Immunochemical studies in Wilson's disease.

The present study deals with a total of 28 cases of Wilson's disease, 50 normal individuals alongwith siblings and parents of eight cases. Male predominance (18 out of 28 cases), a median age of 11 years and universal presence of Kayser-Fleischer (K. F.) ring marked the cardinal features. Furthermore, 11 patients had hepatic-neural presentation while two had only the K. F. Ring without clinical abnormality. Single Radial Immunodiffusion (SRID) as the absolute quantitative procedure revealed a profound deficiency of ceruloplasmin with the levels ranging anywhere between 0.5 mg/dI to 23 mg/dI amongst the patients of Wilson's disease. The data from siblings and parents also revealed deficiency in 15 out of 23 serum samples when subjected to disc electrophoresis-benzidine screening procedure.

Haemagglutination, haemolysin production and serum resistance of proteus and related species isolated from clinical sources.

A total of 148 strains of Proteus and related species comprising of Proteus mirabilis (116), Proteus vulgaris (24), Providentia rettgeri (4), Providentia alcalifaciens (2), Providentia stuarti (1) and Morganella morganii (1), isolated from various sources, were examined for haemagglutination (HA), haemolysin production (HL) and serum resistance (SR). Maximum isolates were obtained from urine (47.30%) and pus (40.54%) and they were multidrug resistant. The sensitivity to Ciprofloxacin was 78.38%, Gentamicin: 62.84%, Cefotaxime: 29.73%, Norfloxacin: 22.97%, Tetracycline: 20.95% and Ampicillin: 6.76%. There were four commonest resistance patterns shown by 58.62% of Proteus mirabilis and 66.67% of Proteus vulgaris strains. Haemagglutination was shown by 91 (61.49%) strains, HL production in 126 (85.14%) strains and SR by 124 (83.78%) isolates. All the three i.e. HA, HL and SR were simultaneously present in 77 (52.27%) strains, any two were present in 40 (27.03%) strains and any one was positive in 30 (20.03%) strains. Thus in as many as 147 (98.32%) isolates, any one or more virulence factors were present. The virulence in commensal pathogen like Proteus is basically a multifactorial phenomenon. The presence of more virulence factors in one strain may increase its pathogenic ability. The evaluation of multiple virulence factors instead of one single parameter will be of greater help in assessing its pathogenic potential.

Rapid detection of enteric fever by coagglutination and countercurrent immunoelectrophoresis.

A total of 463 patients clinically suspected of enteric fever and 100 healthy individuals were investigated by coagglutination (COAG) and countercurrent immunoelectrophoresis (CIEP) for rapid diagnosis of enteric fever. The S. typhi was grown in blood culture in 32 (6.91%) and Widal test was positive in 126 (27.21%) cases. The serum COAG with local antiserum was positive in 165 (35.64%), serum COAG with standard antiserum in 163 (35.21%), Blood culture supernatant (BCS) COAG in 153 (33.05%), serum CIEP for antigen (Ag) in 118 (25.09%), BCS CIEP in 99 (21.39%) while serum CIEP for antibody (Ab) was positive in 34 (7.34%) cases. Only two healthy controls revealed positive COAG result with local antiserum. The sensitivity of all antigen detection tests was 100% except BCS CIEP (98.25%) in the first week of fever and declined rapidly to 75.79% for serum COAG tests, 69.47% for BCS COAG and dramatically to 37.89% for serum CIEP for Ag and 22.11% for BCS CIEP tests during the second week whereas the sensitivity of serum CIEP for Ab detection rose from 17.54% to 23.16% from first to second week of illness. In view of the resulting data, it is suggested that both COAG and CIEP may be employed for the rapid diagnosis of enteric fever in the routine clinical setup.

Comparative evaluation of immunochemical methods for estimation of albumin in microalbuminuria.

Three immuno assays namely radioimmunoassay (RIA), radial immunodiffusion (RID) and rocket immunoelectrophoresis (RIE) were compared for their performance and utility. The accuracy limits of the methods were compared and also between methods using RIA as the reference. Urine samples, from known diabetic patients with albumin concentration ranging from 2.5 mg/l to 120 mg/l were analysed by the three methods. The mean differences were only 0.91 mg/dl and 0.5 mg/dl respectively for RID vs RIA and rocket vs RIA which is not statistically significant. Excellent correlation was seen between RIA and RIE (r = 0.98) and also between RIA and RID (r = 0.97). Compared to RID, RIE required less time and was more precise. RIA is suited for assaying large sample loads yet not suited for laboratories receiving samples occasionally. For a small pathological laboratory with limited facility rocket electrophoresis may be the most suitable method taking into consideration accuracy, time and cost.

Immunochemical studies in multiple myeloma.

