducibility was lost indicating other aspects of the method were also important.
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Sensitivity
The results of the second IDW International Workshop demonstrated that higher ICA titres were reported by laboratories using prolonged incubation with aprotinin (Bonifacio et al., 1988). The 4th Workshop in Auckland further demonstrated this and also the difficulties in detecting antibodies when using insensitive methods. Out of nine laboratories, four were unable to detect antibodies at 20 IU and two gave discrepant results on duplicates. One laboratory was unable to detect 40 IU and two gave discrepant results (Fig. 4).
Laboratories B,C,D,E,G,I used 20-30 min incubations with standard conditions, laboratory F used protein A, and laboratory H used conditions similax to A (Auckland). 1CA in test sera
Of the 467 normal sera tested 460 (98.5%) showed levels of less than 10 IU, four (0.9%) at 10 IU, two (0.4%) at 20 IU, and one (0.2%) at 320 IU. Of the 100 newly diagnosed diabetic children, five showed levels less than 1.25 IU, one at 5 IU, 11 at 10 IU, 11 at 20 IU, 19 at 40 IU, 25 at 80 IU, 12 at
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160 IU, 14 at 320 IU, two at 640 IU. N o age relationship was seen. Using a dilution equivalent to 10 IU we established a sensitivity of 94% in the disease group, with a specificity of 98.5% compared with the 'normal' control group thus indicating an acceptably small overlap of the two populations. At 20 U the sensitivity was 89% and specificity was 99.4% (Fig. 5) A cut off point of 10 IU was used thereafter. In a group of 1216 first degree relatives, antibodies were detected in 6.3% with no significant difference in the percentages at any age. A m o n g the 611 aged less than 20 years, 93% had levels less than 10 IU, 13 (2.1%) at 10 IU and 30 (4.9%) between 20 and 640 IU.
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116
Discussion and conclusions
Acknowledgements
A m o n g the possible reasons for poor interlaboratory correlation of islet cell antibody levels, factors such as morphologic and antigenic quality of the pancreatic substrate, the specificity and strength of the conjugate, and the microscope efficiency m a y all be important. The use of an internationally accepted standard serum has helped to correlate results from some laboratories with others only where the inherent reproducibility of these methods has been satisfactory. E n h a n c e m e n t of the sensitivity of the m e t h o d can be obtained by prolonged incubation of the test serum with the pancreatic substrate in the presence of a protease inhibitor (Pilcher and EIliott, 1984; Landin-Olsson et al., 1987; Bonifacio et al., 1988). The increased sensitivity of this method permits relatively high serum dilutions to be used thereby avoiding m a n y of the b a c k g r o u n d problems encountered at low serum dilutions, while still permitting detection of low levels of ICA. T h e ability to measure levels of antibody lower than the desired cut off or screening dilution improves the reproducibility of the test at that level by allowing confirmation o f 'borderline' reactions. A review of the recent literature shows that 59-86% of newly diagnosed diabetics have detectable ICA. With this method we find 94% > 10 IU. Whilst it is possible that the presence of I C A in I D D M m a y have a different geographic prevalence, it is much more likely that m a n y of the samples considered to be I C A negative had low levels of the antibody detectable by the m e t h o d used. F r o m the results shown above, it is clearly important to have an assay capable of reliably measuring levels of a n t i b o d y as low as 10 I U in order to assign clinical significance. Laboratories which assign a n t i b o d y 'negative' status to a given serum will often be in error if their lower limit of reproducible result is 20 or m o r e I U and confusion or lack of confidence m a y arise in the use of this test to predict diabetes. The assay described can be used to measure I C A levels d o w n to 0.3 I U and should prove useful in a variety of clinical contexts.
This work was funded by the National Child Health Research Foundation. We wish to thank Professor D. O'Brien for his helpful c o m m e n t s in the preparation of this manuscript, and D i a n a Wright for secretarial assistance.
