Serologic profiling of D variants in donor routine: unveiling the impact on false-negative results and alloimmunization.

The high number of D variants can lead to the unnecessary use of Rh immune globulin, overuse of D- RBC units, and anti-D allommunization. D variant prevalence varies among ethnic groups, and knowledge of the main variants present in a specific population, their behavior in serologic tests, and their impact on clinical practice is crucial to define the best serologic tests for routine use. The present study aimed to explore the serologic profile of D variants and to determine which variants are most associated with false-negative D typing results and alloimmunization. Donor samples were selected in two study periods. During the first period, D typing was performed on a semi-automated instrument in microplates, and weak D tests were conducted in tube or gel tests. In the second period, D typing was carried out using an automated instrument with microplates, and weak D tests were performed in solid phase. Samples from patients typed as D+ with anti-D were also selected. All samples were characterized by molecular testing. A total of 37 RHD variants were identified. Discrepancies and atypical reactivity without anti-D formation were observed in 83.4 percent of the samples, discrepant D typing results between donations were seen in 12.3 percent, and D+ patients with anti-D comprised 4.3 percent. DAR1.2 was the most prevalent variant. Weak D type 38 was responsible for 75 percent of discrepant samples, followed by weak D type 11, predominantly detected by solid phase. Among the D variants related to alloimmunization, DIVa was the most prevalent, which was not recognized by serologic testing; the same was true for DIIIc. The results highlight the importance of selecting tests for donor screening capable of detecting weak D types 38 and 11, especially in populations where these variants are more prevalent. In pre-transfusion testing, it is crucial that D typing reagents demonstrate weak reactivity with DAR variants; having a serologic strategy to recognize DIVa and DIIIc is also valuable.

Genetic diversity of Gerbich alleles in Brazilians reveals an unexpected prevalence of the GE:-2,-3,4 phenotype.

BACKGROUND AND OBJECTIVES: Gerbich (GE) blood group system carries high-frequency antigens and the absence of them leads to rare phenotypes: GE:-2,3,4, GE:-2,-3,4 and GE:-2,-3,-4. Their serological differentiation is limited and misclassification of Gerbich phenotypes may occur, but this can be avoided by molecular characterization. This study aimed to characterize the molecular background responsible for rare Gerbich phenotypes in Brazilian population. MATERIALS AND METHODS: We selected eight samples from patients with anti-Ge, six from their relatives and nine samples with normal expression of Gerbich antigens. Serological tests were performed in gel and red blood cells (RBCs) were tested with anti-Ge2 and anti-Ge3. Monocyte monolayer assay (MMA) was performed. Molecular investigation was performed with allele-specific polymerase chain reaction and DNA sequencing. RESULTS: Patient plasma samples reacted with all commercial RBCs. Patient RBCs showed negative results with anti-Ge2 and anti-Ge3. Using MMA two of eight antibodies were clinically significant. Exon 3 was not amplified in any of the patient samples and in two samples from relatives, suggesting the presence of GE*01.-03/GE*01.-03. By sequencing, we identified the genetic variability that interferes with the definition of deletion breakpoints, thus two options of genetic structure were suggested to be responsible for the GE:-2,-3,4 phenotype. CONCLUSION: This study showed for the first time the genetic diversity of GYPC alleles for carriers of Gerbich-negative phenotypes in a Brazilian population and showed an unexpected prevalence of the GE:-2,-3,4 phenotype. It also demonstrated the importance of using molecular tools to correctly classify Gerbich phenotypes for selection of variants in antigen-matched transfusions.

RHCE diversity among Brazilian patients with sickle cell disease (SCD) and selected groups of blood donors.

BACKGROUND: Several centers have selected Black donors to prevent Rh alloimmunization of patients with sickle cell disease (SCD). As the Brazilian population is considered very admixed and race definition by self-declaration is questionable, this study aimed to compare RHCE diversity among patients with SCD and selected groups of Brazilian blood donors to define which group of donors would be the adequate red cell supply for patients with SCD. METHOD: We compared RHCE allele frequencies between patients with SCD and four groups of Brazilian blood donors: self-declared Black donors (SDB), donors with predominant African genetic markers (AAM), donors with weak D expression (WDD), and random donors (RDs). Variant RHCE alleles were identified using molecular protocols. RESULTS: Among patients with SCD, 47% had at least one variant RHCE, in SDB and WDD this frequency was higher, 53% and 58.6%, respectively. In AAM and in RD the frequencies were 32% and 27.6%, respectively. In patients with SCD and SDB, the most common alleles were RHCE*ce.01, RHCE*ceVS.01, and RHCE*ceVS.02. WDD had a high frequency of RHCE*ceAR and highest frequency of variant RHCE in both alleles, followed by patients with SCD and SDB. CONCLUSION: This study showed that even in an admixed population the selection of SDB donors is the best choice of matching for transfusion support in patients with SCD. For specific RHCE alleles, selection of donors with weak D expression could be a good option.