The study deals with a total of 72 patients with Plasma cell dyscrasias (PCD) selected on the basis of atypical plasmacytosis in the bone marrow aspirate and radiological evidence of osteolytic lesions. Males(48) outnumbered the females (24). Pathological fracture and paraplegia was the commonest presenting symptom encountered in 38 patients. Electrophoresis of serum for 'M' band and Immunoelectrophoretic analysis of the serum revealed IgG myeloma in 40 patients followed by, IgA myeloma(13), Light chain disease (12) and other variants in remaining seven cases. The urinary Bence Jones proteins were detected in a total of 34 cases and was frequently encountered with IgA myeloma (7 out of 13) compared with IgG myeloma (13 out of 40) when analysed in Disc electrophoresis. Kappa light chain was observed in 21 cases and lambda counterpart in nine cases without any clinical significance. One case of solitary myeloma terminated in characteristic multicentric multiple myelomatosis within a span of six months in the sequential follow up study. We recommend the triangular approach to diagnosis of paraproteinemia with a special emphasis on immunoelectrophoresis for typing multiple myeloma and allied disorders along with disc electrophoresis for the demonstration of urinary Bence Jones protein in the routine set up.

Bisalbuminemia: an interesting electrophoretic abnormality--a report of two cases.

We hereby report two cases of Bisalbuminemia for its extreme rarity in Indian literature. In an attempt to look for 'M' component, polyacrylamide gel disc electrophoresis of serum displayed observation. The saline eluates of the slower (variant in our cases) and the faster component showed a reaction of complete identify in the agar gel double diffusion test when tested against monospecific antialbumin antibody. The immunoelectrophoretic analysis also confirmed the antigenic similarity between the two fractions. A wider use of disc electrophoresis perhaps would disclose additional cases of Bisalbuminemia in normals and in disease states.

Analysis of immunoglobulin deficiency cases: a five year study.

A total of 734 serum specimens from various clinical disorders along with 100 control samples from healthy subjects were processed for estimation of serum IgG, IgA and IgM employing single radial immunodiffusion procedure. Immunoglobulin deficiency, either selective or combined was noted in 31 males and 24 females in all age groups. Of the 55 cases encountered it was secondary immunoglobulin deficiency which was seen on a larger scale and encountered in patients with Multiple myeloma (16 out of 32) followed by Leprosy (14 out of 250), Lymphoma (5 out of 43), Malaria (4 out of 137), Burns (4 out of 52), Rheumatoid arthritis (2 out of 69) and non lymphoreticular malignancies (1 out of 41) in decreasing order of frequency. Primary immunoglobulin deficiency was observed in nine cases comprising of six belonging to Idiopathic late onset immunoglobulin deficiency, two of dysgammaglobulineamia and a solitary case of Ataxia telangiectasia. Panimmunoglobulin deficiency was observed in six cases, 11 had a dual deficiency while 38 showed deficiency of an isolated class with selective IgA deficiency in 20 cases. Furthermore, one patient each had total absence of IgG or IgA while IgM was not detectable in seven patients. A high suspicion index along with a regular rapport between the clinician and the laboratory personnel is necessary in the diagnostic set up of immunoglobulin deficiency states.

Evaluation of cell mediated immunity in healthy contacts of leprosy.

The cell mediated immunity (CMI) was studied in 50 healthy contacts of leprosy patients and 50 age & sex matched non-contact controls by lepromin test and leucocyte migration inhibition (LMI) test using phytohaemagglutinin (PHA) and lepromin and its association with other risk factors in contacts was assessed. The lepromin positivity correlated well with LMI results. There was no difference in CMI in I, II and III degree of contacts. Amongst direct contacts lepromin test was positive in 67 per cent as compared to 92 per cent in indirect contacts while in LMIT migration index (MI) was significantly increased (0.66 +/- 0.20) in direct contacts. MI was also significantly increased (0.73 +/- 0.20) contacts of less than two years duration which decreased to 0.51 +/- 0.18 in contacts of more than five years duration. Lepromin positivity also increased from 60 per cent to 100 per cent in these contacts. The specific CMI was significantly suppressed in contacts of LL patients (MI:0.74 +/- 0.21) and BL patients (MI: 0.61 +/- 0.01) as compared to healthy controls. B.C.G. vaccinated individuals showed better CMI response. The findings in the study showed specific unresponsiveness to lepromin in LMI in leprosy contacts of less than two years duration, direct contacts, contacts of lepromatous spectrum of index patients and contacts not vaccinated with B.C.G. emphasizing that CMI status is an important parameter in identifying the contact population at the greater risk of acquiring leprosy.

Species distribution of clinical isolates of Staphylococci.