References Bergua, M., Sole, J., Marion, G., Perez, M.C., Recasens, A., Fernandez, J., Casamitjana, R. and Gomis, R. (1987) Prevalence of islet cell antibodies, insulin antibodies and hyperglycaemia in 2291 schoolchildren. Diabetologia 30, 724. Betterle, C., Zanette, F., Pedini, B., Presotto, F., Rapp, L.B., Moncioni, C.M. and Rigon, F. (1984) Clinical and subclinical organ specific autoimmune manifestations in Type I (insulin-dependent) diabetic patients, and their first degree relatives. Diabetologia 26, 431. Betterle, C., Presotto, F., Pedini, B., Mort, L., Slack, R.S., Zanette, F. and Zanchetta, R. (1987) Islet cell and insulin autoantibodies in organ-specific autoimmune patients. Their behaviour and predictive value for the development of Type 1 (insulin-dependent) diabetes mellitus. A 10 year follow-up study. Diabetologia 30, 292. Bizzaro, A., De Bcllis, A., Marfella, A., Gentile, S., Di Martino, G., Amoresano Paglionico, V. and lacono, G. (1988) Effect of a prednisone and azothiaprine treatment on patients with impaired glucose tolerance. Diabetes Res. Clin. Pract. 5 (suppl. 1), $181. • Boitard, C., Debray-Sachs, M., Chatenoud, L. and Bach, J.-F. (1988a) The effect of cyclosporin A on humoral and cellular immunity in insulin dependent (Type 1) diabetes mellitus. Transplant. Proc. 3 (suppl. 4), 193. Boitard, C., Bonifacio, E., Bottazzo, G.F., Gleichman, H. and Molenaar, J. (1988b) Immunology and diabetes workshop: report of the third international (stage 3) workshop on the standardization of cytoplasmic islet cell antibodies. Diabetologia 31,451. Bonifacio, E., Dawkins, R.L. and Lemmark, A. (1987) Immunology and diabetes workshops: report of the second international workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetoiogia 30, 273. Bonifacio, E., Lcrnmark, A., Dawkins, R.L. and co-authors (1988) Serum exchange and use of dilutions have improved precision of measurement of islet cell antibodies. J. Immunol. Methods 106, 83. Bottazzo, G.F. and Gleichmann, H. (1986) Immunology and Diabetes Workshop. Report of the first international workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetologia 29, 125. Bruining, GJ., Molenaar, J.L., Grobbee, D.E., Hofman, A., Scbeffer, G.J., Bruining. H.A., De Brnyn, A.M. and Valkenburg, H.A. (1989). Ten-year follow-up study of
117 islet-cell antibodies and childhood diabetes mellitus. Lancet i, 1100. Chase, H.P., Voss, M.A., Butler-Simon, N., Hoops, S., O'Brien, D. and Doberson, M.J. (1987) diagnosis of pre-type 1 diabetes. J. Paediatr. 111, 807. Elliott, R.B., Crossley, J.R., Berryman, C.C. and James, A.G. (1981) Partial preservation of p-cell function in children with diabetes. Lancet ii, 1. Gleichmann, H. and Bottazzo, G.F. (1987) Progress toward standardisation of the cytoplasmic islet-cell antibody assay. Diabetes 36, 578. Gleichmann, H., Zorcher, B., Greulich, B., Giles, F.A., Henrichs, HAL, Bertrams, J. and Kolb, H. (1984) Correlation of islet cell antibodies and HLA-DR phenotypes with diabetes mellitus in adults. Diabetologia 27, 90. Gorsuch, A.N., Spencer, K.M., Lister, J., McNally, J.M., Dean, B.M., Bottazzo, G.F. and Cudworth, A.G. (1981) Evidence for a long prediabetic period in Type I (insulin dependent) diabetes mellitus. Lancet ii, 1363. Groop, L.C., Bottazzo, G.F. and Doniach, D. (1986) Islet cell antibodies identify latent Type I diabetes in patients aged 35-75 years at diagnosis. Diabetes 35, 237. lrvine, W.J., Javers, J.S.A., F e e l C.M., Prescott, R.J. and Duncan, UJ.P. (1979) The value of islet cell antibody in predicting secondary failure of oral hypoglycaemic agent therapy in diabetes mellitus. J. Clin. Lab. Immunol. 