The impact of neonatal 17-hydroxyprogesterone cutoff determination in a public newborn screening program for congenital adrenal hyperplasia in Southern Brazil: 3 years' experience.

Congenital adrenal hyperplasia (CAH) occurs due to enzyme defects in adrenal steroidogenesis. The 21-hydroxylase deficiency accounts for 90-95% of cases, triggering accumulation of 17-hydroxyprogesterone (17-OHP). Early diagnosis through neonatal screening allows adequate treatment and reduced mortality. The purpose of the study was to determine 17-OHP cutoffs for the diagnosis of CAH in a public newborn screening program in Southern Brazil. A retrospective, descriptive, cross-sectional study was conducted to analyze 17-OHP levels in dried blood samples collected on filter paper of 317,745 newborns screened at a public newborn screening center from May 2014 to April 2017. Neonatal 17-OHP was measured in DBS samples using a time-resolved fluoroimmunoassay (GSP® kit 3305-0010; PerkinElmer). Different cutoffs were determined and stratified by birth weight. The incidence of CAH was 1:15,887 live births in the state of Rio Grande do Sul, with 20 cases of classical CAH diagnosed during the study period. Most newborns (80.73%) were white, and the prematurity rate was 9.8% in the study population. The combination of different percentiles, 98.5th for birth weight 2001-2500 g and 99.8th for the other birth weight groups, decreased false-positive results and increased specificity compared with current reference values to identify classical CAH cases. The local 17-OHP cutoffs determined were higher than those currently used by this screening program for all birth weight groups. The calculation of reference values from local population data and the combination of percentiles proved to be a valuable tool for proper diagnosis of CAH and reduction in the number of false positives.

Epidemiological profile of congenital hypothyroidism at a southern Brazilian state.

Objective: To determine the incidence of congenital hypothyroidism (CH) over a 10-year period at the Reference Service in Neonatal Screening of the state of Rio Grande do Sul (RSNS-RS). Subjects and methods: Historical cohort study including all newborns screened for CH by the RSNS-RS from January 2008 until December 2017. Data of all newborns with neonatal TSH (neoTSH; heel prick test) values ≥ 9 mIU/L were collected. According to neoTSH values, the newborns were allocated into two groups: Group 1 (G1), comprising newborns with neoTSH ≥ 9 mIU/L and serum TSH (sTSH) < 10 mIU/L, and Group 2 (G2), comprising those with neoTSH ≥ 9 mIU/L and sTSH ≥ 10 mIU/L. Results: Of 1,043,565 newborns screened, 829 (0.08%) had neoTSH values ≥ 9 mIU/L. Of these, 284 (39.3%) had sTSH values < 10 mIU/L and were allocated to the G1 group, while 439 (60.7%) had sTSH ≥ 10 mIU/L and were allocated to the G2 group, and 106 (12.7%) were considered missing data. The overall incidence of CH was 42.1 per 100,000 newborns screened (95% confidence interval [CI] 38.5-45.7/100,000) or 1:2377 screened newborns. The sensibility and specificity of neoTSH ≥ 9 mIU/L were 97% and 11%; of neoTSH 12.6 mUI/L, 73% and 85% respectively. Conclusion: In this population, the incidence of permanent and transitory CH was 1:2377 screened newborns. The neoTSH cutoff value adopted during the study period showed excellent sensibility, which matters for a screening test.

Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women

Abstract Introduction During pregnancy, women are at an increased risk of developing iron-deficiency anemia. Objective The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. Method: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. Results The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. Conclusion Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women.

INTRODUCTION: During pregnancy, women are at an increased risk of developing iron-deficiency anemia. OBJECTIVE: The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. METHOD: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. RESULTS: The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. CONCLUSION: Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Epidemiological profile of congenital hypothyroidism at a southern Brazilian state

