Abstract Background The diagnostic and prognostic relevance of Human Leukocyte Antigen B-27 (HLA-B27) in Axial Spondyloarthritis (AxSpA) is undeniable, with 70% of Ankylosing Spondylitis (AS) patients carrying the B27 gene, contrasted with a mere 4.35% in the general population. Flow cytometry (FC) and Polymerase Chain Reaction (PCR) have emerged as the predominant techniques for routine HLA-B27 typing. While various studies have compared these methods, none have catered to the unique characteristics of the Brazilian demographic. Therefore, this research aims to compare FC and PCR in a Brazilian cohort diagnosed with AxSpA. Methods An analytical cross-sectional study was undertaken involving 62 AxSpA outpatients from a Brazilian University Hospital. Both FC and PCR-SSP assays were utilized to ascertain HLA-B27 typing. The outcomes (either confirming or refuting the allele’s presence) underwent rigorous scrutiny. Agreement between the methodologies was assessed using the kappa statistic. A p-value of < 0.05 was deemed statistically significant. Results Of the participants, 90.3% (n= 56) were HLA-B27 positive according to FC, while 79% (n= 49) were identified as positive using the PCR method. FC exhibited a sensitivity rate of 98% paired with a specificity of 38.5%. The Positive Predictive Value for FC stood at 85.7%, and the Negative Predictive Value was 83.5%. Consequently, the overall accuracy of the FC method was gauged at 85.5%. A kappa coefficient of κ = 0.454 was derived. Conclusions FC demonstrated noteworthy sensitivity and satisfactory accuracy in HLA-B27 detection, albeit with a reduced specificity when contrasted with PCR-SSP. Nevertheless, given its cost-effectiveness and streamlined operation relative to PCR, FC remains a pragmatic option for preliminary screening in clinical practice, especially in low-income regions. To optimize resource allocation, we advocate for a refined algorithm that initiates by assessing the relevance of HLA-B27 typing based on Choosing Wisely recommendations. It then leans on FC, and, if results are negative yet clinical suspicion persists, advances to PCR. This approach aims to balance diagnostic accuracy and financial prudence, particularly in regions contending with escalating medical costs. B 27 B-2 HLAB27 HLAB HLA (HLA-B27 (AxSpA undeniable 70 AS (AS B2 gene 435 4 35 4.35 population (FC (PCR HLA-B2 methods demographic Therefore crosssectional cross sectional 6 Hospital PCRSSP SSP either alleles allele s presence scrutiny statistic pvalue p value 005 0 05 0.0 significant participants 903 90 3 90.3 n= n (n 56 79 49 98 385 38 5 38.5% 857 85 7 85.7% 835 83 83.5% Consequently 855 85.5% 0454 454 0.45 derived detection PCRSSP. SSP. Nevertheless costeffectiveness cost effectiveness practice lowincome low income allocation recommendations persists prudence costs 2 B- HLAB2 (HLA-B2 43 4.3 HLA-B 00 0. 9 90. 38.5 8 85.7 83.5 85.5 045 45 0.4 (HLA-B 4. 38. 85. 83. 04

