Anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis patients

Objective: To detect the anti cyclic citrullinated peptide [Anti-CCP] antibody in rheumatoid arthritis [RA] patients to determine its diagnostic value in Pakistani patients

Study Design: Cross sectional study

Place and Duration of Study: Military Hospital [MH] Rawalpindi, from January 2013 to June 2015

Material and Methods: A total of 58 patients with complications of rheumatoid arthritis were recruited in the study using convenient sampling technique after their informed consent. Age and gender of the patients were recorded. Blood was collected from the patients subjected to ELISA based detection of anti-CCP and latex agglutination test for detection of Rheumatoid Factor [RF]. Data obtained were analyzed using Microsoft excel 2010

Results: Among the fifty eight RA patients, 40% were males and 60% were females. Age ranged between 12 to 80 years [mean age 49.74 +/- 16.81 years] of the males RA patients and was higher as compared to females [mean age 43.2 +/- 16.70 years]. ELISA based detection of anti-CCP antibody showed that about 91 percent of RA patients were positive for anti CCP antibody. About 72% were positive for anti CCP antibody alone, 19 percent were positive for both anti-CCP and RF and 9 percent were positive for RF

Conclusion: The results concluded that a higher percentage of the RA patients are positive for anti-CCP antibody marking its importance as a diagnostic marker. Anti-CCP has more sensitivity as compared to RF in RA patients

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

Correlation of anti C1q antibodies with disease activity in patients with systemic lupus erythematosus

Objective: To study the correlation of anti C1q antibodies with disease activity in patients with systemic lupus erythematosus [SLE]

Study Design: Cross sectional, observational study

Place and Duration of study: The Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi in collaboration with Military Hospital, Rawalpindi, Pakistan Institute of Medical Sciences, Islamabad and Benazir Bhutto Hospital, Rawalpindi, from Jan 2012 to Dec 2013

Material and Methods: Patients with a clinical diagnosis of SLE were included in the study on fulfilling revised American College of Rheumatology [ACR] criteria [1997]. Main outcome measures were SLE disease activity index [SLEDAI] score and anti C1q antibody levels in serum. SLEDAI scores were calculated for each patient on the basis of physical examination, patient interviews and previous clinical records. Anti C1q antibody levels in the serum were determined by enzyme-linked immunosorbent assay [ELISA] and correlated with the SLEDAI scores by calculating Pearson's correlation coefficient 'r'. The cutoff value for anti C1q antibody positivity in the serum was determined by evaluating the serum levels of anti C1q antibodies in 25 healthy subjects and was 12 U/ml

Results: Six male and forty nine female SLE patients with an age range of 16-47 years [mean 34.5 years] and 8-70 years [mean 31.7 years] respectively were studied. The correlation between anti C1q levels and SLEDAI scores in all patients was demonstrated by calculating the correlation coefficient and was not significant [r=0.19, p=0.14]. However, there was an inverse correlation between anti C1q levels and SLEDAI scores in patients with severe disease and this was statistically significant [r=-0.448, p=0.037]. The difference in anti C1q antibody positivity between patients with and without nephritis was not significant. The anti C1q antibody levels correlated poorly with anti double stranded deoxyribonucleic acid [dsDNA] antibody positivity. A significantly higher percentage of patients with evidence of complement consumption was found to be positive for anti C1q antibodies [p=0.01]. This significance was only seen in patients with reduced C3 levels [p=0.04] and not reduced C4 levels [p=0.23] or both [p=0.23]. Anti C1q antibody levels had significant inverse correlation with serum C3 levels. [p=0.007]

Conclusion: A significant inverse correlation was found between SLEDAI scores and serum anti C1q antibody levels in patients with severe SLE. The anti C1q antibody positivity is significantly higher in patients with reduced C3 levels

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. Key Words: Pemphigus vulgaris. Human leukocyte antigen. Pakistan. Haplotype.

Laboratory evidence of autoimmunity in HCV infected patients after interferon treatment

To determine the significance of interferon alpha therapy in emergence of autoantibodies in HCV infected patients in a local population Quasi experimental study Department of Immunology, Armed Forces Institute of Pathology [AFIP] Rawalpindi from Mar 2007 to Oct 2007. A total of 106 HCV infected individuals [not on any antiviral therapy], were screened for laboratory evidence of autoimmunity [ANA, SMA, AMA, Anti LKM, Anti GPC, Anti Thyroid microsomal and RA factor]. HCV infected patients without any laboratory evidence of autoimmunity were included in the study and this population was divided in to a test group [36] who were treated with interferon alpha and a control group [32] who did not receive anti viral treatment during this period. All were retested for the same autoantibodies after a period of 3 months. All autoantibodies were tested by indirect immunofluorescence except RA factor which was tested by agglutination method. Out of 106 patients scarred, 32 showed autoimmunity that were excluded from study. Six denied to participate in study. After 3 months, 61% of the patients showed autoimmunity in study group and frequencies of ASMA, anti-TPO [p 0.036], ANA [p 0.14] and RA were 36%, 25%, 25% and 33% respectively. Control group showed autoimmunity in 37.5% of the patients with frequency of 28%, 15.6%, 6.3% and 3% for ASMA, RA, TPO and ANA respectively. AMA anti-LKM antibodies were not found in both groups. ANA and anti thyroid antibodies emerge in increased frequency in HCV infected patients after treatment with IFN-alpha