2, 23. Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J. (1982) Fading of immunofiuorescence during microscopy: a study of the phenomenon and its remedy. J. lmmunol. Methods 2, 231. Karjaiainen, J. (1988) Prevalence of cytoplasmic islet cell antibodies in a Finnish 3-18 year old general population - a cross-sectional study of 1206 subjects. Diabetes Res. Clin. Pract. 5 (suppl. 1), $254. Kobayashi, T., Sugimoto, T., ltoh, T., Kosaka, K., Tanaka, T., Suwa, S., Sato, K. and Tsuji, K. (1986) The prevalence of islet cell antibodies in Japanese insulin-dependent and non-insulin dependent diabetic patients studied by indirect immunofluorescence and by a new method. Diabetes 35, 335. Landin-Olsson, M., Sundqvist, G. and Lernmark, A. (1987) Prolonged incubation in the two-colour immunofluores-
cence test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus. Diabetologia 30, 327. Maclaren, N.J., Home, G., Spillar, R.P., Barbour, H., Harrison, D. and Duncan, J. (1985) Islet cell autoantibodies (ICA) in U.S. school children. Diabetes 34 (suppl. 1) 335. Mandrup-Poulsen, T., Nerup, J., Stiller, C.R., Mamer, B., Bille, G., Heinilchs, D., Martell, R., Dupre, J., Keown, P.A., Jenner, M.R., Rodger, N.W., Wolfe, B., Graffeniled, B.V. and Binder, C. (1985) Disappearance and reappearance of islet cell cytoplasmic antibodies in cyclosporintreated insulin-dependent diabetics. Lancet i, 599. Pilcher, C. and Elliott, R.B. (1984) Improved sensitivity of islet cell cytoplasmic antibody assay in diabetics. Lancet i. 1352. Pilcher, C.C. and Elliott, R.B. (1990) The ontogeny of islet cell antibodies (ICA) in insulin dependent diabetic children and their immediate family. Immunology of Diabetes IX International Workshop (1988). J. Autoimmun., in press. Sibley, R.K., Sutherland, D.E.R., Goetz, F. and Michael, A.F. (1985) Recurrent diabetes mellitus in the pancreas iso and allograft: a light and electron microscopic and immunohistochemical analysis of four cases. Lab. Invest. 53, 132. Srikanta, S., Ganda, O.P., Rabizadeh, A., Soeldner, J.S. and Eisenbarth, G.S. (1985) First-degree relatives of patients with Type I diabetes mellitus. Islet-cell antibodies and abnormal insulin secretion. New Engl. J. Med. 313, 461. Steel, J.M., Irvine, W.J. and Clarke, B.F. (1980) The significance of pancreatic islet cell antibody and abnormal glucose tolerance during pregnancy. J. Clin. Lab. lmmunol. 4, 83. Sutherland, D.E.R., Sibley, R., Xu, X.-Z., Michael, A., Srikama, S., Taub, F., Najarian, J. and Goetz, F.C. (1984) Twin-totwin pancreas transplantation: reversal and reenactment of the pathogenesis of type 1 diabetes. Transplant. Assoc. Am. Physicians 97, 80. Tam, A.C., Dean, B.M., Schwarz, G., Thomas, J.M., lngram, D., Bottazzo, G.F. and Gale, E.A.M (1988) Predicting insulin dependent diabetes. Lancet i, 845. Vardi, P., Dibella, E.E., Pasquarello, T.J. and Srikanta. S. (1987) Islet cell autoantibodies: Pathobiology and clinical applications (Review). Diabetes Care 10, 645.
