Surveillance for Zika, Chikungunya, and Dengue Virus Incidence and RNAemia in Blood Donors at 4 Brazilian Blood Centers During 2016-2019.

BACKGROUND: Except for public health case reports, the incidence of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) infection are not available to assess the potential blood transfusion safety threat in Brazil. METHODS: Pools of 6 donation samples (MP6) left over from human immunodeficiency virus, hepatitis B virus, and hepatitis C virus nucleic acid testing were combined to create MP18 pools (3 MP6 pools). Samples were tested using the Grifols triplex ZIKV, CHIKV, and DENV real-time transcription mediated amplification assay to estimate prevalence of RNAemia and incidence, and to compare these results to case reports in São Paulo, Belo Horizonte, Recife, and Rio de Janeiro, from April 2016 through June 2019. RESULTS: ZIKV, CHIKV, and DENV RNAemia were found from donors who donated without overt symptoms of infection that would have led to deferral. The highest RNAemic donation prevalence was 1.2% (95% CI, .8%-1.9%) for DENV in Belo Horizonte in May 2019. Arbovirus infections varied by location and time of year, and were not always aligned with annual arbovirus outbreak seasons in different regions of the country. CONCLUSIONS: Testing donations for arboviruses in Brazil can contribute to public health. Transfusion recipients were likely exposed to ZIKV, CHIKV, and DENV viremic blood components during the study period.

Detection of unreported usage of the antiretroviral drug lamivudine in two blood donors.

BACKGROUND: Unreported HIV antiretroviral (ARV) drug usage by blood donors compromises the ability to detect evidence of HIV infection in blood screening tests and represents a risk for blood transfusion safety. Our objective was to determine the frequency of undeclared ARV drug use by blood donors with altered HIV markers. STUDY DESIGN AND METHODS: This was a retrospective cross-sectional analysis of donations that were tested for HIV antibody (ab), antigen (ag), and RNA by chemiluminescent immunoassay and nucleic acid screening tests. Positive samples were retested and were subjected to ARV drug testing by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Of 345,252 blood donations, 361 (0.1%) were positive on initial testing. Samples from 296 (81.9%) of these donations were available for further analysis. The presence of HIV ab/ag and/or RNA was confirmed in 83 (28.0%) of these samples. All 296 bloods were subjected to ARV testing. The ARV drug lamivudine, at 11.3 and 6.7 ng/mL, was detected in 2 of 83 (2.4%) donations that were HIV positive. Other drugs were not detected. CONCLUSION: Unreported ARV usage was identified in two candidates for blood donation. More intensive efforts to educate donors about disclosure and to investigate the extent of this phenomenon in Brazil are needed.

Evaluation of Dengue, Zika virus, and Chikungunya virus transmission by blood components in recipients of haematopoietic stem cell transplantation.

BACKGROUND: Brazil has a high prevalence of arboviruses, especially Dengue (DENV), Zika (ZKV), and Chikungunya (CHKV). OBJECTIVES: To study the risk of DENV, ZKV, and CHKV transmission by blood components in the haematopoietic stem cell transplantation (HSCT) population. METHODS: Prospective cohort of HSCT recipients and donors performed at the Hospital das Clinicas da FMUSP, São Paulo-Brazil. Patients were evaluated by serology and RT-PCR for DENV, ZKV, and CHKV pre-transplantation and once a week until neutrophil grafting. In positive cases (positive RT-PCR and/or serology conversion), an investigation was carried out on the blood components that the patient received to evaluate the possibility of it being transfusion transmitted. RESULTS: A total of 93 patients were included during the study period. The mean age was 52 years with a predominance of males (56.9%). We considered five (5.3%) DENV cases positive by seroconversion in our study. One patient had IgM seroconversion and the other four presented IgG seroconversion to DENV. In the investigation of the blood components, 145 individual samples were analysed. None of the investigated blood components showed a positive RT-PCR. CONCLUSION: We observed a low prevalence of DENV, ZKV, and CHKV in HSCT donors and recipients by serology and RT-PCR, and no case of blood transfusion transmission by RT-PCR.

Improved detection of hepatitis C virus-positive blood donors and determination of infection status.