Identification of laboratory markers of disease activity in rheumatoid arthritis

To identify the laboratory markers of disease activity, by finding relationship of biochemical markers with clinical disease activity measurement in patients suffering from rheumatoid arthritis [RA]. Cross sectional analytical study. Department of Immunology, Armed Forces Institute of Pathology [AFIP], Rawalpindi from January 2009 to January 2010 in collaboration with Fauji Foundation Hospital and Military Hospital Rawalpindi. One hundred patients diagnosed as having rheumatoid arthritis [RA] as per American college of Rheumatology [ACR] revised criteria 1987 and fulfilling the study's inclusion criteria were studied. These patients were assessed clinically according to Simplified Disease Activity Index [SDAI] and divided into three groups which were mild, moderate and severe based on disease activity. These three groups were then assessed for disease activity by Rheumatoid factor [RA factor], Anti Cyclic Citrullinated Peptide antibodies [anti CCP antibodies], Erythrocyte Sedimentation Rate [ESR] and C- Reactive Proteins [CRP]. The association of these laboratory markers with three groups of disease activity was analyzed to detect most sensitive disease activity markers for RA. All the assessed laboratory markers that are RA factor, anti CCP antibodies, ESR and CRP are directly related with RA disease activity and any of them can be used to assess disease activity in RA. However a combination of the tests, analyzed in this study markers maybe used for better prediction of disease activity. The identification of the laboratory markers of disease activity may help physician to diagnose aggressive disease early and evaluate prognosis in RA patients

Human leukocyte antigen class II susceptibility conferring alleles among non-insulin dependent diabetes mellitus patients

To determine the frequency of Human Leukocyte Antigen [HLA] class II susceptibility conferring alleles among type 2 Diabetes mellitus patients, in comparison with healthy controls. Cross-sectional comparative study. Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, from January 2009 to April 2010. Patients with non-insulin dependent Diabetes mellitus meeting World Health Organization criteria were studied. These were compared with age and gender matched healthy control subjects. For each subject [patients as well as controls], DMA was extracted from ethylene diamine tetra-acetate sample and HLA class II DRB1 typing was carried out at allele group level [DRB1*01-DRB1*16] by sequence specific primers. Human leukocyte antigen DRB1 type was determined by agarose gel electrophoresis and results were recorded. Frequencies were determined as number of an allele divided by total number of alleles per group; p-value was computed using Pearson's chi-square test. Among the 100 patients, there were 63 males and 37 females with 68 controls. A total of 13 different HLA DRB1 alleles were detected, with DRB1*15 being the commonest in both the groups. The allele DRB1*13 had statistically significant higher frequency in patient group as compared to controls [p = 0.005]. HLA DRB1*13 was found with a significantly increased frequency in non-insulin dependent Diabetes mellitus

HLA DR beta1 alleles in Pakistani patients with rheumatoid arthritis

To determine frequencies of HLA DR beta1 alleles in rheumatoid arthritis in Pakistani patients. Cross sectional / analytical study. Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi in collaboration with Rheumatology departments of Military Hospital, Rawalpindi and Fauji Foundation Hospital, Rawalpindi, from January 2009 to January 2010. HLA DR beta 1 genotyping of one hundred Pakistani patients, diagnosed as having RA as per American College of Rheumatology revised criteria 1987, was done. HLA DR beta 1 genotyping was carried out at allele group level [DR beta1[asterisk]01-DR beta1[asterisk]16] by sequence specific primers in RA patients. Comparison of HLA DR beta1 allele frequencies between patients and control groups was made using Pearson's chi-square test to find possible association of HLA DR beta1 alleles with RA in Pakistani rheumatoid patients. HLA DR beta1[asterisk]04 was expressed with significantly increased frequency in patients with rheumatoid arthritis [p <0.05]. HLA DR beta1[asterisk]11 was expressed statistically significantly more in control group as compared to rheumatoid patients indicating a possible protective effect. There was no statistically significant difference observed in frequencies of HLA DR beta1 allele [asterisk]01, DR beta1 allele [asterisk]03, DR beta1 allele [asterisk]07, DR beta1 allele [asterisk]08, DR beta1allele [asterisk]09, DR beta1 allele [asterisk]10, DR beta1 allele asterisk12, DR beta1 allele [asterisk]13, DR beta1 allele [asterisk]14, DR beta1 allele [asterisk]15 and DR beta1 allele [asterisk]16 between patients and control groups. The identification of susceptible HLA DR beta1 alleles in Pakistani RA patients may help physicians to make early decisions regarding initiation of early intensive therapy with disease modifying anti rheumatic medicines and biological agents decreasing disability in RA patients

