alpha-1 antitrypsin phenotypes by isoelectric focusing in a metropolitan southern Chinese population.

AIMS/BACKGROUND: alpha-1 antitrypsin (alpha1AT) is an abundant protease inhibitor in human plasma. Its phenotypic variability has been reported to be associated with pulmonary emphysema and chronic liver diseases. However, alpha1AT deficiency is an uncommon condition in the Chinese population. The aim of this study was to describe the phenotypic distribution of alpha1AT in a southern Chinese population. METHODS: A total of 1085 healthy blood donors underwent alpha1AT phenotyping by isoelectric focusing. RESULTS: Two thirds (66.1%) were homozygous for either M1 or M2, whereas 32.6% were heterozygous for two different M phenotypes. The frequency of allelic variants was only 0.007, and deficiency variants were absent. Compared with earlier studies on southern Chinese populations, this study found a lower frequency of M2, and a higher number of allelic variants, including E, L, N, P, and S. This phenomenon can be attributed to population migration and mixing. CONCLUSIONS: An understanding of the alpha1AT pattern is important for evaluating the predisposition of the population to selected clinical diseases.

Heterogeneity of anti-PR3 associated disease in Hong Kong.

Thirty seven patients tested positive for antibody against proteinase 3 (anti-PR3) using either one of the two in-house ELISA methods at the Immunology Laboratory, University of Hong Kong. All except three were Chinese. Systemic vasculitis was diagnosed in 13 patients. However, the positive predictive value of anti-PR3 for Wegener's granulomatosus was low (22%). The commonest presenting feature was respiratory (32%). Both respiratory and renal symptoms were commonly reported in anti-PR3 positive patients as in previous studies. In this largely Chinese population, there is marked variability in the spectrum of diseases associated with anti-PR3; though many had vasculitic diseases, some were diagnosed with other conditions such as inflammatory bowel diseases and respiratory infection. In contrast with series in white people, antimyeloperoxidase rather than anti-PR3 associated disease is more prevalent in Chinese patients.

A latex slide agglutination test for rapid detection of antimyeloperoxidase antibody.

AIM: To develop and test a new latex slide agglutination test (MPO-LSAT) to detect antimyeloperoxidase (anti-MPO) antibody in serum. METHODS: Latex bead coating was adjusted to give maximum sensitivity by attending to latex size, MPO to latex ratio for coupling, ratio of diluted serum to MPO-latex, reaction time and temperature for coupling, and reaction time for agglutination. Inhibition studies were performed using MPO, proteinase 3, bactericidal/permeability increasing protein, and lactoferrin. RESULTS: There was very good correlation between this test and the conventional anti-MPO enzyme linked immunosorbent assay (ELISA): 81% of sera positive in the ELISA were positive by MPO-LSAT. MPO-LSAT results correlated better with IgM anti-MPO than with IgG anti-MPO. CONCLUSIONS: MPO-LSAT is a simple diagnostic test that is potentially useful in the clinical laboratory as a rapid screening tool for vasculitic diseases.

Comparison of counter immunoelectrophoresis with immunoblotting for detection of anti-extractable nuclear antigen antibodies in systemic lupus erythematosus.

Anti-extractable nuclear antigen (ENA) antibodies were assayed by counter immunoelectrophoresis (CIE) and immunoblotting in patients with systemic lupus erythematosus (SLE). We found the two methods showed good concordance rates, the lowest being 67% for anti-SS-A. Immunoblotting was more sensitive in detecting anti-Sm, anti-SS-B and anti-PCNA (proliferating cell nuclear antigen); CIE was more sensitive for anti-nRNP and anti-SS-A. Overall, the prevalence of these anti-ENA antibodies in SLE was increased by 9-20% if immunoblotting was used in addition to CIE. Sera specific for the 52 kDa peptide of the SS-A antigen (anti-52kDa SS-A) were better detected by immunoblotting. Anti-PCNA antibody was found in 6.3% of SLE patients and was associated with active disease and hemolytic anemia. The positive rate of anti-Sm was 9% by CIE and 23.7% by immunoblotting and this antibody was a specific marker for SLE using either method. It was concluded that using immunoblotting in addition to CIE, the overall sensitivity of detection of anti-ENA antibodies in SLE was increased and clinically useful antibodies such as anti-52kDa SS-A and anti-PCNA could be detected.

