Development and Validation of In-house HEp-2 Cell Slides for Detection of Antinuclear Antibodies (ANA) by Indirect Immunofluorescence.

OBJECTIVE: To develop and validate in-house HEp-2 cell slides for the detection of ANA by indirect immunofluorescence. STUDY DESIGN: Cross sectional validation study. Place and Duration of the Study: Department of Immunology, Armed Forces Institute of Pathology Rawalpindi, Punjab, Pakistan, from April to September 2022. METHODOLOGY: This study involved development of in-house HEp-2 cell slides after procuring cell lines, sub-culturing and fixing them on different slides using variety of fixatives under different protocols. After standardisation of procedure, validation of procedure was done by testing sera of 305 patients for ANA detection at 1:40 dilution on in-house HEp-2 cell slides and subsequently on commercial HEp-2 cell slides (gold standard). Indirect immunofluorescence was observed by the two observers working independently and kept blinded from the results interpreted by each other. Data were collected on a pre-designed proforma and then analysed. Sensitivity, specificity, and positive predictive value (PPV) and negative predictive values (NPV) of in-house HEp-2 cell slides were calculated. RESULT: Sera of 305 patients were tested on in-house and commercial HEp-2 cell slides. Sensitivity and specificity of in-house HEp-2 cell slides for ANA detection were 96.92% and 99.58%, respectively. PPV and NPV of in-house HEp-2 cell slides came out to be 98.43% and 99.17% respectively. CONCLUSION: In-house HEp-2 cell slides are as effective as commercial HEp-2 cell slides for the detection of ANA and can be used as cost-effective alternative. KEY WORDS: Antinuclear antibodies (ANA), Human epithelial type-2 (HEp-2), Immunofluorescence.

Comparison of immune response to SARS-COV-2 vaccine in COVID-recovered versus non-infected Individuals.

To determine the antibody levels at 6 months in SARS-CoV-2 vaccinated individuals in COVID-recovered versus non-infected groups to determine the need to administer booster COVID vaccine in each group. Prospective longitudinal study. Pathology Department, Combined Military Hospital, Lahore for a period of eight months from July 2021 to February 2022. Two hundred and thirty three study participants in both COVID recovered and non-infected groups (105 participants in infected group, 128 participants in non-infected group) were subjected to blood sampling at 6 months post-vaccination. Anti-SARS-CoV-2 IgG antibody test was done using Chemiluminescence method. Comparison of antibody levels between COVID-recovered and non-infected groups was made. Results were compiled and statistically analyzed using SPSS version 21. Out of 233 study participants, males were 183 (78%) while females were 50 (22%), mean age being 35.93 years ± 8.298. Mean Anti-SARS-CoV-2 S IgG levels among COVID-recovered group was 1342 U/ml and among non-infected group was 828 U/ml at 6 months post-vaccination. Mean antibody titers in COVID-19 recovered group are higher than in non-infected group at 6 months post-vaccination in both groups.

Aberrant expression of myeloid antigens in patients of acute lymphoblastic leukaemia.

OBJECTIVE: To determine the immunophenotypic pattern and aberrant expression of myeloid antigens in newly diagnosed patients of acute lymphoblastic leukaemia(ALL). METHODS: This descriptive cross-sectional study was carried out in Haematology / Pathology department, Army Medical College, National University of Medical Sciences (NUMS) in collaboration with Immunology and Haematology departments of Armed Forces Institute of Pathology (AFIP), Rawalpindi from 1st January, 2019 to 31st December, 2019. Seventy-three (73) recently diagnosed patients of Acute Lymphoblastic leukaemia of all age groups and both genders were included in the study. A proforma was used to note demographic data. CBC, cytochemical stains and bone marrow examinations were carried out and assessed for morphology and percentage of blasts using a microscope. Flow cytometry was used to perform immunophenotyping on samples of peripheral blood and bone marrow, using a standard panel. RESULTS: The most commonly expressed markers were weak CD45, TdT, CD19, CD10 and HLA-DR. Weak CD45 was present in almost all blast cells and there was no remarkable difference in its positivity among various subtypes of ALL. Myeloid expression was observed in 13 (17.8%) cases. CD13 and CD33 were aberrantly expressed in 11 and 12.3 of all cases of ALL respectively. CONCLUSIONS: Expression of aberrant myeloid CD markers in acute lymphocytic leukaemia has prognostic significance and should be documented during lineage assignment of acute leukaemias while performing immunophenotyping.