ABSTRACT Objective: To determine the incidence of congenital hypothyroidism (CH) over a 10-year period at the Reference Service in Neonatal Screening of the state of Rio Grande do Sul (RSNS-RS). Subjects and methods: Historical cohort study including all newborns screened for CH by the RSNS-RS from January 2008 until December 2017. Data of all newborns with neonatal TSH (neoTSH; heel prick test) values ≥ 9 mIU/L were collected. According to neoTSH values, the newborns were allocated into two groups: Group 1 (G1), comprising newborns with neoTSH ≥ 9 mIU/L and serum TSH (sTSH) < 10 mIU/L, and Group 2 (G2), comprising those with neoTSH ≥ 9 mIU/L and sTSH ≥ 10 mIU/L. Results: Of 1,043,565 newborns screened, 829 (0.08%) had neoTSH values ≥ 9 mIU/L. Of these, 284 (39.3%) had sTSH values < 10 mIU/L and were allocated to the G1 group, while 439 (60.7%) had sTSH ≥ 10 mIU/L and were allocated to the G2 group, and 106 (12.7%) were considered missing data. The overall incidence of CH was 42.1 per 100,000 newborns screened (95% confidence interval [CI] 38.5-45.7/100,000) or 1:2377 screened newborns. The sensibility and specificity of neoTSH ≥ 9 mIU/L were 97% and 11%; of neoTSH 12.6 mUI/L, 73% and 85% respectively. Conclusion: In this population, the incidence of permanent and transitory CH was 1:2377 screened newborns. The neoTSH cutoff value adopted during the study period showed excellent sensibility, which matters for a screening test.

Twenty Years of Neonatal Screening for Sickle Cell Disease in Brazil: The Challenges of a Continental Country with High Genetic Heterogeneity

Abstract Sickle cell disease (SCD) is the most common inherited hematological disease worldwide. The benefits of diagnosis and early intervention have led to the wide dissemination of public health programs worldwide. Through neonatal screening programs, it is possible to reduce morbidity and mortality in the first 5 years of life. The prophylactic use of penicillin, the anti-pneumococcal vaccine and other intensive care, increase the survival and quality of life of people with SCD. The aim of this study is to present the 20-year history of screening for hemoglobinopathies in Brazil and its challenges. From 2001 to 2019, an average of 2,400,000 children were screened per year nationwide, with the coverage being of 82,16%. The screening of 54,9% of newborns is collected up to their 5th day of life. The prevalence of SCD was 1:2,263 newborns; therefore, it was the second most-common disease detected by the program of Brazil, being only after hypothyroidism (1/2,175 live births). The healthcare system should provide the necessary infrastructure to confirm the diagnosis of newborns and to provide appropriate counseling and treatment. The early diagnosis and treatment, as well as the follow-up with a multidisciplinary team, are fundamental to the survival rate and the quality of life of patients.

Development of CYP21A2 Genotyping Assay for the Diagnosis of Congenital Adrenal Hyperplasia.

BACKGROUND: Steroid 21-hydroxylase deficiency due to CYP21A2 gene mutations represents more than 90% of all congenital adrenal hyperplasia cases. This deficiency is screened by measuring levels of 17-hydroxyprogesterone, which may vary, causing false positive or false negative results. In order to assist the diagnosis, molecular methodologies have been employed. This work aimed to perform genotyping assays to detect mutations in the CYP21A2 gene and compare the findings with other population studies. METHODS: The SNaPshot assay was developed to simultaneously detect 12 frequent point mutations in the CYP21A2 gene (p.Arg409Cys, p.Gln319Ter, p.Arg357Trp, p.Leu308PhefsTer6, p.Val237Glu, IVS2-13A/C > G, p.Ile173Asn, p.Pro31Leu, p.Pro454Ser, p.Val282Leu, p.Gly111ValfsTer21 and p.His63Leu). The direct sequencing and multiplex ligation-dependent probe amplification assays were used to confirm point mutations present in the developed method. The latter was also used to search large deletions and gene conversion, complementing the investigation. A total of 166 cases were studied. RESULTS: The SNaPshot assay was successfully developed to detect the 12 mutations. The results of mutation analysis indicated 84 pathogenic alleles in 48 cases, with p.Val282Leu (27.1%) and IVS2-13A/C > G (20.8%) being the most frequently found mutations. Between the findings of this study and those of other South American studies, there were significant differences in frequency for p.Pro31Leu and p.Val282Leu (p < 0.001). A new variant T in IVS2-13A/C > G was identified in two patients via the SNaPshot assay. CONCLUSION: The molecular strategy developed for CYP21A2 gene mutation screening allowed us to detect the principle mutations described around the world. Furthermore, the first Southern Brazilian mutation frequencies concerning the CYP21A2 gene were obtained.

Do Alpha Thalassemia, Fetal Hemoglobin, and the UGT1A1 Polymorphism have an Influence on Serum Bilirubin Levels and Cholelithiasis in Patients with Sickle Cell Disease?