Antigen matching for transfusion support in Brazilian female patients with sickle cell disease to reduce RBC alloimmunization.

BACKGROUND: Red blood cell (RBC) alloimmunization is a complication of patients with sickle cell disease (SCD) and it has a greater impact on pregnancy, leading to a risk of hemolytic disease of the newborn and reducing blood availability for pregnant women. This study proposed to evaluate antigen matching transfusion protocols, aiming to reduce RBC alloimmunization in Brazilian female patients with SCD. METHODS: Samples from female patients with SCD (153) and self-declared Afro-Brazilian donors (307) were genotyped for RBC antigens and RH variants were investigated. The transfusion needs of patients during 1-year period and the number of compatible donors were assessed using three antigen-matching transfusion protocols: prophylactic CEK antigen-matched RBCs, prophylactic extended antigen-matched RBCs, and extended-matched red blood cells (RBCs) only for alloimmunized patients. In addition, RH molecular matching has been proposed for patients carrying variant RHCE. RESULTS: Provision of CEK antigen-matched donors would have been possible in 92.4% of transfusion events while provision of prophylactic extended antigen-matched RBCs would cover 88.7% of the transfusion events. Extended antigen matching for alloimmunized patients would be efficient in 99% of the cases. The presence of partial D in 10 patients increased the need of D-negative donors. Compatible donors could be enough for four of the five patients with altered RHCE genotypes in both alleles. CONCLUSION: In Brazilians, screening African descent donors allows the implementation of prophylactic CEK and extended antigen-matching transfusion protocols to female patients with SCD to reduce RBC alloimmunization; however, the supply of compatible blood can be impaired for patients with Rh variants.

Characterization of RHD alleles present in serologically RHD-negative donors determined by a sensitive microplate technique.

BACKGROUND AND OBJECTIVES: Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D-negative, leading to D alloimmunization of D-negative recipients. The present study aimed to investigate the presence of RHD-positive genotypes in blood donors typed as D-negative by an automated system using the solid-phase methodology as a confirmatory test. METHODS: Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D-negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D-negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo-Immucor®), and the confirmatory test was performed by solid-phase technique using Capture R® technology. A multiplex PCR specific to RHD and RHDψ was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD-positive samples, and a specific PCR was used to identify the D-CE(4-7)-D hybrid gene. RESULTS AND CONCLUSION: No weak D type was found in either screening populations. Additionally, 353 (18·4%) D-negative samples presented previously reported non-functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D-negative.

Correlation among automated scores of agglutination, antigen density by flow cytometry and genetics of D variants.

BACKGROUND: Laboratory testing to identify the molecular basis of serologic weak D phenotypes is recommended to determine whether a pregnant woman or potential transfusion recipient should be managed as RhD-positive or RhD-negative. The variation in D antigen expression on RBCs, different potencies of anti-D typing reagents, lack of standardized test methods, and the subjectivity of interpreting agglutination reactions complicate the detection of D variants. We evaluated the correlation of agglutination scores by an automated immunoassay analyzer with D antigen densities determined by flow cytometry, and D variant types identified by molecular analysis. MATERIALS AND METHODS: We selected 273 blood donor samples with agglutination scores of less than 92 (4+), measured by an automated analyzer (NEO®, Immucor, Norcross, GA, USA). D antigen densities were measured by flow cytometry for 89 samples. Samples were classified as molecularly-determined weak D or partial D variants by multiplex PCR, PCR RFLP and DNA sequencing. RESULTS: All samples with a D antigen density ≥15% had an agglutination score >80 (4+). Agglutination scores for weak D types varied from 10 to 90. Agglutination scores for partial D antigens were graded with scores varying from 60 to 99. D antigen densities varied from 0.55% to 10.67% for weak Ds and 4.1% to 30.5% for partial Ds. DISCUSSION: Our results showed that score values follow a pattern among D variants that could be related to antigen density and to the RhD variant classification.

Novel RHAG allele encoding the Rh(null) phenotype in Brazil.

Rhnull is a rare phenotype characterized by the loss of Rh antigen expression. This phenotype can be related to several molecular backgrounds. In this study, we show a novel allele in a Brazilian pregnant woman encoding the Rhnull phenotype due to a change in RHAG exon2 c.310C>T, which leads to a premature stop codon (Gln104Stop).

A simple approach to screen rare donors in Brazil.

Providing blood units for patients with an antibody to a high-prevalence antigen or with multiple common antibodies is a constant challenge to the blood banks. Finding a compatible donor requires extensive screening, with incurs a large amount of investment. In this article, we share our experience of organizing a rare donor inventory with limited resources, we include the strategy used for finding rare donors, and we share the difficulties found during the implementation of the approach and the results obtained.