111
JIM 05551
A sensitive and reproducible method for the assay of human islet cell antibodies C.C. Pilcher and R.B. Elliott School of Medicine, University of Auckland, Private Bag~Auckland, New Zealand (Received 26 April 1989, revised received 24 October 1989, accepted 8 January 1990)
The islet cell antibody test is increasingly used to predict insulin-dependent diabetes before it is clinically apparent. Four international workshops on the test have shown wide variations in reproducibility, sensitivity and specificity. This illustrates the need for a more clearly defined method. We report a technique which offers both increased sensitivity and reproducibility. Using this method 94% of newly diagnosed diabetic children have ICA levels > 10 international units, whereas only 1.5% of normal children are positive at his level, and 7% of first degree relatives (aged less then 20 years) of_IDDM patients. Key words: Islet cell antibody; lmmunofluorescent procedure; Insulin-dependent diabetes rnellitus; Prediction; Family study; Reproducibility
Introduction The presence of islet cell antibodies (ICA) has been used to predict the onset of insulin-dependent diabetes meUitus (IDDM) in first degree relatives of IDDM (Gorsuch et al., 1981; Betterle et al., 1984; Srikanta et al., 1985; Chase et al., 1987; Tam et al., 1988; Pilcher and Elliott, 1990) and in 'normal' populations (Maclaren et al., 1985; Bergua et al., 1987; Karjalainen, 1988; Bruining et al., 1989) and to differentiate the disease from non-insulin-dependent diabetes mellitus (Irvine et al., 1979; Gleichmann et al., 1984; Groop et al.,
Correspondence to: C.C. Pileher, Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. Abbreviations: ICA, islet cell antibody; IDDM, insulin-dependent diabetes mellitus; IDW, Immunology and Diabetes Workshop; IU, international unit; JDF, Juvenile Diabetes Foundation; PBS, phosphate-buffered saline.
1986; Kobayashi et al., 1986). Other clinical indications for ICA testing, which have been reviewed by Vardi et al. (1987), include predicting the conversion of gestational diabetics to permanent IDDM (Steel et al., 1980) predicting IDDM in patients with other autoimmune endocrinopathies (Betterle et al., 1987) and monitoring immunosuppressive treatment (Elliott et al., 1981; MandrupPoulsen et al., 1985; Sibley et al., 1985, Bizzaro et al., 1988; Boitard et al., 1988) and the immune response following the transplantation of either islet cells or whole pancreas into diabetic patients (Sutherland et al., 1984). The absence of ICA may also help in the clinical interpretation of atypical forms of IDDM. There is wide variation in the precision, sensitivity and specificity of the test. The results of the four international Immunology and Diabetes Workshops (IDW) on standardization of ICA showed within laboratory variations of up to six doubling dilutions, and up to nine doubling dilu-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
112 tions between laboratories (Bottazzo and Gleichman, 1986; Gleichman and Bottazzo, 1987; Boitard et al., 1988; Bonifacio et al., 1988). Not all of these variations could be accounted for by major methodological differences or corrected by conversion to international units (IU), though some laboratories showed increased concordance both between laboratories and over time when the Juvenile Diabetes Foundation (JDF) reference serum was made available (Bonifacio et al., 1988). Many otherwise specific and precise assays had unacceptably low sensitivity at less than 80 IU. Discordance between laboratories was greatest around the limits of detection. The presence of other antibodies such as antinuclear antibodies, pancreatic acinar cell antibodies or high background fluorescence (usually at high serum concentration), can lead to erroneous ICA results or make tests unreliable. We now sl,ow that these difficulties can be overcome by increasing the sensitivity of the test. Using this method, which incorporates a prolonged incubation step in the presence of a protease inhibitor (Pilcher and Elliott, 1984), it has been possible to obtain a level of sensitivity and reproducibility superior to that of any previously published method.
Experimental procedure (1) Pancreata from young group O kidney donors were collected premortem into a beaker in an ice bath. Fatty tissue was first removed before 5 mm cubes of pancreas were cut and then frozen in liquid nitrogen as quickly as possible before being stored in prechilled containers at - 7 0 ° C for up to 3 years. The whole procedure took less than 1 h. Some pancreata were not suitable as morphological changes had occurred (e.g., acinar degeneration), or the islets failed to bind antibody avidly. (2) Glass microscope slides were treated with silicone (see appendix) leaving uncoated 'wells' to permit ten sections per slide without sample mixing, and to speed up the handling time. No adhesive compounds were applied to the slides. (3) The base of the pancreatic block was mounted in OCT Compound (Tissue Tek, Miles
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x
11128
x
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20
40
80
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Fig. 1. Relationship between JDF standard and end point titre. (A) Initial dilution of JDF standard in normal human serum. (B) International units present in each preparation.