BACKGROUND: Increased destruction of erythrocytes in patients with sickle cell disease results in chronic hyperbilirubinemia and leads to the formation of gallstones. OBJECTIVES: The objective of this study was to determine the combined influence of alpha thalassemia, fetal hemoglobin, and the UGT1A1 polymorphism on serum bilirubin levels and cholelithiasis in patients with sickle cell disease. METHODS: We analyzed 72 patients treated in the outpatient hematology unit of the Clinical Hospital of Porto Alegre. The alpha thalassemia trait was determined by multiplex polymerase chain reaction and the polymorphisms of UGT1A1 by capillary electrophoresis with tagged primers. RESULTS: Total and indirect bilirubin levels differed significantly between genotypes TA7/TA7 and TA6/TA6 (p < 0.05). Bilirubin levels were influenced by the UGT1A1 polymorphism but not by alpha thalassemia and fetal hemoglobin. There was no association between cholelithiasis and any of the variables studied. CONCLUSION: These preliminary findings suggest that the UGT1A1 gene can influence serum bilirubin levels in sickle cell anemia and serve as a tool to differentiate an acute hemolytic condition from a pre-existing condition of hyperbilirubinemia.

Characterization of Patients with Chronic Myeloid Leukemia Unresponsive to Tyrosine Kinase Inhibitors Who Underwent Allogeneic Hematopoietic Stem Cell Transplantation.

Background: Tyrosine kinase inhibitors (TKIs) were the first drugs to use an intracellular signaling molecule as a therapeutic target. Unresponsiveness to TKIs limits therapeutic options, making allogeneic hematopoietic stem cell transplantation (HSCT) the only option leading to molecular remission. The aim of this study is to characterize CML patients unresponsive to first- and/or second-generation TKI therapy who underwent HSCT and to describe the main factors associated with treatment failure. Subjects and Methods: Twenty one CML patients who underwent allogeneic HSCT and had previously used first- and/or second-generation TKIs from January 2005 to May 2014. Results: Of the 21 patients, 52.4% were male, with a median age of 49 years (23-65 years) and 85.7% had chronic phase CML at the time of diagnosis; 28.6% showed inadequate treatment adherence to TKI therapy. Thirteen patients were resistant and eight were intolerant to TKIs; additionally, nine did not have T315I mutation. Ten transplantations involved related donors, and more than a half of patients (11) died, three of which due to graft failure. Most patients who survived transplantation were in the chronic phase of disease at the time of HSCT. Conclusion: The population was composed mainly of young age patients at diagnosis, male, white, and coming from areas in the state of Rio Grande do Sul other than Porto Alegre and metropolitan region. Low adherence to TKI therapy may be related to unresponsiveness to treatment, especially in patients with acquired resistance, or this low adherence, together with the presence of molecular changes, may have led to the need for HSCT.

Neonatal screening for congenital adrenal hyperplasia in Southern Brazil: a population based study with 108,409 infants.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder associated with inborn errors of steroid metabolism. 21-hydroxylase enzyme deficiency occurs in 90 to 95% of all cases of CAH, with accumulation of 17 hydroxyprogesterone (17-OHP). Early diagnosis of CAH based on newborn screening is possible before the development of symptoms and allows proper treatment, correct sex assignment, and reduced mortality rates. This study describes the results obtained in the first year of a public CAH screening program in the state of Rio Grande do Sul, Brazil. METHODS: We reviewed the screening database in search of babies with suspected CAH, that is, altered birth-weight adjusted 17-OHP values at screening. The following data were analyzed for this population: screening 17-OHP values, retest 17-OHP values, serum 17-OHP values for those with confirmed CAH on retest, maternal and newborn data, and family history of CAH. For the screening program, 17-OHP levels are determined on dried blood spots obtained in filter paper with GSP solid phase time-resolved immunofluorescence. RESULTS: Of 108,409 newborns screened, eight were diagnosed with CAH (four males, four females). The incidence of CAH in the state was 1:13,551. Six cases were identified as classic salt-wasting CAH and two were cases of virilizing CAH. The positive predictive value (PPV) of the initial screening (before diagnostic confirmation) was 1.6%. The overall rate of false positive results was 0.47%. The number of false positive results was higher among newborns with birth weight < 2000 g. CONCLUSION: The present results support the need for CAH screening by the public health care system in the state, and show that the strategy adopted is adequate. PPV and false positive results were similar to those reported for other states of Brazil with similar ethnic backgrounds.

Flow Cytometry in Detection of Fetal Red Blood Cells and Maternal F Cells to Identify Fetomaternal Hemorrhage.