Measurement of anti-dsDNA: a comparative study of two ELISA and the Crithidia assay.

We compared the measurement of anti-dsDNA by a commercial ELISA test (DIASTAT), an in-house ELISA and the Crithidia luciliae assay in cross-sectional sera samples of 209 systemic lupus erythematosus (SLE) patients and 64 patients with a variety of rheumatological, autoimmune and non-autoimmune diseases in Hong Kong. The Crithidia assay was found to be the least sensitive (17%) but most specific (95%) method for detection of a positive result in SLE patients. The DIASTAT assay has a higher sensitivity (68%) but lower specificity (80%) than the in-house ELISA test (32% sensitivity and 89% specificity). The positive predictive value of the three assays are comparable at 90-92% while DIASTAT had the highest negative predictive value (44%). There was good linear correlation (r = 0.7) between the two ELISAs. ELISA can serve as a useful screening test for anti-dsDNA in SLE patients and doubtful cases can then be confirmed by another method such as radio-immunoassay.

Autoantibody formation after allogeneic bone marrow transplantation: correlation with the reconstitution of CD5+ B cells and occurrence of graft-versus-host disease.

Manifestations of autoimmune diseases are common in patients who have received allogeneic bone marrow transplantation (BMT). Autoantibodies have been reported in these patients but the source and clinical significance of these autoantibodies are still obscure. In the present study the kinetics of autoantibody formation and the reconstitution of CD5+ B cells was followed in 21 patients who were submitted to allogeneic BMT. Anti-nuclear, anti-smooth muscle, anti-neutrophil cytoplasmic antibodies, anti-reticulin and rheumatoid factor were found at a frequency of 25%, 17%, 24%, 22% and 10% respectively after BMT. Anti-double stranded DNA levels were mildly elevated in 15% of samples. The screening for anti-extractable nuclear antigen, anti-mitochondrial, anti-gastric parietal cell, anti-proteinase III, anti-myeloperoxidase, anti-lactoferrin antibodies was negative. The percentage and absolute count of CD5+ B cells increased with time after allogeneic BMT. Those patients with anti-nuclear or anti-smooth muscle antibodies had significantly higher CD5+ B cell counts than those without these two antibodies. Correlations of CD5+ B cell counts with other autoantibodies were negative. Acute graft-versus-host disease (GVHD) occurred in eight of the patients and chronic GVHD in four patients, but the frequency of autoantibodies had no relationship with the occurrence of acute or chronic GVHD.

Case report: anti-proteinase 3 antibody activity in a patient with primary sclerosing cholangitis: clinical remission following ursodeoxycholic acid therapy.

Primary sclerosing cholangitis is rare among Chinese. We report on a 71 year old male patient who presented with clinical features consistent with the disorder. Subsequent investigations confirmed the diagnosis. The patient was found to have anti-neutrophil cytoplasmic antibodies with specificity against proteinase 3. Treatment with ursodeoxycholic acid resulted in clinical remission and disappearance of the antibodies.

Ulcerative colitis and antineutrophil cytoplasmic antibodies in Hong Kong Chinese.

Inflammatory bowel diseases are known to be rare among the Chinese. The diagnosis of ulcerative colitis has been difficult in some of the Asian countries where infective colitis is more prevalent. Twenty-three Hong Kong Chinese patients diagnosed to have ulcerative colitis were reviewed. The symptoms were relatively mild and extraintestinal manifestation had been rare. Patients responded well to steroid therapy and sulfasalazine. Three patients in this series were found to have cyst and/or trophozoites of Entamoeba histolytica in stool. In this series, 19 patients were tested for antineutrophil cytoplasmic antibody (ANCA). Fourteen patients (73.5%) were positive, of which six (31.5%) showed a perinuclear staining pattern and eight (42%) demonstrated a cytoplasmic pattern. Five patients (26.5%) were negative for any ANCA, and none was positive for both. Sera of these patients were also tested for anti-alpha granules, anti-myeloperoxidase, and anti-lactoferrin activities. None was positive. Control sera collected from 16 patients with irritable bowel syndrome were all negative for the tests. In conclusion, testing of ANCAs may help in making the diagnosis of idiopathic inflammatory bowel disease in difficult situations.