BACKGROUND: The Kell blood group system is clinically important in transfusion medicine, particularly in patients with antibodies specific to Kell antigens. To date, genetic variations of the Kell metallo-endopeptidase (KEL) gene among Thai populations remain unknown OBJECTIVE: This study aimed to determine the frequencies of KEL*03 and KEL*04 alleles among Thai blood donors using an in-house polymerase chain reaction-sequence-specific primer (PCR-SSP) method. METHODS: Blood samples obtained from 805 unrelated central Thai blood donors at a blood bank in Pathumthani, Thailand, from March 2023 to June 2023, were typed for Kpª and Kp b antigens using the column agglutination test, and the results for 400 samples were confirmed using DNA sequencing. A PCR-SSP method was developed to detect the KEL*03 and KEL*04 alleles, and genotyping results were validated using known DNA controls. DNA samples obtained from Thai donors in central (n = 2529), northern (n = 300), and southern (n = 427) Thailand were also genotyped using PCR-SSP for comparison. RESULTS: All 805 (100%) donors had the Kp(a−b+) phenotype. The PCR-SSP genotyping results agreed with the column agglutination test and DNA sequencing. All 3256 Thai blood donors had the homozygous KEL*04/KEL*04 genotype. Frequencies of the KEL*03 and KEL*04 alleles among Thai donors differed significantly from those of Japanese, Native American, South African, Brazilian, Swiss, and German populations. CONCLUSION: This study found a 100% KEL*04 allele frequency in three Thai populations. These data could provide information on KEL*03 and KEL*04 allele frequencies to estimate the risk of alloimmunisation in Thai populations What this study adds: This study demonstrates that in-house PCR-SSP can be used to determine KEL*03 and KEL*04 alleles to predict Kpª and Kp b antigens. Even though only homozygous KEL*04/KEL*04 genotypes were found among Thai donor populations, the established PCR-SSP method may be useful for estimating the risk of alloimmunisation in other populations.

Abstract Breast cancer (BC) is the leading cause of death by this disease in women worldwide. Among the factors involved in tumorigenesis, long non-coding RNAs (lncRNAs) and their differential expression have been associated. Differences in gene expression may be triggered by variations in DNA sequence, including single nucleotide polymorphisms (SNPs). In the present study, we analyzed the rs527616 (C>G), located in the lncRNA AQP4-AS1, using PCR-SSP in 306 BC patients and 312 controls, from a Brazilian population. In the BC group, the frequency found for CG heterozygotes was above the expected and the overdominant model is the best one to explain our results (OR: 1.70, IC 95%: 1.23-2.34, P<0.001). Furthermore, the SNP were associated with age at BC diagnosis and the risk genotype more frequent in the older age group. According to TCGA data, AQP4-AS1 is down-regulated in BC tissue, and the overexpression is associated with better prognoses, including Luminal A, HER2-, stage 1 of disease and smaller tumor. In conclusion, the CG genotype is associated with increased susceptibility in the southern Brazilian population. This SNP is mapped in the lncRNA AQP4-AS1, showing differential expression in BC samples. Based on these results, we emphasize the potential of the role of AQP4-AS1 in cancer.

ABSTRACT Background: Immunological life-threatening complications frequently occur in post-hematopoietic stem cell transplantation (HSCT), despite matching recipient and donor (R/D) pairs for classical human leukocyte antigens (HLA). Studies have shown that R/D non-HLA disparities within the major histocompatibility complex (MHC) are associated with adverse effects post-HSCT. Methods: We investigated the impact of mismatches of single-nucleotide polymorphisms (SNPs) in C4A/C4B genes, for showing the highest diversity in the MHC gamma block, on 238 patients who underwent HLA 10/10 unrelated donor (URD) HSCT. The endpoints were acute graft-versus-host disease (aGVHD), chronic graft-versus-host disease (cGVHD) and mortality. One hundred and twenty-nine R/D pairs had 23 C4-SNPs typed by PCR-SSP (Gamma-Type™v.1.0), and 109 R/D pairs had these 23 SNPs identified by next-generation sequencing (NGS) using the Illumina platform. Results: The percentage of patients who received HSC from HLA 10/10 donors with 1-7 mismatches was 42.9%. The R/D pairs were considered C4 mismatched when bearing at least one disparity. These mismatches were not found to be risk factors for aGVHD, cGVHD or mortality after unrelated HSCT when SNPs were analyzed together (matched or mm ≥ 1), independently or according to the percentage of incompatibilities (full match for 23 SNPs; 1-3 mm and >3 mm). An exception was the association between 1-3 mismatches at the composite of SNPs C13193/T14952/T19588 with the development of aGVHD (P = 0.012) and with grades III-IV of this disease (P = 0.004). Conclusion: Our data are not consistent with the hypothesis that disparities in C4A/C4B SNPs increase the risks of post-HSCT adverse effects for the endpoints investigated in this study.