Accurate detection and quantitation of fetomaternal hemorrhage (FMH) is critical to the obstetric management of rhesus D alloimmunization in Rh-negative pregnant women. The flow cytometry is based on the detection of fetal red blood cells using a monoclonal anti-HbF antibody, and is the method most indicated for this estimation. The objective of this study was to quantify fetal red blood cell levels of pregnant women using flow cytometry. We analyzed 101 peripheral blood samples from Rh-negative and Rh-positive women, whose mean age was 24 years (20-32 years), after vaginal delivery or cesarean section. Our study showed that 53% of pregnant women had fetal red blood cells levels <2.0 mL, 31% between 2.0-3.9 mL, 16% between 4.0-15.0 mL, and 1% >15.0 mL. Accurate quantitation of fetal red blood cells is necessary to determine the appropriate dose of anti-D (RHD) immunoglobulin to be administered to pregnant or postpartum women.

Evaluation of the Genetic Screening Processor for the Performance of Newborn Screening Tests

Abstract The collection of dried blood spots (DBSs) on filter paper has been a powerful tool in newborn screening (NBS) programs and in other fields. However, filter paper has been associated with some level of imprecision due to the filter paper matrix effect. In order to minimize measurement variations, these interferences should be evaluated by NBS assays. The aim of this study is to evaluate the performance of genetic screening processor (GSP) equipment in comparison with a widely used AutoDELFIA and to discuss the limitations and advantages of this new technology in NBS. We evaluated the performance of 3 NBS assays in DBS using GSP in comparison with AutoDELFIA. To determine the inaccuracy and the intra-assay precision, a comparative study and a replication experiment were performed. In the comparative study, human thyroid-stimulating hormone (hTSH) assay showed the highest correlation coefficient, followed by 17α-OH-progesterone and immunoreactive trypsinogen (IRT) assays. The results of the present study suggest that the GSP equipment and kits are suitable for implementation and have acceptable performance for NBS routine. Genetic screening processor assay tends to underestimate hTSH and IRT concentrations in the clinically relevant range when compared to AutoDELFIA assays. More studies are necessary to reevaluate cutoff values. Furthermore, the equipment has advantages when compared with AutoDELFIA, such as methodology with more specificity, reduction in the processing time, and randomized routine. This helps promoting faster dynamic technical processes and faster report generation.

Neutrophil CD64 expression as an important diagnostic marker of infection and sepsis in hospital patients.

INTRODUCTION: Infection and sepsis are major health problems. Therefore, the need for improved diagnostic indicators, as well as for better therapeutic monitors in the treatment of infection, remains, since the current diagnostic tools have low specificity and passed through minimal changes in the last two decades. OBJECTIVE: The aim of this study was to establish the correlation of neutrophil CD64 with indicators of infection and sepsis. METHODS: We established the correlation of the neutrophil CD64 expression with the following variables: complete white blood count, band count, neutrophils, C-reactive protein (CRP), cultures, flags released by automated hematology analyzers and clinical groups. Accordingly clinical groups were divided into two: patients "without clinical or laboratory evidence of infection or inflammatory process" and "clinical or laboratory evidence of a systemic inflammatory response (SIRS) and systemic sepsis" based upon identification of organisms by culture. We analyzed 93 whole blood samples anticoagulated with K3EDTA of patients admitted in the Intensive Care Unit (ICU) of a community hospital. RESULTS: The expression CD64 was statistically significant with clinical groups, flags, and neutrophils and was not significantly correlated with total count of white blood cells and cultures. CONCLUSION: Our results indicate that high expression of CD64 is an indicator important in the diagnosis of infection and sepsis.

Estudo comparativo entre metodologias de triagem para detecção de hemoglobina S em bancos de sangue / Comparative study between methods of screening for detection of hemoglobin S in the blood banks

INTRODUÇÃO: O traço falciforme é a presença em heterozigose da hemoglobina S (HbS). A partir de junho de 2004, por meio da RDC 153/04, tornou-se obrigatória a triagem de hemoglobinas anormais em doadores de sangue. OBJETIVO: O objetivo deste estudo foi a comparação de diferentes metodologias de triagem utilizadas nos bancos de sangue para a detecção da HbS. Material e método: No período de abril de 2007 a abril de 2008, foram realizados três métodos de detecção de HbS em 4.108 doadores de sangue aptos que se apresentaram ao banco de sangue do Hospital Universitário de Santa Maria (HUSM). O estudo comparativo entre as metodologias incluiu os testes de solubilidade e de gel-centrifugação, tendo como referência de positividade a presença de HbS na eletroforese de hemoglobina. RESULTADOS: Dos 4.108 doadores estudados, 23 (0,56 por cento) apresentaram resultado positivo para HbS e dois (0,05 por cento) para HbC. Das amostras positivas para HbS detectadas por eletroforese qualitativa, 22 (95,6 por cento) foram detectadas pelo teste de solubilidade e 20 (86,9 por cento) pelo de gel-centrifugação. CONCLUSÃO: A eletroforese de hemoglobinas representou a melhor metodologia na identificação de hemoglobinas variantes e, portanto, deve ser valorizada quando se trata de diagnóstico para triagens em bancos de sangue pelo seu grau de sensibilidade, minimizando ao máximo os resultados falsos negativos e garantindo a qualidade do sangue que estará sendo utilizado.