BACKGROUND: In low-risk populations, variability in the sensitivity of current serological tests for Hepatitis C virus (HCV) blood donor screening may lead to the presence of false-positive results. This contributes to the unnecessary loss of blood donor samples as well as to difficulty in accurate donor counselling. The present study determined the optimal cut-off value of a chemiluminescent immunoassay for identification of HCV-reactive blood donors. STUDY DESIGN AND METHODS: In a retrospective cross-sectional analysis of 193 973 blood donations, 578 samples that were positive for HCV antibody in a chemiluminescent immunoassay and/or RNA screening tests were identified. Blood from 379 of these positive samples was available for retesting by a second confirmatory HCV immunoassay followed by a receiver operating characteristic (ROC) curve analysis. Donors were also recalled for a new analysis. RESULTS: Only 71 (18.7%) blood samples remained HCV-positive upon retesting, while 233 (61.5%) now tested negative and 75 (19.8%) yielding indeterminate results. A signal to cutoff ratio ≥4.32 was determined as the best differential threshold between a positive and negative result, increasing the positive predictive value from 27.3% to 66.7%. CONCLUSION: Using a higher threshold for an HCV-positive blood sample enhances the chemiluminescent immunoassay screening test´s accuracy and helps to improve donor counselling and notification processes.

External quality control program in infectious diseases screening at laboratories and blood banks in Latin America: an analysis of the past 5 years.

Objective: To evaluate the screening of blood samples for infectious disease markers at laboratories and blood banks in Latin America per the findings of an External Quality Assessment Program (EQAP). Methods: This qualitative analysis used data from the EQAP coordinated by the Fundação Pro Sangue Hemocentro de São Paulo with the support of the Pan American Health Organization to assess the performance of blood screening for infectious diseases from 2014 to 2018 in Latin America. Each participating laboratory or blood bank received an identical blind panel with 24 blood samples with variable reactivity for all the screening parameters. Panels were processed at each participating facility and results were returned to the Fundação Pro Sangue Hemocentro de São Paulo for individual and joint analyses. Two types of discrepant results were potential failures: false positive results (FPRs) and false nonreactive results (FNRRs). Results: A total of 23 136 samples were evaluated. Global rates of FPR, FNRR, and concordant results were 0.3%, 1.0% and 98.7%, respectively. Seven FNRRs were found for HBsAg (1.0%), 12 for syphilis (2.6%), and 21 for Chagas disease (2.9%). No FNRRs were found for the HIV, HCV, and HTLV viruses. The average accuracy of all the laboratories and blood banks participating in the EQAP during the study period was 99.5% (standard deviation, 0.5%). Conclusion: The findings of this qualitative analysis are positive for blood safety in Latin America, with an average accuracy of 99.5% among the participating laboratories and blood banks. This report reflects an important improvement in blood bank serological screening EQAP-PAHO report since the 2003.

The impact of immediate adverse donation reactions on the return of volunteers undergoing platelet apheresis.

INTRODUCTION: Immediate adverse reactions experienced during donation decrease return rates among whole-blood donors, but little is known about this effect among platelet apheresis donors. We investigated the impact of immediate adverse reactions on the return rates of volunteer apheresis platelet donors. METHODS: In a sample of 4108 consecutive platelet apheresis donors seen from August 2016 through June 2019, we evaluated whether immediate adverse reactions were associated with returning for a subsequent platelet apheresis donation within a 12-month period. We used propensity score matching to compare donors with and without adverse reactions. RESULTS: An immediate adverse reaction occurred in 312 (7.6%) donors; 98.5% were mild, and 0.3% were severe. Of the original 4108 platelet apheresis donors, only 3211 (72.3%) returned for a subsequent donation within 12 months. Experiencing an immediate adverse reaction during the donation process significantly decreased the return rate for a subsequent donation [HR= 0.74 (0.63-0.87)], especially among female donors [HR= 0.70 (0.53-0.93)], donors aged < 30 years [HR= 0.71 (0.54-0.94)], with a high school educational level [0.63 (0.49-0.81)], donors donating for the first time [HR= 0.73 (0.59-0.90)], and repeat donors with a previous platelet apheresis donation more than 180 days prior [HR= 0.68 (0.50-0.93)]. CONCLUSION: Even mild adverse events reduce the return rates for a subsequent donation among platelet apheresis donors. Female donors, younger donors, and first-time donors are at higher risk of not returning after an immediate adverse reaction. Preventing the incidence of immediate adverse reactions during platelet apheresis donation may increase the rate of donor retention.

Survival among children and adults treated with granulocyte transfusions: Twenty years' experience at a Brazilian blood center.