HLA-DQ2 and HLA-DQ8 Alleles in Celiac Disease Patients and Healthy Controls.

OBJECTIVE: To compare HLA-DQ2 and HLA-DQ8 alleles between celiac disease patients and healthy control group. STUDY DESIGN: Observational cross-sectional study. PLACE AND DURATION OF STUDY: Department of Immunology, Armed Forces Institute of Pathology (AFIP), from April to December 2018. METHODOLOGY: Subjects were included: 100 celiac disease patients selected by non-probability consecutive sampling, and 100 healthy subjects. After collecting peripheral blood in EDTA tubes, chromosomal DNA was extracted and amplified, using sequence specific primers. Post-amplification electrophoresis was performed on two per cent agarose gel, followed by ethidium bromide staining; and specific band patterns were recorded under ultraviolet illumination to determine the HLA-DQ alleles. The subtypes of HLA-DQ2, i.e. HLA-DQ2.5 and HLA-DQ2.2 were also assessed. Frequency, percentage, mean and SD were calculated. Post-stratification Chi-square test was applied. RESULTS: The mean age of celiac disease group and healthy subjects was 14.79 ± 5.32 years and 14.71 ± 5.21 years, respectively. The frequency of HLA-DQ2 and HLA-DQ8 among celiac disease patients was 93% and 4%, respectively. Among HLA-DQ2 positive, HLA-DQ2.5 and HLA-DQ2.2 were found in 92% and 8%, respectively. Statistically significant difference (p <0.05) was observed between the celiac disease patients and healthy group. There was no significant difference observed among different age groups and gender (p >0.05). CONCLUSION: HLA-DQ2 detection reliably diagnoses celiac disease among all age groups and either gender. It can be used as an effective marker for early diagnosis of celiac disease instead of invasive procedures such as intestinal biopsy. The diagnosis can be pin-pointed by presence of HLA-DQ2.5. Key Words: Celiac disease, HLA-DQ2, HLA-DQ8, HLA-DQ2.5, HLA-DQ2.2.

Next Generation Sequencing-Based Germline Panel Testing for Breast and Ovarian Cancers in Pakistan.

BACKGROUND: Pathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype. METHODS: Peripheral blood of 24 women, diagnosed with breast and epithelial ovarian cancers, was taken from the recruited cases with the consent of performing germline genetic testing. DNA was isolated from the peripheral blood and subjected to indexed BRCA Panel libraries. Targeted NGS was performed for all coding regions and splicing sites of BRCA1 and BRCA2 genes using AmpliSeq for Illumina BRCA Panel and Illumina MiSeq sequencer (placed at AFIP). Analysis of the sequencing results has been done by using Illumina bioinformatics tools. RESULTS: We detected 421 variants having a quality score of 100 in all cases under study. The list of identified variants in BRCA1 and BRCA2 genes was narrowed down after filtering out those which did not pass q30 and those with a minor allele frequency (MAF) > 0.05 based on gnomAD browser. To classify these variants, clinical significance was predicted using external curated databases. As a result, we interpreted (n = 4) 16.7% pathogenic variants in BRCA1 and (n = 6) 25% variants of uncertain significance (VUS) in both genes. Descriptive statistics depicted that the age and BMI of BRCA positive cases are less than BRCA negative cases. CONCLUSION: Our findings exhibit an initial report for the NGS based cancer genetic testing in Pakistan.  This will enable us to pursue screening and diagnosis of hereditary BRCA mutation utilizing the latest state-of-the-art technique locally available in Pakistan ultimately resulting in targeted cancer therapy.

Comparison of Sequence Specific Primers in the Next Generation Sequencing in Human Leukocyte Antigen Typing for Transplant Recipients.