Lymphocyte subsets and serum immunoglobulins in patients with premature ovarian failure before and after oestrogen replacement.

We have previously demonstrated that in patients with oestrogen deficiency, there was a significant reduction in the ratio of CD4+ to CD8+ lymphocytes. To test our hypothesis that oestrogen deficiency was the cause of the changes in lymphocyte subsets, we have studied the lymphocyte subsets and serum immunoglobulin levels in 35 patients with idiopathic premature ovarian failure before and after oestrogen replacement therapy. Before oestrogen replacement the lymphocyte subsets in peripheral blood were enumerated with indirect immunofluorescence techniques using monoclonal antibodies. The serum immunoglobulins (Ig) G, A and M were measured with nephelometry. After oestrogen replacement therapy, the tests were repeated around day 7-11 of the 2nd, 4th and 24th month. There was no significant change in any of these parameters by the 2nd or 4th months. After 2 years of oestrogen replacement, there was a significant decrease in the percentage of CD8+ cells, a significant increase in the CD4:CD8 ratio and a significant increase in the mean serum IgG concentration. There was no significant change in the other parameters. Our results support the hypothesis that oestrogen deficiency is the cause for the changes in the lymphocyte subsets.

Low predictive value of c-ANCA and anti-alpha-granule antibody for vasculitis in Hong Kong.

One thousand sera, not previously tested for ANCA, were assayed for C-ANCA and anti-alpha-granule antibody. There was low concordance (23%) between the two assays in terms of positivity. Each assay gave a positive predictive value for systemic vasculitis excluding SLE and other connective-tissue diseases) of not more than 20%.

Antilactoferrin antibody in systemic lupus erythematosus.

Lactoferrin is a secondary granule protein of neutrophils. Seventy-nine systemic lupus erythematosus patients who fulfilled the ARA criteria for classification were tested for antibody against human lactoferrin (LF-ab) by ELISA. Thirty-one of these (39.2%) demonstrated elevated levels. There was significant correlation between LF-ab positivity and disease duration. Clinical flare was common with positive LF-ab (P less than 0.05). Disease manifestations were independent of antibody status except for an increased incidence of lymphadenopathy and crescentic gomerulonephritis among those who had LF-ab. No consistent immunofluorescence pattern could be demonstrated on alcohol-fixed neutrophils for the LF-ab positive sera. It is suggested that LF-ab is related to lupus activity, and can be useful as a marker for disease monitoring.

Antinuclear antibody detection using streptavidin-biotin-peroxidase complex on HEp-2 cell substrate.

The streptavidin-biotin-peroxidase complex (SABC) technique was compared to conventional indirect immunofluorescence (IIF) for the detection of anti-nuclear antibody (ANA) on HEp-2 cell substrate. SABC showed higher specificity and predictive value and gave more reproducible titres and clearer staining patterns than IIF in sera from a series of rheumatic disease patients. Sera from 80 patients with various types of rheumatic diseases and 20 without rheumatic disease were further tested using the SABC method. All systemic lupus erythematosus (SLE) sera were positive. The overall sensitivity was 95%, specificity 90% and predictive value 97% for rheumatic disease. The rim pattern was associated with SLE and mixed connective tissue disease. The nucleolar/homogeneous pattern was associated with scleroderma and SLE in remission. ANA titre and staining pattern have limited value in the clinical assessment of rheumatic disease; however, ANA has very high sensitivity for SLE and remains an excellent screening test.

Antineutrophil cytoplasmic antibody and gallium lung scintiscan in usual interstitial pneumonitis.

A 2 year old Chinese boy was reported in whom there was an association between usual interstitial pneumonitis (UIP) and serum antineutrophil cytoplasmic antibody of the classical cytoplasmic type (cANCA). The concentration of cANCA reflected disease activity and decreased during a 6-week course of prednisolone. The gallium lung scintiscan index correlated with his disease activity. Both the serum cANCA concentration and the gallium lung scan could be used to monitor responses to treatment in this patient with UIP.

Distinction between antinuclear antibody and P-ANCA.

