T cell reactivity to Collagen II as a possible prognostic marker in patients with rheumatoid arthritis.

OBJECTIVE: To compare the frequency and the functional state of the collagen II reactive T cells with disease activity in rheumatoid arthritis patients and healthy controls. METHODS: This case-control cross-sectional study was carried out at the Department of Immunology; Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from June to October 2014. Rheumatologist from Rehmat Noor Rheumatology Clinic, a private health facility of the city, was requested to send in patients with clinical diagnosis of rheumatoid arthritis. Samples were obtained and relevant investigations were carried out. Data were compared with a group of age and gender-matched healthy subjects. T cell proliferative response was assessed against bovine collagen II by measuring incorporation of bromodeoxyuridine into deoxyribonucleic acid of proliferating cells and by expression of CD25 on proliferating cells as percentage of CD3+/bromodeoxyuridine+ and CD3+/CD25+ T-cells, respectively. Among the patients, the frequency of T cells with disease activity was compared. Patients were classified into groups of mild, moderate and severe disease and frequency of CD3+/bromodeoxyuridine+, frequency of CD3+/CD25+ cells, mean fluorescent intensity of bromodeoxyuridine-fluorescein isothiocyanate and mean fluorescent intensity of CD25-fluorescein isothiocyanate were compared in the groups. RESULTS: Of the 60 subjects, 30(50%)were patients and 30(50%) were controls. Of the patients, 5(16.66%) were males and 25(83.33%) were females with an overall mean age of 42±12 years. The mean age of the controls was 41±9.28 years. Mean disease duration of the patients was 10.5 ± 4.2 years. Percentage of CD3+/CD25+ cells and CD3+/bromodeoxyuridine+ cells stimulated with collagen II, in patients was much higher than the controls(p<0.05).Statistically significant differences were observed when frequency of CD3+/bromodeoxyuridine+ cells and CD3+/CD25+ cells was compared among the mild, moderate and severe patient groups (p<0.05). CONCLUSIONS: Collagen II was found to be an important auto antigen in joints of rheumatoid arthritis patients.

Optimization of an in-house PCR method for the detection of HLA-B*27 alleles.

The association of spondyloarthropathies with different alleles of human leukocyte antigen (HLA) B*27 is well established. Different subtypes of HLA-B*27 may be linked with different ethnic groups, distinct clinical manifestations, specific age of onset and different prognoses. Polymerase chain reaction with sequence specific primers (PCR-SSP) is the most frequently adapted molecular method used for the recognition of HLA-B*27-specific DNA sequences. The aim of the present study was to standarise an in-house protocol of PCR-SSP for HLA-B*27 allele detection for use in the Armed Forces Institute of Pathology (AFIP), Pakistan, with consideration of its cost effectiveness. A total of 49 individual samples were included, comprising 10 transplant samples determined to be HLA-B*27-negative by PCR-SSP and 39 HLA-B*27-positive samples determined by flow cytometry, obtained from patients who were symptomatic and referred for HLA-B*27 testing. By altering each variable individually, an in-house PCR-SSP protocol was optimized to amplify common HLA-B*27 alleles (2701-2721, 2723-2730). To discriminate B*27 from all other HLA-B alleles, a low-resolution HLA-B typing set with a 96 PCR-SSP primer mixture was used in conjunction. Among the 39 HLA-B*27-positive specimens, 31 (79%) were detected as positive by PCR-SSP, with the remaining samples failing due to a sub-optimized protocol and/or low DNA concentration. Additionally, there was complete concordance between flow cytometry and in-house PCR, and the sensitivity and specificity of the PCR-SSP were determined to be 100%. In conclusion, in-house SSP-PCR is, standard method for the detection of HLA-B*27 alleles. The determination of associations between specific HLA-B*27 alleles and AS may aid to identify individuals at higher risk of developing the disease. Furthermore, the identification of individuals at risk may aid to adapt preventive strategies.

Clinico-Haematological Features of Paroxysmal Nocturnal Haemoglobinuria.

The aim of this study was to determine the frequency of various clinico-haematological features in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). It was an observational study carried out from October 2008 - January 2016. All the patients of PNH, diagnosed on the basis of clinical and laboratory findings and confirmed by CD55 and CD59 deficiency on red cells by means of flow cytometry, were included in the study. A total of 22 patients were diagnosed which included 18 (81.8%) males and 4 (18.1%) females. Median age was 27 years. Pallor, fever, fatigability and haemoglobinuria were the most common clinical features. Pancytopenia was seen in 13 (59.09%) and hypocellular marrow was found in 14 (63.6%) patients. One patient presented with Budd Chiari syndrome.

Chronic granulomatous disease.

Chronic granulomatous disease is a rare inherited disorder characterised by inability of phagocytes to generate reactive oxygen species needed for intracellular killing of phagocytosed microorganisms. We report the case of an 8-month-old male child with recurrent chest infections and perianal abscess that had no response to conventional antibiotic treatment. His two elder brothers died due to similar complaints at the ages of 4 and 5 months. Four elder sisters were healthy and alive. This history indicated that the patient might have X-linked chronic granulomatous disease. A definite absence of superoxide activity in the patient's granulocytes detected by dihydrorhodamine test and nitroblue tetrazolium dye reduction test confirmed this diagnosis.

HLA- DR Alleles in Pakistani Patients of Pemphigus Vulgaris.