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Abstract Background Alloimmunization is a major problem in transfusion practice due to the clinical complications of the patients and the difficulty of choosing a unit of compatible blood product. Serological methods are widely used in blood banks, but they not always determine the phenotype. Thus, genotyping is an important complement to the serology tool as it allows one to predict the phenotype from deoxyribonucleic acid (DNA) with high accuracy. Objective To compare the centrifugation gel, microarray, Restriction Fragment Length Polymorphismone PCR (PCR-RFLP) and Sequence-Specific Primer PCR (PCR-SSP) techniques, in terms of cost, reaction time and reliability of the results. Methods The RHCE, Kidd, Kell and Duffy blood group systems were chosen to determine the approximate cost of each technique, considering the reagents used in both methods and considering only one sample. The time required for the development of each reaction was obtained at the Maringa Regional Blood Center and Immunogenetics Laboratory at the State University of Maringa. Data from Microarray reactions were obtained at the Campinas Blood Center. The results of phenotyping and genotyping of the 16 samples were compiled in a spreadsheet and compared. Results The PCR-SSP was more economical compared to other methods, and the serological method was faster than the molecular methods. However, all methods proved to be effective and safe in the detection of erythrocyte antigens. Conclusion Analyzing the advantages and limitations of the molecular and serological methods tested in this study, we note that both are important and complementary. However, the choice of a methodology depends on the reality and needs of each health service.

Abstract INTRODUCTION: HPA polymorphism has been associated with HCV presence and fibrosis progression in chronic hepatitis C. However, it is unknown if there is an association between HPA-1 polymorphism and hepatocellular carcinoma (HCC). Therefore, this study aimed to evaluate HPA-1 polymorphism in the presence of HCC. METHODS: PCR-SSP was used to perform HPA genotyping on 76 HCV-infected patients. RESULTS: There was no association between patients with and without HCC. There was significant difference in HPA-1 genotypic frequency distribution between HCC and F1/F2 fibrosis degree. CONCLUSIONS: The HPA-1a/1b polymorphism appears to be more associated with liver damage progression than with HCC presence.

RESUMEN La investigación en Inmunogenética brinda información acerca de marcadores genéticos asociados con enfermedades autoinmunes, como el Lupus Eritematoso Sistémico (LES), se puede observar entonces ciertos factores de riesgo o protección hacia la enfermedad en una población determinada. OBJETIVO: Determinar la asociación genética entre los polimorfismos del Complejo Principal de Histocompatibilidad (CPH) representados por los loci HLA-DRB1 y HLA-DQB1 con la susceptibilidad a LES. METODOLOGÍA: Se trabajó con 85 pacientes lúpicos y 85 pacientes sin la enfermedad; se obtuvo DNA humano a partir de sangre periférica, se realizó un PCR-SSP de baja y alta resolución para tipificar molecularmente a los loci HLA-DRB1 y HLA-DQB1. Se determinó las frecuencias alélicas, las cuales fueron asociadas con ambas muestras mediante el uso del Odds Ratio, a un nivel de significancia del 5 %. RESULTADOS: Los resultados del PCR-SSP de baja resolución muestran que ningún alelo HLA tiene un rol predisponente, se observó que el alelo HLA-DRB1*04 presenta un rol protector OR=0,49 (p=0,03). Los resultados por PCR-SSP de alta resolución muestran que los alelos HLA-DRB1*03:01 (OR=18,3; p=0,007), DRB1*04:04 (OR=4,2; p=0,009), DRB1*09:01 (OR=18,3; p=0,007), HLA-DQB1*03:03 (OR=18,8; p=0,006) y DQB1*02:01 (OR=21,2; p=0,003) son factores de riesgo. Se evidenció que los alelos HLA-DRB1*08:02 (OR=0,42; p=0,003) y HLA-DQB1*04:02 (OR=0,50; p=0,02) son de carácter protector. CONCLUSIONES: Los alelos que representan riesgo de padecer LES en la muestra estudiada son HLA-DRB1*03:01, 04:04, 09:01 y HLA-DQB1*03:03, 02:01. Los alelos que tiene un carácter protector a la enfermedad son HLA-DRB1*08:02 y HLA-DQB1*04:02.