OBJECTIVE: To compare two human leukocyte antigen (HLA) typing methods, namely sequence specific primers (SSP) and next generation sequencing (NGS) for alleles concordance Study Design: Descriptive study. PLACE AND DURATION OF STUDY: Immunology department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 2019 to May 2020. METHODOLOGY: A total of 48 subjects, including 30 males and 18 females, were typed by NGS at 7 loci, making a total of 672 types loci. SSP typing was done for 276 loci among these. Comparison was made at SSP level of low resolution. NGS typing was done with Illumina's MiSeq instrument using Omixon HLA holotype 7 loci kit and analysis done with HLA twin software. SSP typing was done with micro SSP kit from onelambda. Statistical analysis was done using statistical package for social sciences (SPSS) version 24.0. RESULTS: Among the 672 NGS types loci and 276 SSP types loci, there were mismatches at one B locus and one C locus, whereby NGS computed HLA-B*58:01 and HLA-C*12:02 while SSP detected HLA-B*57 and HLA-C*05, respectively. At remaining 274 loci, HLA typing fully matched at low resolution, making concordance rate 99.3%. Commonest alleles detected by NGS were HLA-A*02:01, B*51:01, C*07:02, DPB1*04:01, DQA1*01:03, DQB1*02:01 and DRB1*13:01. CONCLUSION: Batch testing, high throughput, improved accuracy, more loci coverage, maximum gene coverage including all exons and introns and high-resolution typing confer significant advantages to next generation sequencing over old methods of HLA typing. This technique is suitable for high throughput laboratories. High running cost hampers its routine implementation in 3rd world countries. Key Words: Human leukocyte antigen, Next generation sequencing, High resolution.

Frequency of Panel Reactive Antibodies (PRA) among Renal Transplant Recipients and its Effect Modifiers.

OBJECTIVE: To determine frequency of panel reactive antibodies among renal transplant recipients and its effect modifiers. STUDY DESIGN:   A cross-sectional study. PLACE AND DURATION OF STUDY: Department of Immunology, Armed Forces Institute of Pathology from October 2016 to October 2017. METHODOLOGY: One hundred and sixty-two (162) patients, who were referred to Department of Immunology for pre-transplant workup for kidney transplantation of both genders and Pakistani nationality. Informed consents were taken and detailed history were recorded. Frequency and percentages were calculated for panel reactive antibodies, blood transfusion, pregnancy and previous transplant were noted and Chi-square test was applied. RESULTS: One hundred and sixty-two (162) patients including 141 males and 21 females were analysed and 48 patients (30%) were positive for panel reactive antibodies (PRA). Of 141 male patients analyzed, 35 were positive for PRA, which were about 25%. Twenty-one females were tested for PRA and 13 female patients were positive that is about 62% of the analysed population. Out of the total 141 males, 20 (14%) had blood transfusion and of these 11 (55%) were positive for PRA. Without history of transfusion, only 9 (7%) were positive for PRA. Out of 21 females, 10 were positive for blood transfusion, out of which 6 (60%) were positive for PRA. Without history of blood transfusion, 7 (64%) were positive for PRA. Out of 21 females, 20 had history of pregnancy. Out of whom, 13 (65%) were positive for PRA. Two patients (one male and one female) were with history of previous transplant and both were positive for PRA. CONCLUSION: A significant number of patients were sensitised with panel reactive antibodies waiting for renal transplant. The PRA was more common in recipients who were prone to effect modifiers such as pregnancy, blood transfusion and re-transplant. These risk factors were mostly present in combination, which also suggests their synergistic effects on PRA synthesis. Key Words: Blood transfusion, Effect modifiers, Panel reactive antibodies, Pregnancy, Kidney transplant, Re-transplant.

Impact of Homozygous C4A Deficiency on Clinical Presentation of Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the association of C4A null allele (C4AQ0) with systemic lupus erythematosus (SLE) and determine the clinical presentation of SLE in relation to C4A null allele. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Pathology (AFIP), Rawalpindi, Immunology Department, from December 2018 to December 2019. METHODOLOGY: Patients referred to AFIP, who fulfilled American College of Rheumatology (ACR) criteria of 1997 for diagnosis of SLE were included in the study. Approval from the Institutional Ethical Review Board was taken. C4A and C4B null alleles were determined in 66 SLE patients and 40 age- and gender-matched healthy controls by polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP). Various clinical features and laboratory findings in the SLE patients were analysed in relation with C4A null allele. RESULTS: The mean age of the study population was 30.56 ±10.08 years. C4A null allele was detected in 7 (10.6%) patients; whereas, C4B null allele was detected in only two (3%) patients. SLE patients with C4A null allele had increased incidence of arthritis (100%) and renal damage (85.7%); compared to those with normal C4A allele, 57.6% and 32%, respectively. Fisher's Exact test revealed strong association of C4A null allele with arthritis and renal damage, (p = 0.039 and 0.01, respectively). CONCLUSION: Homozygous absence of C4A alleles was encountered in 10.6% of Pakistani patients of SLE and is closely related with clinical features of arthritis and renal damage. Knowledge of C4A null allele in SLE patients at diagnosis can predict disease course. Key Words: SLE, C4A null alleles, C4AQ0, Homozygous C4A deficiency.