How do we identify RHD variants using a practical molecular approach?

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.

DARC (Duffy) and BCAM (Lutheran) reduced expression in thyroid cancer.

Duffy or DARC (Duffy Antigen Receptor for Chemokines) is a glycosylated membrane protein that selectively binds angiogenic chemokines. Previous in vivo and in vitro studies of DARC function in cancer have associated DARC over expression with better prognosis, decreased metastatic potential, and inhibition of tumor-associated neovascularization. Another carcinogenesis-associated antigen is Lutheran or BCAM (basal cell adhesion molecule), a surface glycoprotein that acts as a receptor for the extracellular matrix protein, laminin. BCAM is a protein related to tumor progression; and, its over expression is associated with skin, ovarian and pancreatic cancers. We explored DARC and BCAM functions and investigated whether or not their expressions were altered in thyroid cancer. The expression of DARC and BCAM were evaluated by quantitative real-time PCR (qPCR) in a set of 18 normal thyroid tissues (NT), 15 follicular adenomas (FTA), 17 follicular carcinomas (FTC), and 122 papillary thyroid carcinomas (PTC), including 78 classical (CVPTC) and 44 follicular variant (FVPTC). RNA was isolated, reverse transcribed to cDNA, and used in qPCR reactions containing SYBR Green. The relative expression value was calculated using ribosomal protein S8 as an internal control. When we compared benign (NT and FTA) versus malignant samples (FTC, CVPTC and FVPTC) we observed a significant decrease of DARC and BCAM relative expression in malignant cases. Additionally, we correlated clinic-pathological features (tumor size, presence of metastasis, presence of lymphocyte infiltrate) with DARC and BCAM expression. We found a diminished expression of DARC in PTC samples, which was correlated with tumor size and presence of a lymphocyte infiltrate. We, also, found a correlation between decreased BCAM expression and tumor size or presence of metastasis. DARC and BCAM expression was associated with pathogenesis of thyroid carcinoma and correlated with clinical-pathological features.

From the investigation of RHD-CE hybrid genes to the recognition of RHCE variants and RHD zygosity. Expanding the analysis by QMPSF in Brazilian donors and in patients with sickle cell disease.

BACKGROUND: Hybrid genes are responsible for the formation of Rh variants and are common in patients with sickle cell disease (SCD). However, it is not usually possible to detect them by conventional molecular protocols. In the present study, hybrid genes were investigated using the Quantitative Multiplex Polymerase chain reaction of Short Fluorescent Fragments (QMPSF), a molecular protocol that quantifies the copy number of RHD and RHCE exons. In addition, we explored additional relevant information obtained with QMPSF, such as recognition of variant RHCE and RHD zygosity. MATERIALS AND METHODS: Three groups of subjects were selected for the study: patients with SCD, self-declared African descent donors (SDA), and D-negative donors. RHD and RHCE hybrids genes were investigated by the QMPSF method. Real-time multiplex polymerase chain reaction (PCR) assay was used to confirm the copy number of the RHD in two samples. Cloning was performed to investigate the allele. Relative RhD antigen density was investigated by flow cytometry, and RhCE phenotyping was performed with both tube and gel methods. RESULTS: In the 507 samples analysed, hybrid allele frequencies were found in 20.08% of patients with SCD, in 18.22% of individuals in the SDA group, and 3.67% of D-negative donors. The SCD and SDA groups had a higher frequency of hybrid alleles, most commonly involving exon 8, with which we found an association with c.733C>G, a common polymorphism observed in individuals of African descent. Of note, two patients with SCD were shown to carry three gene copies, as confirmed by quantitative PCR; no increase in D expression was observed in these patients. In addition, the QMPSF guided the investigation of 144 RHCE variants and RHD zygosity, and two novel alleles were identified. DISCUSSION: The QMPSF was shown to identify hybrid alleles involved in altered Rh phenotypes in Brazilian donors and patients with SCD. The association of the hybrid RHCE-D(8)-CE allele with c.733C>G suggests this hybrid allele may be used as a marker to detect the most frequent variants found in patients with SCD.

Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells.

BACKGROUND: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection. METHODS: The present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined. RESULTS: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05). CONCLUSIONS: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.

Increasing rate of anti-SARS-CoV-2 antibodies between the first and second waves of COVID-19 in São Paulo, Brazil: A cross-sectional blood donors-based study.