BACKGROUND: It remains controversial whether granulocyte transfusions as a supportive treatment improve survival in patients with febrile neutropenia or granulocyte dysfunctions. We describe survival rates subsequent to granulocyte transfusions in pediatric and adults patients treated at a major blood center in Brazil. MATERIAL AND METHODS: We retrospectively reviewed the clinical charts of pediatric and adult patients treated with granulocyte transfusions at our institution from January 2000 to October 2019. We assessed demographic characteristics, clinical features, indications for transfusion, units transfused, dose of granulocytes administered and survival rates 30 and 100 days after the initial transfusion. RESULTS: We identified 64 pediatric and 67 adult patients treated with 262 granulocyte transfusions. An optimal dose (> 0.6 × 109 granulocytes per kilogram per transfused unit) was available for transfusion in 80.4 % of pediatric patients but in only 19.6 % of adults (p = 0.017). Thirty days after their first granulocyte transfusion, 38 (59.4 %) pediatric and 61 (91 %) adult patients had died. Patients receiving the optimal dose of granulocytes had better survival outcomes, but even among this sub-group, adults were more likely to die than were children either at 30 days (OR = 8.67, 95 %CI 2.69-34.9) or 100 days (OR = 6.27, 95 %CI 1.86-25.9) after their initial granulocyte transfusion. CONCLUSION: Survival rates following granulocyte transfusion varied by the dose transfused and were higher in children than in adults.

Impact of HLA-G +3142C>G on the development of antibodies to blood group systems other than the Rh and Kell among sensitized patients with sickle cell disease.

BACKGROUND: Patients' inflammatory history is an important factor underlying red blood cell (RBC) alloimmunization, which is a frequent transfusion complication among individuals with sickle cell disease (SCD). HLA-G has been associated with different inflammatory and auto - immune diseases. Our goal was to verify whether the HLA-G + 3142 C>G and 14-bp Ins/Del variations are associated with RBC antibody development among SCD patients. METHODS: This was a single-center case-control study. SCD patients were randomly selected for the study and divided into two groups: 'Alloimmunized' and 'Nonalloimmunized' depending on the presence of irregular antibodies. The 'Alloimmunized'group was further divided into two subgroups according to the presence of only antibodies against the Rh and Kell blood group systems or the existence of antibodies to antigens of the other blood group systems. RESULTS: A total of 213 patients were included in the study (110 alloimmunized and 103 non-alloimmunized). The 'Alloimmunized' and 'Non-alloimmunized' groups did not differ statistically regarding the HLA-G + 14 bp Ins/Del ( p = 0.494) and + 3142 C>G ( p = 0.334). Individuals who had only antibodies against the Rh and Kell antigens had a frequency of HLA-G + 3142GG genotype almost twice as high compared to the groupwith antibodies against less immunogenic antigens ( p = 0.043). CONCLUSIONS: The genotype frequency of HLA-G + 3142 C>G differs among alloimmunized SCD patients, depending on the presence of antibodies against low immunogenic RBC antigens. This highlights a possible role played by the HLA-G molecule in the RBC alloimmunization process.

Variant genotypes associated with reduced expression of RhCE antigens among Brazilian blood donors.

BACKGROUND: The genetic diversity of the RHCE gene locus has been explored in diverse populations of different racial backgrounds. Data referring to the diversity of RHCE encoding weakened expression of C, c, E, and e in multiethnic populations is still incomplete. METHODS: Samples from Brazilian blood donors presenting reduced expression of C, c, E, or e on gel method were selected for the study. All exons and flanking introns of RHCE were genotyped though direct Sanger sequencing for the included donors. RESULTS: Sixty-six donors were included: 23 with weak C, 22 with weak c, 6 with weak E, 14 with weak e, and 1 with weak c and E. Among the samples with weak C, the following altered RH*C were encountered: RHCE*CeMA (n = 3), RHCE*Ce941C (n = 1), and RHCE*CeVA (n = 1). RHD*D-CE(4-7)-D was detected in six cases, RHCE*CE was presumably present in five cases, and seven cases were unexplained. Two altered alleles underlay the weak c phenotype: RHCE*ceJAL (n = 20) and RHCE*ce340T (n = 2), and two altered RHCE justified weak e: RHCE*ceMO (n = 6) and RHCE*ceJAL (n = 8). Three variant RHCE were associated with weak E: RHCE*cEJU (n = 4), RHCE*cE382C (n = 1), and RHCE*cEIV (n = 1). The RHCE*cE905A justified one case of weak c and E. CONCLUSION: We describe the distribution of RHCE variants found in association with weak expression of C, c, E, and e in blood donors of multiethnic origin, which differs in comparison to that previously reported for people of African or Caucasian descent.

Correlation between SARS-COV-2 antibody screening by immunoassay and neutralizing antibody testing.

