HLA Class I, II, and III Polymorphism in Italian Patients With Sarcoidosis: Methods

Subjects
We studied 107 patients (64 female and 43 male) with newly recognized, biopsy specimen-proven sarcoidosis admitted to our departments (Pavia and Padova) from May 1988 to April 1991. The mean age at the onset of the disease was 36.08 years (39.3 years for female subjects and 31.13 years for male subjects), ranging from 18 to 64 years. As controls, the HLA phenotype frequencies obtained in 510 healthy individuals from continental Italy and reported in the Data Book Addendum—Anthropological Analysis, from the 11th Histocompatibility Workshop book were employed. BF allotyping was performed in 382, and C4A and C4B allotyping in 370 healthy northern Italian control subjects.
Assessment of Sarcoidosis
Patients with sarcoid were assessed by bronchoalveolar lavage, Ga lung scanning with computer-assisted score expressed Ga uptake, pulmonary function tests, and serum angiotensin-converting enzyme activity. The chest radiograph was evaluated according to De Remee and patients were assigned to stage I (bilateral hilar lymphoadenopathy [BHL], II (BHL and diffuse pulmonary involvement), or III (diffuse pulmonary involvement only). Typing for HLA class I and II antigens was performed in all patients with a set of sera, whose reliability was proven during the Tenth International Histocompatibility Workshop, using the National Institutes of Health microlymphocytotoxicity method. The technique of resetting with sheep erythrocytes (SRBC) was employed to remove T cells and obtain enriched В-cell suspensions, as previously described.
HLA Class III Typing
BF allotyping was carried out by high-voltage agarose electrophoresis of sera on 1 percent w/v agarose (Seakem, Maine) followed by immunofixation, as described by Alper and colleagues. For the BF nomenclature, see Geserick et al.
C4 allotyping was performed on serum samples carefully stored at — 80°C until the assay was carried out. Samples were treated overnight at 21°C with neuraminidase from Clostridium perfringens (Sigma type VIII, St. Louis, Mo) at a final concentration of 5 U/ml serum. Electrophoresis and immunofixation of the C4 bands achieved using anti-C4 complement (Atlantic Antibodies, Scarborough, Maine) were performed as described by Awdeh and Alper. Functional detection of the C4 bands was performed using an overlay of sensitized SRBC and C4-deficient guinea pig serum in agarose. For the C4 nomenclature, see MaufFet al.
Statistical Analysis
Associations between HLA markers and sarcoidosis were analyzed by 2×2 contingency tables, x2 with Yates’ correction and Fishers probability values were calculated for all the defined HLA specificities and, when necessary, p values were calculated for the degrees of freedom. In addition, probability values were corrected for multiple comparisons (number of antigens tested) (pc).
For the HLA complement markers, we are able to detect all the alleles presented in the articles of Geserick and colleagues and of Mauff and colleagues.
Relative risk (RR) was calculated according to Woolf’s method, reviewed by Svejgaard and colleagues.

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