Influence of gender on disease activity in patients suffering from rheumatoid arthritis

To study the influence of gender on disease activity in patients of rheumatoid arthritis. One hundred patients suffering from Rheumatoid Arthritis [RA], fulfilling the American College of Rheumatology [ACR] revised criteria 1987 and study's inclusion criteria were randomly selected for this study. The influence of gender on disease activity was measured in patients suffering from Rheumatoid Arthritis by the help of Erythrocyte Sedimentation Rate [ESR], C- Reactive Proteins [CRP], Rheumatoid factor [RA factor] and Anti Cyclic Citrullinated Peptide [anti CCP] antibodies. Age and duration of illness in both genders were also compared to find statistically significant differences. The female to male ratio was 7.3:1. Mean age in males was 53 years and in female patients 43 years. Duration of illness was found to have no significant difference in male and female patients. A relative increase in ESR, CRP, RA factor and anti CCP antibodies was observed in the males when compared to females. The median value of ESR in males was 61 mm/hour and in females was 31 mm/hour. The median value of CRP was 31 rng/dl in males and 9.2 mg/dl in females. The median values of anti CCP antibodies were 100 U/ml for the male group and 4.7 U/ml for the female groups respectively. The differences in these parameters between the two groups were found to be statistically significant [p value <0.05]. The median values of RA factor was 43 IU/ml in males and 10 lU/ml in females and the difference was not statistically significant [p value .072]. The prevalence of RA in females is found to be high in this study. Male patients have a significantly later onset of RA and presented with severe disease as compared to female patients suffering from RA

Diagnostic application of smooth muscle antibodies in autoimmune hepatitis

To measure the level of C-reactive protein and find its association with the glycaemic status [fasting plasma glucose, glycosylated haemoglobin] of metabolically normal and diabetic albino rats. Total 60 Albino rats were included [normal n=30; diabetic n=30]. Plasma glucose levels were determined by using glucose oxidase method while determination of total Hb and glycosylated hemoglobin [HbA[1]c] was done by diagnostic kit that uses weak cation-exchange resin to bind Hb. The% HbA[1]c was determined by measuring the absorbance at 415 nm of the glycosylated hemoglobin fraction and the total hemoglobin fraction. The ratio of the two absorbances gave the% of HbA[1]c. C-reactive protein was measured by the ELISA kit. Significant difference was found in the values of fasting glucose, of the normal and diabetic groups [p<0.05] but no significant difference was present in the values of HbA[1]c of both groups. There was no significant difference in the values of C-reactive protein of the normal and diabetic groups. Short duration hyperglycemia has no role in producing inflammation and raising the levels of bioinflammatory marker C-reactive protein

Suppression of humoral immune response due to psychological stress in survivors of large scale natural disaster

To study the impact of psychological stress following a natural disaster on specific immunological parameters. The study was carried out over a 3 month period [Feb 2006 to May 2006] at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan. Physically uninjured male adults between 15-60 years of age, with no prior history of an active physical or psychological disorder, who witnessed the earthquake on 8[th] Oct, 2005 in Pakistan but escaped physical injury, were included in the study. Age and gender matched healthy adults were also studied as control group. Analysis of haemoglobin, total leucocyte count, lymphocyte count, lymphocyte subsets, IgG, IgA, IgM levels, C reactive protein and nitrobluetetrazolium [NBT] dye reduction test was carried out on both the groups. Psychologically distressed individuals had increased CD 3+ cells [p=0.02], increased CD4:CD8 ration [p=0.04], reduced CD19+ cells count [p=0.03] and IgG levels [p=0.01]. Neutrophil oxidative burst activity without stimulation was increased [p=0.02]. Psychological stress consequent to exposure to a natural disaster can suppress humoral immune response

HLA Frequencies in Pakistani Population in comparison with Caucasian, oriental and Negroid population