OBJECTIVE: To determine frequency of HLA-DR alleles in Pakistani patients of pemphigus vulgaris in comparison with local healthy controls. STUDY DESIGN: Cross-sectional, comparative study. PLACE AND DURATION OF STUDY: Department of Immunology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from January 2011 to January 2014. METHODOLOGY: Twenty eight patients with biopsy proven diagnosis of pemphigus vulgaris referred from Department of Dermatology, Military Hospital, Rawalpindi were included. Patients were compared with a group of 150 unrelated local healthy subjects. DNA was extracted from peripheral blood collected in Tri-potassium EDTA. HLA-DRB1 typing was carried out on allele level (DRB1*01--DRB1*16) using SSP (sequence specific primers). HLA type was determined by agarose gel electrophoresis and results recorded. Phenotype frequency of various alleles among patient group and control group was calculated by direct counting and significance of their association was determined by Fisher's exact test/ Chi square test. RESULTS: A total of 12 male and 16 female patients, with age ranging from 21 to 34 (mean 23.4 years) were genotyped for HLA-DRB1 loci. A statistically significant association of the disease with HLA-DRB1*04 was observed (50% versus 20.7% in controls, p < 0.05). CONCLUSION: There is a strong association of HLA-DRB1*04 with pemphigus vulgaris in Pakistani population.

HLA-DR alleles among Pakistani patients of coeliac disease.

OBJECTIVES: To investigate whether certain DR alleles might also contribute to the genetic susceptibility among Coeliac disease patients in Pakistan. METHODS: The case-control study was conducted at the Military Hospital, Rawalpindi, from October 2011 to January 2012, and analysed 25 children diagnosed to have coeliac disease as per the criteria set by the European Society of Paediatric Gastroenterology and Nutrition, which included histopathological alterations in duodenal biopsies, clinical response to gluten withdrawal, and presence of anti-endomyseal antibodies. Patients were compared with a group of 150 healthy subjects. Dioxyribonucleic acid was extracted from peripheral blood collected in ethylenediaminetetraacetic acid.K3. Human leukocyte antigen DRB1 typing was carried out on allele level (DRB1*01--DRB1*16) using sequence specific primers. Human leukocyte antigen type was determined by agarose gel electrophoresis and results were recorded. Phenotype frequency of various alleles among the patient group and the control group was calculated by direct counting, and significance of their association was determined by Fisher Exact Test. RESULTS: A total of 11 (44%) female paediatric coeliac patients in age range 1-9 (mean 7.2 +/- 4.8 years) and 14 (56%) male paediatric patients in the age range 6-14 (mean 8.6 +/- 5.1 years) were genotyped for HLA-DRB1 loci. A statistically significant positive association of the disease with HLA-DRB1*03 (n = 23; 92% versus n=31; 21% in controls, p < 0.01) was observed. CONCLUSION: HLA-DRB1*03 is associated with increased risk of developing coeliac disease.

Evaluation of serum transferrin receptor concentration in diagnosing and differentiating iron deficiency anaemia from anaemia of chronic disorders.

OBJECTIVE: To diagnose and differentiate iron deficiency anaemia (IDA) from anaemia of chronic disorders (ACD) using serum concentration of soluble transferrin receptors (sTfR). METHODS: One hundred and seventy six adult anaemic patients were diagnosed on bone marrow examination as IDA and ACD in the Department of Haematology, Armed Forces Institute of Pathology, Rawalpindi from November 2001 to May 2003. They were further evaluated with sTfR, serum iron, total iron binding capacity (TIBC) and serum ferritin. These biochemical investigations were compared with results of bone marrow iron status, which served as gold standard. Absence of stainable iron in the bone marrow was diagnostic of iron deficiency, whereas abundance of iron along with decreased siderocytes and sideroblasts was considered diagnostic of ACD. Data was collected on a proforma and analysed using software SPSS (version 11.0) and t-test was used to test the statistical significance. Specificity, sensitivity positive and negative predictive value of the sTfR test was calculated. RESULTS: Out of 176 patients studied, 90 (51.1%) were diagnosed as ACD whereas 86 (48.8%) as IDA. The mean +/- SD sTfR levels in IDA patients was 9.68 +/- 2.48 mg/I, whereas mean +/- SD sTfR levels in ACD patients was 2.96 +/- 1.28 mg/I, thus clearly separating the two categories of anaemic patients. Both the sensitivity and specificity of sTfR in IDA was found to be 100%, whereas in ACD, these were 66.6% and 100% respectively. The positive and negative predictive value, in case of IDA was 100%, whereas in ACD it was 100% and 74.1% respectively. The results of serum iron, TIBC and serum ferritin correlated well in IDA, with a fall in serum iron, raised TIBC and decreased serum ferritin, except in few cases in which concomitant inflammatory conditions resulted in falsely high serum ferritin level. Serum iron and TIBC were not useful in cases of ACD. However, the serum ferritin cutoff level of 90 ng/ml was evaluated which virtually excludes IDA, and found this highly effective in cases of IDA alongwith chronic inflammatory conditions. CONCLUSION: The results show that in case of simple IDA, sTfR concentration is significantly raised and it has a very high test efficiency in this condition. However in case of ACD the positive predictive value is high (100%) but the negative predictive value is compromised (74.1%). It is therefore a reliable laboratory index of IDA and in distinguishing IDA from ACD).