Clinical and Laboratory Characteristics of Primary Immunodeficiency Patients from a Tertiary Care Center in Pakistan.

OBJECTIVE: The aim of this study was to describe and identify clinical presentation of primary immunodeficiency disorders (PIDs). Characteristic quantitative and qualitative immunological abnormalities have been described which help in establishing a definitive PID diagnosis. METHODS: This was a cross sectional study conducted in the Immunology department of the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from Jan 2016 to Dec 2018. Sixty patients of different PIDs including humoral defects, combined immunodeficiency, phagocytic defects and other miscellaneous disorders, were diagnosed over a period of 3 years in our institute. Their clinical presentation and laboratory data are presented in this study. RESULTS: In 3 years, 40 (66%) males and 20 (33%) females were diagnosed, with 13 (21.6%) patients of humoral deficiency, 22 (36.6%) of severe combined immunodeficiency, 18 (30%) of phagocytic defects and 7 (11.6%) of other miscellaneous disorders. Maximum patients belonged to Punjab province, i.e., 23 (38.3%). Their mean age for initiation of symptoms was 7±12.6 months, while diagnosis was made at mean age of 26±39.28 months, in all groups combined. Respiratory infections were commonest presentation, in 46 (76.6%) patients. Also 46 (76.6%) patients had consanguineous parents. Presence of family history of PID in 27 (45%) patients was not associated with an earlier diagnosis (p 0.955). Each group of patients carried characteristic laboratory findings. CONCLUSIONS: PIDs should be suspected in offsprings with warning signs coming from consanguineous parents. There is a need to introduce genetic diagnosis of PIDs in order to timely diagnose less characteristic PID presentations.

Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses.

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3, and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.

Distribution of HLA-B*27 Subtypes in Patients with Ankylosing Spondylitis in Local Population.

OBJECTIVE: To compare the distribution of HLA-B*27 subtypes in healthy controls and in ankylosing spondylitis (AS) patients of different ethnic groups from Pakistan. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from April 2016 to October 2017. METHODOLOGY: Forty-nine HLA-B*27 positive, unrelated AS patients and 18 HLA-B*27 positive healthy BMT/renal transplant donors were selected for this study. Typing of the HLA-B27 alleles was performed by the polymerase chain reaction-sequence-specific primer (PCR-SSP). RESULTS: There was a wide number of HLA-B*27 subtypes and an elevated frequency of the B*2707 allele in the AS patients. The allele B*2706 seems to have a protective role in the population studied because it was found only in the healthy controls. HLA-B*27:03 and 07 were found predominant subtypes in Punjabis and Pathans, respectively. CONCLUSION: There were no significant differences for the distribution of B*27 subtypes between patients and controls (p >0.05).

Significance of Testing Anti-Thyroid Autoantibodies in Patients with Deranged Thyroid Profile.

BACKGROUND: We hypothesized that anti-thyroid antibodies are more often positive in individuals with deranged thyroid profile. METHODS: This prospective cohort was done in Immunology Department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from Jan 2017 to Oct 2017. All the samples that were referred to us for testing anti-thyroid antibodies (anti-TPO or anti-TG antibodies) and thyroid profile were included in the study. There were no exclusion criteria. Tests for anti-thyroid antibodies were performed by ELISA and thyroid profile by chemiluminescence. SPSS 23.0 was used for statistical analysis. RESULTS: Over a course of a ten-month study period, we received a total of 316 serum samples for anti-TPO/TG antibodies along with thyroid profile testing (TSH). These included 115 males (36.4%) and 201 females (63.6%). Their age ranged from 3 to 89 years (mean ± SD, 42.22 ± 18.09). Anti-TPO antibodies were more often positive when TSH was deranged (p value 0.001). Anti-TPO antibodies are more often raised in females, in terms of both prevalence (p 0.001) and mean rank (p 0.002). CONCLUSION: As anti-thyroid antibodies are more often present when TSH is deranged, such individuals should be screened for anti-thyroid antibodies. This importance of screening is compounded by the fact that anti-thyroid antibodies may be positive in a significant percentage of elderly people.