ABSTRACT Immunogenetics research provides information on genetic markers associated with autoimmune diseases such as systemic lupus erythematosus (SLE), you can then observe certain risk factors or protection to the disease in a given population. To determine the genetic association between polymorphisms ofthe Major Histocompatibility Complex loci represented by the HLA-DRB1 and HLA-DQB1 with susceptibility to SLE. METHODOLOGY: We worked with 85 lupus patients and 85 patients without the disease; Human DNA was obtained from peripheral blood, PCR-SSP low and high resolution molecularly performed to establish the loci HLA-DRB1 and HLA-DQB1. Allele frequencies, which were associated with both samples using the Odds Ratio at a level of significance of 5% were determined. RESULTS: Results of PCR-SSP low-resolution HLA show that no predisposing allele plays a role, we observed that HLA-DRB1*04 allele has a protective role OR=0.49 (p=0.03). The PCR-SSP results of high resolution show that the HLA-DRB1*03:01 alleles (OR=18.3; p=0.007), DRB1*04:04 (OR=4.2; p=0.009), DRB1*09:01 (OR=18.3; p=0.007), HLA-DQB1*03:03 (OR=18.8; p=0.006) and DQB1 *02:01 (OR=21.2; p=0.003) are risk factors. We demonstrated that HLA-DRB1 *08:02 alleles (OR=0.42; p=0.003) and HLA-DQB 1*04:02 (OR=0.50; p=0.02) are ofa protective nature. CONCLUSIONS: The alleles representing LES risk in the study sample are HLA-DRB1*03:01, *04:04, *09:01 and HLA-DQB1*03:03, *02:01. The alleles having a protective character to the disease are HLA-DRB 1 *08:02 and HLA-DQB1 *04:02.

RESUMO INTRODUÇÃO: Os antígenos leucocitários humanos (HLA) são moléculas que apresentam antígenos ao sistema imune; a presença ou a ausência deles é descrita como fator influente em algumas doenças. O HLA-B*27 é um polimorfismo do HLA que tem sido associado à maior predisposição à espondilite anquilosante (EA) e a outras espondiloartropatias. A detecção do HLA-B*27 é utilizada como ferramenta diagnóstica e prognóstica nesses casos, assim como no diagnóstico diferencial de outras doenças. OBJETIVO: Padronizar a metodologia de reação em cadeia da polimerase-iniciador específico (PCR-SSP) para utilização no Laboratório de Imunogenética da Universidade Estadual de Maringá (UEM), considerando sua especificidade e seu custo-benefício. MATERIAL E MÉTODOS: Foram utilizados 30 indivíduos comprovadamente sem EA positivos para o alelo HLA-B*27 e 10 indivíduos negativos, testados previamente por PCR-oligonucleotídeo específico (PCR-SSO) e, neste estudo, por PCR-SSP. RESULTADOS: Cem por cento dos pacientes testados tiveram seus resultados confirmados, mesmo com diferentes subtipos. CONCLUSÃO: Tendo em vista a alta reprodutibilidade e o amplo espectro de subtipos que abrange, concluiu-se que a técnica de identificação de PCR-SSP para o HLA-B*27 pode ser utilizada como auxílio diagnóstico de rotina para espondiloartropatias.