Scientific), using dry ice to prevent thawing of the tissue. 4 # m sections were cut at - 2 0 ° C, placed on the unsiliconised portions of the slide and fan-dried at room temperature (RT) for approximately 1 h before immediate use or storage at - 7 0 ° C in an histology slide box for up to 1 month. On removal from - 7 0 o C, the slides were fan-dried to remove moisture. (4) Serum samples were initially diluted 1/32 (see Fig. 1) in phosphate-buffered saline (PBS) (NaC1 0.137 M; N a 2 H P O 4 0.008 M; KH2PO 4 0.0015 M; KC1 0.0027 M; pH 7.3) containing an aprotinin concentration of 2500 k l U / m l (Trasylol, Bayer) to inhibit the proteolytic activity of the exocrine pancreas. The 1 / 3 2 dilution (equivalent to 10 IU) was initially used for screening, and those demonstrating antibodies at the screening level were tritrated to end point. (5) 25 #1 of diluted serum were pipetted onto the pancreatic section and incubated in a small, airtight, horizontal chamber saturated with water vapour at room temperature for 24 h. The samples were then rinsed with PBS and the sections soaked in fresh PBS for 10 rain with occasional gentle manual agitation. (6) Without allowing the sections to dry, excess moisture was removed by giving the slide a sharp flip, then 20/~1 of rhodamine-labelled anti-human IgG (Atlantic Antibodies) diluted in PBS were applied to cover each entire pancreatic section. An optimum dilution of 1/500 was established for our conditions. The slides were incubated as above for 30 min at RT, washed as above, then mounted
113 TABLE I CHARACTERISTICS OF THE STUDY GROUPS Category Newly diagnosed IDDM children First degree relatives of IDDM patients Normal school children
Number
Age
100 335 276 605
5 months-14 years 2 weeks-9 years 10-19 years 20-75 years
467
dried. The siliconised slides were fixed by heating to 200 ° C for 2 h. 'Wells' for sections were created using a desiliconising solution of 15 N N a O H in glycerol at a ratio of 1 : 1. This was applied within circles scribed with a diamond pen. After 24 h the slides were rinsed and dried ready for use. Brief washing with detergent permitted re-usage of these slides many times.
5-15 years Results
with a coverslip and Dabco mounting medium (Johnson et al., 1982) to minimise fading. (7) The sections were observed on an Olympus epifluorescence microscope using a 100 W mercury vapour lamp, x 2 0 UV objective and rhodamine filters. Islet cytoplasmic fluorescence intensity was scored over an arbitrary scale of 5, and end-point titres assigned only if a negative was preceded by a weak positive. Where an antibody was only weakly detectable at the screening dilution, the result was confirmed by repeating the test together with a lower dilution. The weak reaction should then be preceded by a stronger reaction. (8) Conversion of end-point titres to IU is essential for interlaboratory comparisons. T o accomplish this, the J D F standard (80 U) was serially double diluted in a normal human serum found to have less than 0.3 IU and no other interfering factors. Each of these was then titrated to end point and the relationship between the J D F standard diluted in this way and the titre was shown to be linear (Fig. 1). Thereafter, the J D F standard (or an in-house standard calibrated against the J D F standard) was subsequently titrated to end point together with only two predilutions (1/8, 1/64) which normally plot as a straight line, and allows the conversion of titre to international units in this range. (9) Sera from newly diagnosed I D D M children, first degree relatives of insulin-dependent subjects, and normal children with characteristics shown in Table I were measured for ICA.