INTRODUCTION: The sickle cell trait is the presence of hemoglobin S (HbS) in heterozygosity. According to RDC regulation 153/04, abnormal hemoglobin screening has become mandatory in blood donation samples since June 2004. OBJECTIVE: The aim of this study was to compare different screening methods used in blood banks for HbS detection. Material and method: From April 2007 to April 2008, three HbS detection methods were applied in 4,108 suitable blood samples from the blood bank of the University Hospital of Santa Maria (HUSM). The comparative study among the methods comprised solubility tests and gel-centrifuge (ID-HbS). Furthermore, the positivity reference was the presence of HbS on hemoglobin electrophoresis. RESULTS: Twenty-three (0.56 percent) out of 4,108 samples showed positivity for HbS and two (0.05 percent) showed positivity for HbC. Twenty-two (95.6 percent) out of 23 HbS positive samples determined through qualitative electrophoresis were detected by solubility test and 20 (86.9 percent) were detected by gel-centrifugation test. CONCLUSION: Hemoglobin electrophoresis proved the best method in the identification of hemoglobin variants and, therefore, worthwhile when it comes to diagnostic screening in blood banks due to its high sensitivity, which keeps false-negative results to a minimum and ensures blood quality.

Prevalence of UGT1A1 gene polymorphism in patients with hemolytic anemia in southern Brazil.

The aim of the work was to determine the variation of UGT1A1 genotypes in patients with hemolytic anemia in the southern Brazil. Three hundred twenty-three patients with hemolytic anemia were genotyped for UGT1A1 along with 232 controls. Allelic and genotypic distribution did not differ among studied groups. The TA7/TA7 genotype presented a frequency that ranged from 3.2% to 18.0% (nonsignificant). Alleles TA5 and TA8 were also found in the sample, even though southern Brazil is a major Caucasoid region. Genotype prevalence was very similar to those of African origins, reflecting the diversity of ethnic origins and the high degree of admixture in southern Brazil. Further studies should be conducted to correlate the modulating role of UGT1A1 polymorphism with the clinical conditions of each patient with hemolytic anemia.

Glucose-6-phosphate-dehydrogenase deficiency and its correlation with other risk factors in jaundiced newborns in Southern Brazil.

OBJECTIVE: To evaluate the correlation between glucose-6-phosphate-dehydrogenase (G6PD) deficiency and neonatal jaundice. METHODS: Prospective, observational case-control study was conducted on 490 newborns admitted to Hospital de Clínicas de Porto Alegre for phototherapy, who all experienced 35 or more weeks of gestation, from March to December 2007. Enzymatic screening of G6PD activity was performed, followed by PCR. RESULTS: There was prevalence of 4.6% and a boy-girl ratio of 3:1 in jaundiced newborns. No jaundiced neonate with ABO incompatibility presented G6PD deficiency, and no Mediterranean mutation was found. A higher proportion of deficiency was observed in Afro-descendants. There was no association with UGT1A1 variants. CONCLUSIONS: G6PD deficiency is not related to severe hyperbilirubinemia and considering the high miscegenation in this area of Brazil, other gene interactions should be investigated.

Polymorphic variants of UGT1A1 in neonatal jaundice in southern Brazil.

Alterations in the hepatic conjugation of bilirubin due to uridyl-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) polymorphisms have been proposed as risk factors to neonatal jaundice. Herein, we estimated the frequency of genotypes of the promoter region of UGT1A1 gene in newborns and evaluated its association with severe hyperbilirubinemia. Prospective study of cases and controls including all newborns admitted for phototherapy at HCPA, Brazil, during 9 months; 490 babies were enrolled and PCR was performed. Polymorphic genotypes were detected in 16% of the patients and 7 of the 10 possible genotypes were identified with higher prevalence of polymorphisms in Afro-descendants. In this sample, the variants of UGT1A1 were not associated to severe hyperbilirubinemia; other genic factors should be sought in this high miscegenation area of Brazil.