ABSTRACT INTRODUCTION: Human leukocyte antigens (HLA) are molecules that present antigen to the immune system; their presence or absence have been described as an influential factor in some diseases. HLA-B*27 is an HLA polymorphism that has been associated with increased susceptibility to ankylosing spondylitis (AS) and other spondyloarthropaty. The detection of HLA-B*27 has been used as diagnostic and prognostic tool in these cases, as well as in the differential diagnosis of other diseases. OBJECTIVE: Standardize the single specific primer-polymerase chain reaction (PCR-SSP) methodology for use in the Immunogenetics Laboratory of the Universidade Estadual de Maringá (UEM), considering its specificity and cost-effectiveness. MATERIAL AND METHODS: A total of 30 individuals without AS positive for HLA-B*27 allele and 10 AS negative individuals, were previously tested by PCR-sequence specific oligonucleotide (PCR-SSO) and, in this study, by PCR-SSP. RESULTS: One hundred percent of the patients tested confirmed their results, even with different subtypes. CONCLUSION: Considering the high reproducibility and the broad spectrum of subtypes covering, it was concluded that the PCR-SSP identifying method for HLA-B*27 can be used as a routine diagnostic tool for spondyloarthropathies.

Abstract Killer Cell Immunoglobulin-like Receptors (KIR) have been used as good markers for the study of genetic predisposition in many diseases and in human genetic population dynamics. In this context, we have investigated the genetic diversity of KIR genes and their main HLA class I ligands in Saudi population and compared the data with other studies of neighboring populations. One hundred and fourteen randomly selected healthy Saudi subjects were genotyped for the presence or absence of 16 KIR genes and their HLA-C1, -C2, -Bw4Thr80 and Bw4Ile80 groups, using a PCR-SSP technique. The results show the occurrence of the framework genes (3DL2, 3DL3 and 2DL4) and the pseudogenes (2DP1 and 3DP1) at highest frequencies. All inhibitory KIR (iKIR) genes appeared at higher frequencies than activating genes (aKIR), except for 2DS4 with a frequency of 90.35%. A total of 55 different genotypes were observed appearing at different frequencies, where 12 are considered novel. Two haplotypes were characterized, AA and Bx (BB and AB), which were observed in 24.5% and 75.5% respectively of the studied group. The frequencies of iKIR + HLA associations were found to be much higher than aKIR + HLA. KIR genes frequencies in the Saudi population are comparable with other Middle Eastern and North African populations.

Backgound and aim: There are some common genetic features between celiac disease (CD) and diabetes mellitus type 1 (DM). However, the genetic risk factors have not been fully clarified for CD and the co-occurrence of CD and DM. KIR (killer immunoglobulin-like receptor) genes regulate the cytolitic activity of NK-cells and T lymphocytes. The aim of this study is to evaluate the contribution of KIR genes, KIR ligands, and combinations of KIR/KIR ligands on the genetic predisposition to CD and co-occurrence of CD and DM. Material and methods: Forty six patients with CD (n = 46), 20 patients with CD+DM (n = 20), and 60 healthy controls (n = 60) were included in this study. KIR genes and KIR ligands were investigated with PCR-ssOP and PCR-ssP in all subjects, respectively. Results: This study showed that while the telomeric KIR genes (2DS5 and 3DS1), and combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso- (p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively) were observed more frequently in patients with CD than in controls, the 2DS5, 3DS1 KIR genes, C1 ligand, and combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso- (p = 0.002, p = 0.004, p = 0.036, p < 0.001, and p = 0.007, respectively) were observed more frequently in patients with CD+DM than in controls. Conclusions: The results of this study indicated that some KIR genes, KIR ligands, and KIR/KIR ligand interactions may be responsible for a predisposition to CD and the coexistence of CD and DM. For development of coexisting CD and DM, the 2DS5 and 3DS1 genes, C1 ligand, and combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso- were found to be risk factors.