Reproducibility A plot of the end point titres of the J D F reference serum (80 IU) over 12 weeks demonstrates the between run variation (Fig. 2), and the threshold of sensitivity (0.32 IU + 1 dilution, i.e., 0.16-0.63). Participants of stage 2 of the IDW standardization of ICA received coded samples which included the standard serum serially doubly diluted and sent in duplicate. A second distribution of the same samples (recoded) allowed within run, between run and between laboratory analyses. Fig. 3, from the report of the second workshop by Bonifacio et al. (1988) (reproduced with permission from the Journal of Immunological Methods) demonstrates the reproducibility of the method. G o o d reproducibility was achieved by some laboratories, but with significantly lower sensitivity than the reported method. Laboratories I,J,M,N,P incorporated a prolonged incubation step, but reproducibility was lost indicating other aspects of the method were also important.
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Sensitivity
The results of the second IDW International Workshop demonstrated that higher ICA titres were reported by laboratories using prolonged incubation with aprotinin (Bonifacio et al., 1988). The 4th Workshop in Auckland further demonstrated this and also the difficulties in detecting antibodies when using insensitive methods. Out of nine laboratories, four were unable to detect antibodies at 20 IU and two gave discrepant results on duplicates. One laboratory was unable to detect 40 IU and two gave discrepant results (Fig. 4).
Laboratories B,C,D,E,G,I used 20-30 min incubations with standard conditions, laboratory F used protein A, and laboratory H used conditions similax to A (Auckland). 1CA in test sera
Of the 467 normal sera tested 460 (98.5%) showed levels of less than 10 IU, four (0.9%) at 10 IU, two (0.4%) at 20 IU, and one (0.2%) at 320 IU. Of the 100 newly diagnosed diabetic children, five showed levels less than 1.25 IU, one at 5 IU, 11 at 10 IU, 11 at 20 IU, 19 at 40 IU, 25 at 80 IU, 12 at
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160 IU, 14 at 320 IU, two at 640 IU. N o age relationship was seen. Using a dilution equivalent to 10 IU we established a sensitivity of 94% in the disease group, with a specificity of 98.5% compared with the 'normal' control group thus indicating an acceptably small overlap of the two populations. At 20 U the sensitivity was 89% and specificity was 99.4% (Fig. 5) A cut off point of 10 IU was used thereafter. In a group of 1216 first degree relatives, antibodies were detected in 6.3% with no significant difference in the percentages at any age. A m o n g the 611 aged less than 20 years, 93% had levels less than 10 IU, 13 (2.1%) at 10 IU and 30 (4.9%) between 20 and 640 IU.
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116
Discussion and conclusions
Acknowledgements
A m o n g the possible reasons for poor interlaboratory correlation of islet cell antibody levels, factors such as morphologic and antigenic quality of the pancreatic substrate, the specificity and strength of the conjugate, and the microscope efficiency m a y all be important. The use of an internationally accepted standard serum has helped to correlate results from some laboratories with others only where the inherent reproducibility of these methods has been satisfactory. E n h a n c e m e n t of the sensitivity of the m e t h o d can be obtained by prolonged incubation of the test serum with the pancreatic substrate in the presence of a protease inhibitor (Pilcher and EIliott, 1984; Landin-Olsson et al., 1987; Bonifacio et al., 1988). The increased sensitivity of this method permits relatively high serum dilutions to be used thereby avoiding m a n y of the b a c k g r o u n d problems encountered at low serum dilutions, while still permitting detection of low levels of ICA. T h e ability to measure levels of antibody lower than the desired cut off or screening dilution improves the reproducibility of the test at that level by allowing confirmation o f 'borderline' reactions. A review of the recent literature shows that 59-86% of newly diagnosed diabetics have detectable ICA. With this method we find 94% > 10 IU. Whilst it is possible that the presence of I C A in I D D M m a y have a different geographic prevalence, it is much more likely that m a n y of the samples considered to be I C A negative had low levels of the antibody detectable by the m e t h o d used. F r o m the results shown above, it is clearly important to have an assay capable of reliably measuring levels of a n t i b o d y as low as 10 I U in order to assign clinical significance. Laboratories which assign a n t i b o d y 'negative' status to a given serum will often be in error if their lower limit of reproducible result is 20 or m o r e I U and confusion or lack of confidence m a y arise in the use of this test to predict diabetes. The assay described can be used to measure I C A levels d o w n to 0.3 I U and should prove useful in a variety of clinical contexts.