The main objective of the study was to determine HLA-A, B, and DR frequencies in Pakistani population and their comparison with HLA frequencies reported for caucasian, oriental and negroid population. Material and methods: Five thousand prospective donors/recipients of renal/bone marrow transplant who had undergone HLA typing were included in the study. All subjects were of pure Pakistani origin. They included 1245 recipients [996 males, 249 females] and 3755 donors [2740 males, 1015 females]. The HLA antigens were tested with two stage NIH micro-lymphocytotoxicity assay by using Terasaki plates. Separated T and B lymphocytes were used for class I and Class II antigens detection. Relative antigen frequencies were determined and used to calculate the gene frequencies. The HLA class I antigens A2, A11, A24[9], A1, A26[10], A3, A28, A33[19], B51[5], B35, B8, B57[17], B60[40], B44[12], B7, B61[40] were the most commonly detected antigens for the HLA class I A and B subclasses. In case of HLA-DR, DR3, DR11[5], DR7, DR15[2] were the most frequently found antigens. These antigen frequencies were compared with the antigen frequencies reported for the Caucasians, Orientals and Negroid population. Comparison of common frequencies with other populations indicates that Pakistani population is nearer to Caucasians and Orientals

Frequency of HLA DR2 in patients with severe and very severe Aplastic anaemia

To determine the frequency of HLA DR2 in Pakistani patients with severe and very severe aplastic anaemia. Introduction: In many cases aplastic anaemia is mediated by the immune mechanisms. Increased frequency of certain HLA haplotypes in patients with autoimmune diseases have led to the investigation of HLA subtypes in aplastic anaemia. HLA DR2 was found to be the most frequently encountered allele in aplastic anaemia. It has been reported that patients of aplastic anaemia, who possess HLA DR2 show a good response to immunosuppressive treatment. This study has been designed to establish frequency of HLA DR2 in patients of aplastic anaemia in our population. Setting: Armed Forces Institute of Pathology and Armed Forces Bone Marrow Transplant Centre, Rawalpindi-Pakistan. Materials and Fifty two cases of aplastic anaemia diagnosed at AFIP/AFBMTC during last 03 years [March 2001 to December 2003] were included in the study. Laboratory investigations to establish the diagnosis included blood complete picture, reticulocyte count, bone marrow aspiration and bone marrow trephine biopsy. Cytogenetic studies were carried out in selected cases to exclude possibility of hypoplastic myelodysplastic syndrome/Fanconi's anaemia. LAP score, ham's test, sucrose lysis test, urine for haemosiderin and CD59 analysis were carried out in suspected cases to exclude paroxysmal nocturnal haemoglobinuria. All cases were tested for HLA DR2 by standard National Institute of Health two stage microlymphocytotoxicity assay. Out of 52 patients, 35 were males and 17 were females [M: F 2:1]. Median age of the patients was 17 years [3-35 years]. Twenty eight [54%] of the patients were of severe aplastic anaemia and 24 [46%] were of very severe aplastic anaemia. HLA DR2 was positive in 31[60%] patients compared to 4,1% in healthy population [p. 0.007]. An increased frequency of HLA DR2 is also seen in Pakistani patients of aplastic anaemia which is associated with a good response to immunosuppressive therapy

Lymphocyte subpopulations in pakistani patients with acid fast bacilli positive and acid fast bacilli negative pulmonary tuberculosis

This study was done to comparethe lymphocyte subpopulations in the peripheral blood of patients of acid fast bacilli positive active pulmonary tuberculosis with similar patients who were negative for acid fast bacilli on sputum examination. After exposure to Mycobacterium tuberculosis, only 15% of the infected individuals develop active tuberculosis while about 90% can control and contain the infection successfully. This ability to control the infections is the function of T lymphocytes. Different profiles of lymphocyte subpopulation possibly reflect the immune status of the individual against tuberculosis infections. This study was carried out to compare the lymphocytes subpopulations in the peripheral blood of patients of AFB negative tuberculosis. A total of forty adult patients of pulmonary tuberculosis having positive sputum smears for acid fast bacilli along with fifteen acid fast bacilli negative patients were included in the study. Peripheral blood samples from the patients of both groups were collected aseptically in EDTA containers. A panel of monoclonal antibodies was used to delineate different lymphocyte subsets by flow cytometry. Haemoglobin levels, total leucocyte counts, absolute neutrophil counts and absolute lymphocyte counts were determined by haematology autoanalyser. Total CD4 count was not significantly different in the two groups but activated CD4+ T cell number was significantly high in smear positive patients. Smear positive patients also showed significantly increased total number of CDS positive T lymphocytes but activated CDS lymphocyte number was significantly decreased. Total number as well as activated fraction of gamma dejta T cells was markedly enhanced in smear negative group of patients. Activation status of CD8+ T lymphocytes along with increased number of gamma delta cells [absolute number as well as activated fraction] make important contributions in limiting the extent of disease in pulmonary tuberculosis

Evaluation of serum transferrin recceptor concentration in diagnosing and differentiating Iron deficiency anaemia from anaemia of chronic disorders

To diagnose and differentiate iron deficiency anaemia [IDA] from anaemia of chronic disorders [ACD] using serum concentration of soluble transferrin receptors [sTfR]. 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. 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/l, whereas mean + SD sTfR levels in ACD patients was 2.96 + 1.28 mg/l, 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. 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