Contexto Doença celíaca é uma enteropatia autoimmune desencadeada pela ingestão de gluten em indivíduos geneticamente suscetíveis. Essa suscetibilidade genética está associada a dois conjuntos de alelos, DQA1*05 - DQB1*02 e DQA1*03 - DQB1*03:02, que codificam moléculas MHC de classe II DQ2 e DQ8, respectivamente. Aproximadamente 90%-95% dos pacientes celíacos são HLA-DQ2 positivos, e metade dos restantes são HLA-DQ8 positivos. No diagnóstico da doença celíaca, a ausência desses alelos DQA e DQB específicos possui um elevado valor preditivo negativo. Objetivo Nosso objetivo foi melhorar a detecção de alguns alelos predisponentes para doença celíaca, combinando a simplicidade e sensibilidade da técnica de PCR em tempo real (qPCR) e análise da curva de melting com a especificidade dos primers de sequência específica. Métodos Primers de sequência específica para DQA1*05 (DQ2), DQB1*02 (DQ2), e DQA1*03 (DQ8) foram usados para testar a presença de cada alelo em reações independentes. Primers para Hormônio de Crescimento Humano foram usados como controle interno. Em paralelo, foi usado um protocolo de PCR-SSP como um método de referência para validar nossos resultados positivos. Resultados Das 329 amostras testadas, 187 (56.8%) foram positivas para os alelos HLA predisponentes, usando as duas técnicas. Essas 187 amostras positivas foram subdivididas em 114 (61.0%) positivas para apenas um alelo, 68 (36.3%) para dois alelos e apenas 5 (2.7%) para os três alelos. Conclusão Os resultados obtidos pela técnica de qPCR mostraram-se altamente confiáveis, sem resultados discordantes quando comparados àqueles obtidos pelo método PCR-SSP.

Background Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value. Objective Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR) and melting curve analysis with the specificity of sequence-specific primers (SSP). Methods Amplifications of sequence-specific primers for DQA1*05 (DQ2), DQB1*02 (DQ2), and DQA1*03 (DQ8) were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results. Results Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%). One hundred fourteen samples (61%) were positive for a single allele, 68 (36.3%) for two alleles, and only 5 (2.7%) for three alleles. Conclusion Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.

Introducción: La asociación del HLA-B27 y las Espondiloartritis, ha hecho evidente que la tipificación del HLA-B27 sea considerada como un apoyo en el diagnóstico de estas enfermedades. Los métodos más empleados para la determinación del antígeno HLA-B27 en los laboratorios clínicos y en investigación son: la microlinfocitotoxicidad (MCTX), la citometría de flujo digital (CMFd), la citometría de flujo análoga (CMFa) y la reacción en cadena de la polimerasa con primers de secuencia específicos (PCR-SSP). Objetivo: Comparar MCTX con la CMFd, la CMFa con la CMFd, y la técnica de CMFd frente a PCR-SSP. Métodos: Se analizaron 4109 solicitudes de HLA-B27 en población con manifestaciones sugestivas de EAS remitidas entre 2009 y 2012 al Hospital Militar Central y al Instituto de Referencia Andino. Se evaluaron las frecuencias obtenidas por Chi cuadrado (X2); para estimar la concordancia metodológica se utilizó el Coeficiente de Correlación Intraclase (CCI). Los análisis se realizaron con el paquete estadístico SPSS V18. Resultados: Al evaluar 467 datos por la técnica de CMFa frente a PCR-SSP, la CMFa mostró 239 resultados entre positivos y en rango indeterminado, de los cuales, luego de ser confirmados PCRSSP, solo 213 demostraron la expresión de HLA-B27 (p<0.05). Se obtuvieron 208 resultados realizados por CMFd y PCR-SSP simultáneamente, observándose una alta correspondencia entre estas técnicas (p<0.05). Para evaluar la concordancia entre la MCTX y CMFd se analizaron 34 datos, revelando un 100% de correspondencia entre esta dos metodologías (CCI=1,p<0.05). Conclusión: La citometría de flujo digital es un método rápido que presenta un desempeño altamente confiable para la identificación de HLA-B27, resultados que se recomiendan confirmar por PCR SSP.