Leukocyte Adhesion Deficiency Type 1 with Low Expression of CD 11b.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare autosomal recessive disorder caused by mutations in the gene that codes for CD18, the beta chain of beta-2 integrins, located on the long arm of chromosome 21. This defect results in failure of leukocyte migration to the site of infection due to the absence of surface integrins. Leukocyte adhesion deficiency should be suspected in any patient with recurrent infections, impaired wound healing, history of delayed umbilical cord separation, periodontitis, leukocytosis, recurrent soft tissue and oral infections. Diagnosis is based on the analysis of neutrophils for the surface expression of CD18, CD11a, CD11b and CD11c by flow cytometry. Here, we present a 55-day male infant with umbilical cord separation on the 10th day of life and no history of infection, who was identified with LAD-1 with low expression of CD11b. The purpose of performing LAD flow cytometric analysis in this patient was to screen him for LAD-1 as his elder brother had LAD-1 and one elder sister died undiagnosed with recurrent skin and chest infections at 8 months of age.

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.

Determination of Specificity and Pattern of Antinuclear Antibodies (ANA) in Systemic Rheumatic Disease Patients Positive for ANA Testing.

OBJECTIVE: To determine probability of finding antinuclear antibodies (ANA) and anti extractable nuclear antigens (ENA) positive samples and associating ANA patterns with anti-ENA reactivities among a consecutive cohort of samples of systemic rheumatic disease patients referred for ANA testing. STUDY DESIGN: Prospective cohort study. PLACE AND DURATION OF STUDY: Immunology Department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from January to June 2016. METHODOLOGY: All the samples referred for ANA testing with clinical suspicion of systemic rheumatic disease were included. After screening, ANA positive samples were subjected to anti-ENA antibodies testing (including anti-SSA, antiSSB, anti-Sm, anti-RNP, anti-SCL-70 and anti-Jo-1 antibodies) and ANA pattern and titer determination. RESULTS: Of 4,347 samples received, 397 were positive for ANA (9%). Of 397, 96 (24%) samples positive on ENA screen were tested for anti-ENA reactivity. Anti-SSA antibodies were found in 59 samples. Commonest ANA patterns were coarse and fine speckled (43 and 22 samples of 81 tested), while majority of samples carried ANA in titers of 1:40 and 1:80 (22 and 18 samples of 81 tested). No specific ANA pattern was associated with any particular anti-ENA reactivity. CONCLUSION: Among samples/patients referred for investigations of autoimmune disorders, probability of finding positive ANA is approximately 9%. Of these 9%, about 24% also show reactivity against ENA. Commonest ANA pattern is coarse speckled and majority of such patients carry ANA in titers ranging from 1:40 to 1:80. Commonest ENA reactivity was against SSA.

Tumor protein 53 mutations mapping to its tertiary structure with in-silico prediction of neoepitopic vaccine candidates in bone tumors.

OBJECTIVE: Mapping of tp 53 mutations in bone cancers present in COSMIC database to its secondary and tertiary structure with in silico prediction of newly formed HLA binding epitopes as candidates for synthetic peptide vaccine. Mutations in bone cancers present in COSMIC database were listed and manually induced in wt p53 FASTA sequence. Wt p53 secondary structure was predicted. Template identified and tertiary structure of wt p53 was modelled in Cn3D followed by individual mutations mapping onto this model. HLA class I binding affinity was determined for mutated sequences to determine any newly binding peptide sequences. 62 missense mutations were identified. After predicting secondary structure, template was identified as PDB ID 1MZR for tertiary structure modelling. Mutations were highlighted that showed most of mutations in DNA binding region of tp 53 tetramer. Wt p53 had 19 HLA class I binders whereas in 62 mutated sequences we identified 18 neobinders not present in wt sequence. Neoepitopes identified serve as candidates for individualized anti-cancer peptide vaccine therapy.

Identification of HLA Class I Misreads/Dropouts Using Serological Typing, in Comparison with DNA-based Typing.

Serology and DNA techniques are employed for Human Leukocyte Antigen (HLA) typing in different transplant centers. Results may not always correlate well and may need retyping with different technique. All the patients (with aplastic anemia, thalassemia, and immunodeficiency) and their donors, requiring HLA typing for bone marrow transplant were enrolled in the study. Serological HLA typing was done by complement-dependent lymphocytotoxicity while DNA-based typing was done with sequence specific primers (SSP). Serology identified 167 HLA A and 165 HLA B antigens while SSP in same samples identified 181 HLA A and 184 HLA B alleles. A11 and B51 were the commonest antigens/alleles by both methods. There were a total of 21 misreads and 32 dropouts on serology, for both HLA A and B loci with HLA A32, B52 and B61 being the most ambiguous antigens. Inherent limitations of serological techniques warrant careful interpretation or use of DNA-based methods for resolution of ambiguous typing.