Species identification of 103 strains of staphylococci isolated from various clinical specimens revealed as many as eight species. S. aureus was the commonest isolate (57.28%) followed by S. epidermidis (17.47%). S. hominis (8.73%), S. saprophyticus (6.79%), S. intermedius, S. capitis and S. cohni (1.94% each) in decreasing order. They were mostly isolated from pus (41.74%) followed by urine (22.33%) and blood (18.44%) accordingly. Of the total isolates, 65 were coagulase positive while 38 isolates were coagulase negative. Phase typing of S. aureus strains revealed that 49 isolates were typable of which 17 isolates belonged to phase group III, 16 to phase group 1, 4 to group II and 3 belonged to group V while 9 strains could not be grouped. The antibiotic resistance pattern showed maximum resistance to penicillin (63.10%) while resistance was minimum with gentamycin (8.73%). Resistance to ampicillin (51.45%), cephazoline (51.45%), erythromycin (31.06+) and cloxacillin (18.44%) was low to moderate. The increasing recognition of pathogenic potential of various species of staphylococci and emergence of drug resistance amongst them denotes the need to adopt better laboratory procedures to identify and understand the diversity of staphylococci isolated from clinical material.

Seroprevalence of syphilis by TPHA test.

A total of 1074 sera obtained from clinically suspected cases of Syphilis and various risk groups were screened for antitreponemal antibodies by Treponema pallidum Haemagglutination (TPHA) test and cardiolipin antibodies by the Venereal Disease Research Laboratory (VDRL) tests. The TPHA test was positive in 291 (27.09) per cent. The TPHA was positive in 53.31 per cent (161 out of 302) patients of primary syphilis, 54.17 per cent (26 out of 48) of secondary, 32.76 per cent (19 out of 58) of tertiary syphilis, 17.12 per cent (19 out of 111) of other Sexually Transmitted Disease (STD) patients, 25.32 per cent (60 out of 237) pregnant women and 1.89 per cent (6 out of 318) healthy individuals. TPHA was found to be superior to VDRL test in all the study groups. Almost a total agreement was seen between the TPHA test and VDRL test with a titre of 1 in 16 & above; while in as many as 32.39 per cent sera with VDRL titre of 1 in 8 TPHA was negative indicating that the VDRL titres above 1 in 8 should be considered as true reactives to minimize the biological false positive reactions.

Sequential study of IgG antibody response in immunized rabbit and development of immunization protocol for raising monospecific antibody.

The present study was planned to develop an immunization protocol to decide the bleeding modalities for harvesting anti IgG antibody from the immunized rabbit. A fourteen dose immunization protocol (four primary and ten boosters) of the purified human IgG spread over the one calendar year was executed. The antibody titre estimated by Reverse Single Radial Immunodiffusion displayed a six-phased pattern. The titre following the initial immunization ranged between nil to 1.02, characteristic of the primary response while the titres after 7th and 8th boosters (phase V) ranged between 0.5 to 3.87 consistent with secondary response. Phase II, III, IV had moderately elevated titres. The antibody titre amongst the six phases reached to its peak generally by the 12th day after the last dose of protocol and it took about 60 days to reach to its basal level. Administration of antigen with the higher levels of residual antibody did not produce high titre antibody and is probably ascribed to elimination of antigen through an immunecomplex mechanism. Based on the data we recommend that 15 batches. (3 per phase, phase I to phase V) with a total yield of 100 to 120 ml of serum can be procured from one immunised animal over the span of one calendar year and that should make the programme cost effective.

Study of diphtheria antibody levels in healthy population.

Diphtheria antibody levels were analysed in 200 healthy individuals by Indirect Haemagglutination test. The antibody titres in the age groups 1-5 and 6-10 years were well above the protective level. The titre declined with increase in age. In 11-20 years of age about 17.5 per cent, in 21-30 years of age 52.5 per cent and in persons above 30 years of age 82.5 per cent people had antibodies below protective level. Our results are comparable to studies from India and abroad. Emphasis is given for reconsideration of immunisation programme in higher age group.

Reverse single radial immunodiffusion for estimation of titre of anti IgG antibody.

Reverse Single Radial Immunodiffusion (SRID) for estimating titre of anti IgG antisera is reported. Unlike the conventional radial immunodiffusion, the antigen (IgG) is held immobile in the gel while the antibody (Anti IgG) diffuses radially from the well (7 microliters) and the diameter of the resulting immuneprecipitates after immunodiffusion at 4 degrees C for 24 hr, represents a linear correlation with the antibody titre. The procedure was standardised by an extensive trial and error employing different concentrations of human IgG in the gel (60-240 micrograms) against varying dilutions of the standard antibody (titre: 3.8 mg/ml). The best results were obtained at 80 micrograms of IgG in the gel. The locally raised rabbit anti IgG antisera displayed a distinctive titre pattern under optimised conditions. Technical reproducibility, high-sensitivity threshold (0.25 mg/ml), simultaneous visual scrutiny of several antibody batches at a glance and ability to assess the shelf life of the stored antisera are its distinct assets.