This work was funded by the National Child Health Research Foundation. We wish to thank Professor D. O'Brien for his helpful c o m m e n t s in the preparation of this manuscript, and D i a n a Wright for secretarial assistance.
References Bergua, M., Sole, J., Marion, G., Perez, M.C., Recasens, A., Fernandez, J., Casamitjana, R. and Gomis, R. (1987) Prevalence of islet cell antibodies, insulin antibodies and hyperglycaemia in 2291 schoolchildren. Diabetologia 30, 724. Betterle, C., Zanette, F., Pedini, B., Presotto, F., Rapp, L.B., Moncioni, C.M. and Rigon, F. (1984) Clinical and subclinical organ specific autoimmune manifestations in Type I (insulin-dependent) diabetic patients, and their first degree relatives. Diabetologia 26, 431. Betterle, C., Presotto, F., Pedini, B., Mort, L., Slack, R.S., Zanette, F. and Zanchetta, R. (1987) Islet cell and insulin autoantibodies in organ-specific autoimmune patients. Their behaviour and predictive value for the development of Type 1 (insulin-dependent) diabetes mellitus. A 10 year follow-up study. Diabetologia 30, 292. Bizzaro, A., De Bcllis, A., Marfella, A., Gentile, S., Di Martino, G., Amoresano Paglionico, V. and lacono, G. (1988) Effect of a prednisone and azothiaprine treatment on patients with impaired glucose tolerance. Diabetes Res. Clin. Pract. 5 (suppl. 1), $181. • Boitard, C., Debray-Sachs, M., Chatenoud, L. and Bach, J.-F. (1988a) The effect of cyclosporin A on humoral and cellular immunity in insulin dependent (Type 1) diabetes mellitus. Transplant. Proc. 3 (suppl. 4), 193. Boitard, C., Bonifacio, E., Bottazzo, G.F., Gleichman, H. and Molenaar, J. (1988b) Immunology and diabetes workshop: report of the third international (stage 3) workshop on the standardization of cytoplasmic islet cell antibodies. Diabetologia 31,451. Bonifacio, E., Dawkins, R.L. and Lemmark, A. (1987) Immunology and diabetes workshops: report of the second international workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetoiogia 30, 273. Bonifacio, E., Lcrnmark, A., Dawkins, R.L. and co-authors (1988) Serum exchange and use of dilutions have improved precision of measurement of islet cell antibodies. J. Immunol. Methods 106, 83. Bottazzo, G.F. and Gleichmann, H. (1986) Immunology and Diabetes Workshop. Report of the first international workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetologia 29, 125. Bruining, GJ., Molenaar, J.L., Grobbee, D.E., Hofman, A., Scbeffer, G.J., Bruining. H.A., De Brnyn, A.M. and Valkenburg, H.A. (1989). Ten-year follow-up study of
117 islet-cell antibodies and childhood diabetes mellitus. Lancet i, 1100. Chase, H.P., Voss, M.A., Butler-Simon, N., Hoops, S., O'Brien, D. and Doberson, M.J. (1987) diagnosis of pre-type 1 diabetes. J. Paediatr. 111, 807. Elliott, R.B., Crossley, J.R., Berryman, C.C. and James, A.G. (1981) Partial preservation of p-cell function in children with diabetes. Lancet ii, 1. Gleichmann, H. and Bottazzo, G.F. (1987) Progress toward standardisation of the cytoplasmic islet-cell antibody assay. Diabetes 36, 578. Gleichmann, H., Zorcher, B., Greulich, B., Giles, F.A., Henrichs, HAL, Bertrams, J. and Kolb, H. (1984) Correlation of islet cell antibodies and HLA-DR phenotypes with diabetes mellitus in adults. Diabetologia 27, 90. Gorsuch, A.N., Spencer, K.M., Lister, J., McNally, J.M., Dean, B.M., Bottazzo, G.F. and Cudworth, A.G. (1981) Evidence for a long prediabetic period in Type I (insulin dependent) diabetes mellitus. Lancet ii, 1363. Groop, L.C., Bottazzo, G.F. and Doniach, D. (1986) Islet cell antibodies identify latent Type I diabetes in patients aged 35-75 years at diagnosis. Diabetes 35, 237. lrvine, W.J., Javers, J.S.A., F e e l C.M., Prescott, R.J. and Duncan, UJ.P. (1979) The value of islet cell antibody in predicting secondary failure of oral hypoglycaemic agent therapy in diabetes mellitus. J. Clin. Lab. Immunol. 