Introduction: The association between HLA-B27 and spondyloarthritis has made clear the fact that identification of HLA-B27 antigen is considered as a support in the diagnosis of these diseases. The most commonly used methods for determination of the HLA-B27 antigen in clinical laboratories as well as in their research, are microlymphocytotoxicity (MCTX), digital flow cytometry (CMFd), analogous flow cytometry (CMFa) and the Single Specific Primer-Polymerase Chain Reaction (PCRSSP). Objective: compare the CMFd against MCTX, CMFa against CMFd and CMFd against PCR-SSP. Methods: 4109 requests for HLA-B27 were analyzed with manifestations suggestive of SpA submitted between 2009 and 2012 at Hospital Militar Central and Instituto de Referencia Andino. To analyze the frequencies Chi square (X2) was evaluated; to estimate the methodological concordance the intraclass correlation coefficient (ICC) was used. All proposed analyzes were performed with SPSS V18. Results: 467 data obtained by CMFa versus PCR-SSP evaluated the CMFA showed 239 results between positive and indeterminate range, which, after being confirmed by molecular biology (PCRSSP), only 213 showed the expression of HLA-B27 (p <0.05). PCR-SSP and CMFd performed 208 results simultaneously, showing a high correlation between these techniques (p <0.05). To evaluate the correlation between CMFd and MCTX, 34 data were analyzed, revealing a 100% match on the positive results from these two methodologies (ICC = 1, p <0.05). Conclusion: The digital flow cytometry is a rapid method that presents a highly reliable for the initial identification of HLA-B27; results confirmed by PCR SSP recommend performance.

Introdução: a associação do HLA-B27 e as Espondilartrite, evidenciou que a tipificação do HLAB27 seja considerada como um suporte no diagnóstico dessas doenças. Os métodos mais usados para a determinação do antígeno HLA-B27 nos laboratórios clínicos e no investigação são: a microlinphocitotoxicity (MCTX), a citometria de fluxo digital (CMFd), a citometria de fluxo análoga (CMFa) e a reação em cadeia de a polimerasa com primers de sequência específicos (PCR-SSP). Objetivo: Comparar MCTX com a CMFd, a CMFa com a CMFd, e a técnica de CMFd com PCRSSP. Métodos: 4109 solicitudes de HLA-B27 em população com manifestações sugestivas de EAS remitidas entre 2009 e 2012 ao Hospital Militar e ao Instituto de Referencia Andino, foram analisadas. Avaliaram-se as frequências obtidas por Chi quadrado (X2); para estimar a concordância metodológica foi utilizado o Coeficiente de Correlação Intraclasse (CCI). Os análises estão feitos com o paquete estadístico SPSS V18. Resultados: A CMFa mostrou 239 resultados entre positivos e em rango indeterminado quando avaliou-se 467 dados com a técnica de CMFa com PCR-SSP. Só 213 deles demostraram a expressão de HLA-27 (p<0.05), depois de ser confirmados PCR-SSP. Foram obtidos 208 resultados por CMFd y PCR-SSP em simultâneo, com uma alta correspondência entre estas técnicas (p<0.05). Para avaliar a concordância entre MCTX y CMFd analisaram-se 34 dados, revelando um 100% de correspondência entre as duas metodologias (CCI=, p<0.05). Conclusão: A citometria de fluxo é um método rápido que tem um desempeno muito confiável para a identificação de HLA-B27, resultados recomendados para confirmar por PCR SSP.

Objective:To investigate the relationship between the polymorphism of human leukocyte antigen (HLA)-DRB1 and the susceptibility and repellency of drug use combined with HIV infection in Chinese.Methods:A total of 213 unrelated healthy people, 41 HIV-infected drug users, 24 HIV-uninfected drug users, and 64 HIV-infected non-drug users were recruited. Their HLA-DRB1 allele frequencies were analyzed by PCR-SSP and allele distribution was analyzed.Results:Compared with healthy controls, in drug users, the frequencies of HLA-DRB1 *0401-041, *1001 were significantly higher; in HIV-infected patients, the frequencies of HLA-DRB1 *0101-0103, *0401-0411, *1001 were significantly higher, while the frequencies of DRB1 *1501-1502, *1101-1105, *1301-1302, DRB4, DRB5 were significantly lower; in HIV-infected drug users, the frequencies of HLA-DRB1 *0101-0103, *0401-0411, *0801-0806, *1001, *1401/1404/1405 were significantly higher, while the frequencies of DRB1 *1301/1302, 1501-1502, DRB5 were significantly lower.Conclusion:There is close relationship between the polymorphism of HLA-DRB1 alleles and drug use with HIV infection, which plays an important role in elucidating the pathogenesis and providing the basis for therapeutics and prophylaxis of patients with drug use and HIV infection.