BACKGROUND: The efficacy of convalescent plasma (CP), an alternative for the treatment of COVID-19, depends on high titers of neutralizing antibodies (nAbs), but assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clinical characteristics of CP donors. STUDY DESIGN AND METHODS: A total of 214 CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies (IgG) using two commercial immunoassays: EUROIMMUN (ELISA) and Abbott (Chemiluminescence). Quantification of nAbs was performed using the Cytopathic Effect-based Virus Neutralization test. Three criteria for identifying donors with nAbs ≥ 1:160 were tested: - C1: Curve ROC; - C2: Conditional decision tree considering only the IA results and - C3: Conditional decision tree including both the IA results and the clinical variables. RESULTS: The performance of the immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers ≥1:160. Regarding the studied criteria for identifying CP donors with high nAbs titers: (a) C1 showed 76.1% accuracy if S/CO = 4.65, (b) C2 presented 76.1% accuracy if S/CO ≥4.57 and (c) C3 had 71.6% accuracy if S/CO was ≥4.57 or if S/CO was between 2.68-4.57 and the last COVID-19-related symptoms were recent (within 19 days). CONCLUSION: SARS-CoV-2 IgG immunoassays (S/CO) can be used to predict high anti-SARS-CoV-2 nAbs titers. This study has proposed different criteria for identifying donors with ≥1:160 nAbs titers, all with high efficacy.

10-year analysis of human immunodeficiency virus incidence in first-time and repeat donors in Brazil.

BACKGROUND AND OBJECTIVES: Incidence in first-time and repeat blood donors is an important measure of transfusion-transmitted HIV infection (TT-HIV) risk. This study assessed HIV incidence over time at four large blood centres in Brazil. MATERIALS AND METHODS: Donations were screened and confirmed using serological assays for HIV from 2007 to 2016, and additionally screened by nucleic acid testing from 2011 forward. Limiting antigen (LAg) avidity testing was conducted on HIV seroreactive samples from first-time donors to classify whether an infection was recently acquired. We calculated incidence in first-time donors using the mean duration of recent infection and in repeat donors using classical methods. Time and demographic trends were assessed using Poisson regression. RESULTS: Over the 10-year period, HIV incidence in first-time donors was highest in Recife (45·1/100 000 person-years (105 py)) followed by São Paulo (32·2/105 py) and then Belo Horizonte (23·3/105 py), and in repeat donors was highest in Recife (33·2/105 py), Belo Horizonte (27·5/105 py) and São Paulo (17·0/105 py). Results from Rio de Janeiro were available from 2013 to 2016 with incidence in first-time donors of 35·9/105 py and repeat donors from 2011 to 2016 of 29·2/105 py. Incidence varied by other donor demographics. When incidence was considered in 2-year intervals, no significant trend was evident. Overall residual risk of TT-HIV was 5·46 and 7·41 per million units of pRBC and FFP transfused, respectively. CONCLUSION: HIV incidence in both first-time and repeat donors varied by region in Brazil. Clear secular trends were not evident.

Hepatitis B virus among hematopoietic stem cell transplant recipients: Antiviral impact in seroconversion, engraftment, and mortality in a Latin American center.

BACKGROUND: Hepatitis B virus (HBV) infection is a worldwide concern with a broad distribution. In immunosuppressed populations, such as solid organ and hematopoietic stem cell transplant (HSCT) recipients, it can reactivate leading to acute hepatic failure. Different risk factors are known for higher rates of reactivation, and entecavir, tenofovir, and lamivudine are often used for prophylaxis and treatment. However, data regarding the impact of antiviral drugs in neutrophil and platelet engraftment are still unknown and concern the management of viral hepatitis post-HSCT. METHODS: We performed a single-center, retrospective, observational study reviewing medical records of patients referred for hematopoietic stem cell transplant from 2010 to 2017, which were also HBV infected, aiming to describe outcomes related to antiviral treatment and also the impact on platelet and neutrophil recovery after transplant. A secondary goal consisted of analyzing the impact of HBV infection in early and late mortality post-HSCT. The study included patients with positive blood bank screening for hepatitis B infection (HBsAg, Anti-HBc or HBV-NAT), confirmed later on by a laboratory routine serology. RESULTS: A total of 1132 hematopoietic stem cell recipients were assessed between 2010 and 2017. Eighty-six patients were confirmed to have HBV infection, of which six were HBsAg-positive, 20 were isolated anti-HBc-positive, and 60 had resolved infection (anti-HBc-positive and anti-HBs-positive). With regard to prophylaxis, 19 patients underwent HSCT on HBV antiviral therapy or prophylaxis: two were HBeAg-positive, three were HBeAg-negative and HBV-DNA was only detectable in three of them. Moreover, one patient had an occult HBV infection. Regarding therapy, 9 patients were on entecavir, 6 patients on lamivudine, two on tenofovir, and two of them on a combination of tenofovir + lamivudine due to HIV co-infection. Reverse seroconversion was not identified in any patients receiving antiviral therapy or prophylaxis, but it was detected in one patient with occult hepatitis B and another with resolved infection. No severe side effects led to therapy discontinuation in the treated group, which also did not have any significant delay in neutrophil or platelet engraftment when compared to patients without antiviral therapy. In addition, the only factors associated with increased mortality were transplant onset after 50 years, allogeneic transplant and myeloablative conditioning regimens. Interestingly, the presence of HBsAg or detectable HBV-DNA was not related to worse outcomes, neither the use of rituximab. In multivariate analysis, the use of antiviral therapy, the occurrence of graft-versus-host disease or CMV reactivation also was not linked to increased mortality. CONCLUSIONS: To sum up, HBV serology, ALT, and HBV-DNA monitoring are essential to detect hepatic flares earlier, even in populations with chronic inactive hepatitis, due to the possibility of later seroconversion. HBV infection was not related to increased 2-year mortality post-transplant. Antiviral prophylaxis did not cause any important clinical or laboratory side effects that could demand discontinuation, and its use was not associated with later neutrophil and platelet engraftments.