2, 23. Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J. (1982) Fading of immunofiuorescence during microscopy: a study of the phenomenon and its remedy. J. lmmunol. Methods 2, 231. Karjaiainen, J. (1988) Prevalence of cytoplasmic islet cell antibodies in a Finnish 3-18 year old general population - a cross-sectional study of 1206 subjects. Diabetes Res. Clin. Pract. 5 (suppl. 1), $254. Kobayashi, T., Sugimoto, T., ltoh, T., Kosaka, K., Tanaka, T., Suwa, S., Sato, K. and Tsuji, K. (1986) The prevalence of islet cell antibodies in Japanese insulin-dependent and non-insulin dependent diabetic patients studied by indirect immunofluorescence and by a new method. Diabetes 35, 335. Landin-Olsson, M., Sundqvist, G. and Lernmark, A. (1987) Prolonged incubation in the two-colour immunofluores-
cence test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus. Diabetologia 30, 327. Maclaren, N.J., Home, G., Spillar, R.P., Barbour, H., Harrison, D. and Duncan, J. (1985) Islet cell autoantibodies (ICA) in U.S. school children. Diabetes 34 (suppl. 1) 335. Mandrup-Poulsen, T., Nerup, J., Stiller, C.R., Mamer, B., Bille, G., Heinilchs, D., Martell, R., Dupre, J., Keown, P.A., Jenner, M.R., Rodger, N.W., Wolfe, B., Graffeniled, B.V. and Binder, C. (1985) Disappearance and reappearance of islet cell cytoplasmic antibodies in cyclosporintreated insulin-dependent diabetics. Lancet i, 599. Pilcher, C. and Elliott, R.B. (1984) Improved sensitivity of islet cell cytoplasmic antibody assay in diabetics. Lancet i. 1352. Pilcher, C.C. and Elliott, R.B. (1990) The ontogeny of islet cell antibodies (ICA) in insulin dependent diabetic children and their immediate family. Immunology of Diabetes IX International Workshop (1988). J. Autoimmun., in press. Sibley, R.K., Sutherland, D.E.R., Goetz, F. and Michael, A.F. (1985) Recurrent diabetes mellitus in the pancreas iso and allograft: a light and electron microscopic and immunohistochemical analysis of four cases. Lab. Invest. 53, 132. Srikanta, S., Ganda, O.P., Rabizadeh, A., Soeldner, J.S. and Eisenbarth, G.S. (1985) First-degree relatives of patients with Type I diabetes mellitus. Islet-cell antibodies and abnormal insulin secretion. New Engl. J. Med. 313, 461. Steel, J.M., Irvine, W.J. and Clarke, B.F. (1980) The significance of pancreatic islet cell antibody and abnormal glucose tolerance during pregnancy. J. Clin. Lab. lmmunol. 4, 83. Sutherland, D.E.R., Sibley, R., Xu, X.-Z., Michael, A., Srikama, S., Taub, F., Najarian, J. and Goetz, F.C. (1984) Twin-totwin pancreas transplantation: reversal and reenactment of the pathogenesis of type 1 diabetes. Transplant. Assoc. Am. Physicians 97, 80. Tam, A.C., Dean, B.M., Schwarz, G., Thomas, J.M., lngram, D., Bottazzo, G.F. and Gale, E.A.M (1988) Predicting insulin dependent diabetes. Lancet i, 845. Vardi, P., Dibella, E.E., Pasquarello, T.J. and Srikanta. S. (1987) Islet cell autoantibodies: Pathobiology and clinical applications (Review). Diabetes Care 10, 645.