Objetivos: Identificar biomarcadores de susceptibilidad para AIJ poliarticular y AR de instalación temprana por estudio del polimorfismos de MHC/HLA-DRB1* y PTPN22. Materiales y métodos: Se realizó un estudio de casos y controles con una relación 1:2. Todos los sujetos de investigación y los controles provinieron de una corta anidada perteneciente a un proyecto institucional; 30 pacientes con AIJ y 30 con AR de instalación temprana. Como controles se estudiaron 60 individuos sanos. El ADN se obtuvo por salting out modificado. La tipificación de los alelos MHC/DRB1* se realizó por PCR-SSP y en el polimorfismo (C1858T) del sistema PTPN22 se utilizó PCR-RTq. Resultados: Para AIJ Poliarticular, el alelo DRB1*0404 se asoció con susceptibilidad (OR=10.82; p<0.05), en el grupo con AR de instalación temprana, DRB1*0101 se mostró como marcador de susceptibilidad (OR=4.04; p<0.05). Se destaca que el alelo HLA-DRB1*0701 aparece como marcador protector para ambas patologías (OR=0,15; p<0,05). El polimorfismo del SNP (C1858T) PTPN22 no se asoció con AIJ Poliarticular. En contraste, en AR de instalación temprana, el Alelo CC se asoció con protección p<0.05. En el mismo grupo, CT/TT se mostró como un marcador de susceptibilidad <0.05. El análisis de la secuencia aminoacídica 70QRRAA74 del epítope compartido se asoció con susceptibilidad para ambas entidades (p<0.05) y la secuencia 70DRRGQ74 con protección en ambos grupos de pacientes (p<0.05). Conclusión: Se destaca que en la asociación con la secuencia del epítope compartido, la ubicación del tipo de aminoácido y posición del mismo define probable asociación como marcador molecular de susceptibilidad en ambas entidades. Los polimorfismos compartidos sugieren un origen genético común para ambas entidades.

Objectives: To identify polymorphisms of MHCIHLA-DRB1* and PTPN22 systems as a genetic biomarker of susceptibility to JIA poliarticular and early installation RA. Material and methods; This was a pilot case control study with a relation of 1:2. Patients and control individuals involved in this study were selected from a nested cohort from an institutional previous RAIJIA project. The sample was represented by thirty patients with JIA and 30 diagnosed with early installation RA. Sixty unrelated healthy individuals were involved as a control DNA Isolation was obtained by a modified salting out technique. The oligotyping of the MHCIDRB1* alleles was performed by PCR-SSP and the typing of the PTPN22 polymorphism was done by RT-PCR. Results: The DRB1*0404 allele was associated with susceptibility to JIA(OR=10.82, P<0.05). In the early installation RA group the DRB1*0101 allele was showed as a marker of susceptibility to JIA patients (OR=4.04, P<0.05). It is noteworthy that the HLA-DRB1*0701 appears as a possible protective marker for both diseases (OR=015, p<0.05). The polymorphism of (C1858T)PTPN22 was not associated with poliarticular JIA. In contrast, in the early installation RA group of patients, the CC PTPN22 polymorphism was found to be as a protective marker (p<0.05). On the other hand, the amino acid sequence 70QRRAA74 of the share epitope was a marker for susceptibility to both entities (p<0.05) By contrast, the sequence 70DRRGQ74 of the same epitope was showed as a possible marker for protection on both entities (p<0.05.). Conclusion: The model that was used for searching association between the shared epitope -region 70-74 of the DRB1* alleles and these two entities showed the importance of the location and also the type of amino acid in those positions. The polymorphisms found as molecular markers of susceptibility for both entities suggested a common origin and could suggest its probable roll as a molecular marker of susceptibility.