To differentiate between perinuclear immunofluorescence staining of antinuclear antibody (ANA) and the perinuclear form of anti-neutrophil cytoplasmic antibody (P-ANCA), the pattern after formaldehyde vapour fixation of normal human neutrophils was compared with that of standard ethanol fixation. Fifteen out of 17 myeloperoxidase antibody positive sera showed cytoplasmic staining on formaldehyde vapour fixed cells; 30 of the 32 ANA positive samples became negative or gave weak nuclear staining on the same substrate. Formaldehyde vapour fixation is a simple, useful technique for differentiating between ANA and P-ANCA.

Chronic granulomatous disease: a different pattern in Hong Kong?

From July 1988 to December 1989, six boys with chronic granulomatous disease were diagnosed in our institutions. Their clinical features were reviewed in order to delineate the pattern of infections which seems to have both similarities and differences when compared with published reports of Caucasian patients. The most striking differences was the lack of skin sepsis and chronic lymphadenitis in our six patients. Gram-negative organisms were the commonest pathogens while Staphylococci sp. were not isolated. Clinical features which should alert one to the diagnosis were also highlighted. Prophylactic co-trimoxazole was effective in reducing the frequency of bacterial infections. Early diagnosis is not only essential for optimal patient management but also for genetic counselling for the extended family.

Defective neutrophil and lymphocyte function in leucocyte adhesion deficiency.

We report a Chinese girl with the moderate phenotype of leucocyte adhesion deficiency (LAD), presenting with persistent omphalitis and recurrent soft tissue infections. She had subnormal adhesion-dependent neutrophil functions, such as chemotaxis and chemiluminescence response to a particulate stimulant (opsonised zymosan). Despite her adequate humoral response to documented herpes simplex virus type 1, parainfluenza type 2 and adenovirus infection in vivo, there was marked impairment in the generation of plaque-forming cells (PFC) driven by pokeweed mitogen (PWM) in vitro. IgM PFC were less severely affected than IgG and IgA PFC, probably because IgM production is less dependent on T cell help than IgA and IgG production. The patient's B cells and accessory cells had reduced function compared with the control subsets, while helper function of her CD4+ cells was virtually absent in the PWM-driven PFC assay. She also had marked defect in natural killer cell activity. The proliferation of her lymphocytes was normal to several plant lectins, including phytohaemagglutinin, concanavalin A and PWM, but markedly defective to OKT3.

Lymphocyte subsets in patients with oestrogen deficiency.

We have previously shown that in patients with idiopathic premature ovarian failure there were significant changes in lymphocyte subsets. To test our hypothesis that these changes were due to oestrogen deficiency we studied lymphocyte subsets in patients with oestrogen deficiency due to other causes. Blood was taken for serum oestradiol, lymphocyte counts and lymphocyte subset counts (CD2+, CD4+, CD8+ and B cells) before oestrogen replacement in 19 patients with gonadal dysgenesis, 22 patients with hypothalamic-pituitary failure and 24 healthy female control subjects. The CD4:CD8 ratio in both groups of patients was significantly lower than that in the normal control subjects while the percentages and counts of lymphocytes and CD8+ cells were significantly higher. There was a significant positive correlation between the serum oestradiol level and the CD4:CD8 ratio. These findings support the hypothesis that the changes in lymphocyte subsets are due to oestrogen deficiency.

A controlled trial of bestatin in hydatidiform mole.

A prospective randomized controlled trial was conducted to study whether Bestatin, an immunomodifier, can reduce the incidence of persistent gestational trophoblastic disease in patients with hydatidiform mole. A group of 21 patients (Bestatin group) received 30 mg Bestatin daily after evacuation of the hydatidiform mole. A second group of 23 patients (control group) did not receive any drug. Blood was taken for white cell counts, differential counts, lymphocyte subset counts (CD2+, CD4+, CD8+ and B cells) and natural killer cell activity before evacuation of the hydatidiform moles. The tests were repeated every 4 weeks after evacuation until the serum beta subunit of human chorionic gonadotropin (beta hCG) had returned to normal or until the patient had to receive chemotherapy because of persistent gestational trophoblastic disease. There was no significant difference in the age of the patients, the pre-evacuation serum beta hCG, or the gestational age between the two groups. Chemotherapy was needed by 6 patients in the Bestatin group (28.6%) and 3 patients in the control group (13%) because of persistent gestational trophoblastic disease. There was no significant difference in any of the immunological parameters between the two groups before or after evacuation. We conclude that Bestatin at this dosage does not improve the immunological functions or clinical outcome in patients with hydatidiform mole.