BACKGROUND: SARS-CoV-2 infections rapidly spread along with Brazilian territory with heterogeneous transmission and mortality rates, mostly depending on region and period. Investigation of SARS-CoV-2 antibodies is an important tool to understand virus circulation. Given that blood donors are a representative casuistic of a healthy population, the authors evaluated the seroprevalence of IgG and IgM COVID-19 antibodies in 2,806 blood donors from a blood bank located in São Paulo, Brazil. METHODS: Aiming to evaluate viral behavior over time, the authors selected samples from blood donors who donated in June and October 2020, and February 2021. To determine whether socio-demographic features affected the seroprevalence, the authors analyzed samples from three different regions from São Paulo (capital, metropolitan and countryside regions) and evaluated predictors as gender, age, educational level, race, and use of public transportation. RESULTS: As expected, the authors observed that seroprevalence increased over time. Seroprevalence was greater in São Paulo city compared to metropolitan and countryside regions, being smallest in the countryside. Characteristics associated with a lower percentage of antibodies were age above 50 years, higher educational level, self-declared Caucasian, and use of individual transportation. CONCLUSION: In conclusion, blood donors' samples proved to accurately reflect virus circulation in the healthy population.

ABI3 ectopic expression reduces in vitro and in vivo cell growth properties while inducing senescence.

BACKGROUND: Mounting evidence has indicated that ABI3 (ABI family member 3) function as a tumor suppressor gene, although the molecular mechanism by which ABI3 acts remains largely unknown. METHODS: The present study investigated ABI3 expression in a large panel of benign and malignant thyroid tumors and explored a correlation between the expression of ABI3 and its potential partner ABI3-binding protein (ABI3BP). We next explored the biological effects of ABI3 ectopic expression in thyroid and colon carcinoma cell lines, in which its expression was reduced or absent. RESULTS: We not only observed that ABI3 expression is reduced or lost in most carcinomas but also that there is a positive correlation between ABI3 and ABI3BP expression. Ectopic expression of ABI3 was sufficient to lead to a lower transforming activity, reduced tumor in vitro growth properties, suppressed in vitro anchorage-independent growth and in vivo tumor formation while, cellular senescence increased. These responses were accompanied by the up-regulation of the cell cycle inhibitor p21 WAF1 and reduced ERK phosphorylation and E2F1 expression. CONCLUSIONS: Our result links ABI3 to the pathogenesis and progression of some cancers and suggests that ABI3 or its pathway might have interest as therapeutic target. These results also suggest that the pathways through which ABI3 works should be further characterized.

Antibody indexes in COVID-19 convalescent plasma donors: Unanswered questions.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by high contagiousness, as well as variable clinical manifestations and immune responses. The antibody response to SARS-CoV-2 is directly related to viral clearance and the antibodies' ability to neutralize the virus and confer long-term immunity. Nevertheless, the response can also be associated with disease severity and evolution. This study correlated the clinical characteristics of convalescent COVID-19 patients with immunoglobulin A (IgA) and IgG anti-SARS-CoV-2 antibodies. METHODS: This study included 51 COVID-19 health care professionals who were candidates for convalescent plasma donation from April to June 2020. The subjects had symptomatic COVID-19 with a polymerase chain reaction-confirmed diagnosis. We measured anti-SARS-CoV-2 IgA and IgG antibodies after symptom recovery, and the subjects were classified as having mild, moderate, or severe symptoms. RESULTS: Anti-SARS-CoV-2 antibodies were positive in most patients (90.2%). The antibody indexes for IgA and IgG did not differ significantly between patients presenting with mild or moderate symptoms. However, they were significantly higher in patients with severe symptoms. CONCLUSIONS: Our study showed an association between higher antibody indexes and severe COVID-19 cases, and several hypotheses regarding the association of the antibody dynamics and severity of the disease in SARS-CoV-2 infection have been raised, although many questions remain unanswered.

Optimization of red blood cell unit storage during SARS-COV-2 Pandemic: adopting new strategies to ensure supply in a decentralized blood bank in Brazil.

INTRODUCTION: As coronavirus disease-2019 (COVID-19) spread worldwide and social restrictions were intensified, difficulties in blood supply were expected to result in a shortage of blood donors, logistic issues and a change in blood consumption. Consequences could be detrimental to the meeting of the blood supply demand, especially in a decentralized blood bank in the State of São Paulo responsible for providing blood to more than 100 hospitals, mostly of the public health system. Aiming to minimize negative effects and focusing on maintenance of the blood supply, a different approach was discussed and adopted. MATERIALS AND METHODS: Briefly, strategies were related to monitoring and promoting measures to achieve a positive RBC unit balance. Thus, the number of donors, transfusions, RBC unit inventory, RBC unit loss and RBC units within up to 5 days from the expiration date were evaluated. RESULTS: Several strategies were adopted to ensure sufficient availability of RBC units: blood donation was improved with social media and extra blood collections, a restrictive transfusion protocol was implemented, a new logistic process to use RBC units closer to the expiration date was established and non-isogroup transfusions were avoided. CONCLUSION: Altogether, described strategies were crucial to optimize blood storage during the pandemic. Investing in monitoring and logistics contributed to a positive RBC unit balance and conserving these strategies could be useful.