Using droplet digital PCR to screen for rare blood donors: Proof of principle.

BACKGROUND: Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). METHODS: Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1-5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. RESULTS: DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. CONCLUSION: The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Effectiveness of strategies to screen for blood donors with RH variants in a mixed population.

INTRODUCTION: Patients with RH variants presenting antibodies directed to RH high frequency antigens or multiple RH antibodies might, in some occasions, be better served with RH genotype-matched units, requiring screening for RH variants among blood donors. To date, strategies to identify donors with RH variants were restricted to selecting individuals of African descent based on self-reported race, what can be inaccurate in racially mixed population. Our goal was to: 1) Screen for donors with RH variants in a mixed population using self-declared race and Rh phenotype as selection criteria; and 2) Verify if including the Duffy null genotype in the screening algorithm increases its effectiveness. METHODS: Brazilian donors were included if self-declared as black and phenotyped as R0r or R1r. All individuals were genotyped for RHCE exons 1, 5, 6 and 7 and for the FY*B c.-67 T > C polymorphism in order to determine the Duffy null genotype. RHD variants were searched for in cases of altered RHCE. RESULTS: Among 2500 blood donors, 217 fulfilled the inclusion criteria and were enrolled. Fifty-three (24.4 %) had a predicted clinically relevant Rh phenotype (partial antigens or lack of high frequency antigens). Twelve donors (5.5 %) had a predicted RhCE phenotype lacking either hrB or hrS. Most cases with predicted lack of high frequency antigens (66.7 %) occurred in donors with the Duffy null genotype. CONCLUSION: Selecting donors based on self-declared race, Rh phenotype and Duffy null genotype is feasible and effective in identifying RH variants lacking Rh high frequency antigens among racially mixed donors.

Are different motivations and social capital score associated with return behaviour among Brazilian voluntary non-remunerated blood donors?

BACKGROUND: We examined the association between social capital score, motivator factors and demographic and donation characteristics and donor return at three Brazilian blood centres in Recife, São Paulo and Belo Horizonte. MATERIAL AND METHODS: A total of 5974 donors were interviewed about motivation factors to donate and cognitive and structural social capital just before an effective donation in three Brazilians blood centres in 2009. We assessed the return to a new donation within 2 years for each of these donors. Demographic and donation characteristics, motivators and scores of social capital and their association with donors' return were assessed. RESULTS: Overall, 3123 (52.3%) of the study subjects returned for a blood donation at least once. Predictors of donors' return were male gender (adjusted odds ratio [AOR] = 1.6, 1.3-1.9, for replacement and AOR = 1.3, 1.2-1.6, for community donors), previous donation (AOR = 2.7, 2.3-3.3, for replacement and AOR = 2.9, 2.5-3.5, for community donors) and high altruism (AOR = 1.3, 1.1-1.7, for replacement and AOR = 1.2, 1.0-1.5, for community donors). Altruism was the only motivator associated with return behaviour. Donors from Recife and São Paulo were more likely to return for replacement and/or for community donations than donors from Belo Horizonte. There was no association between social capital score and donor return behaviour. CONCLUSION: The likelihood to return for a subsequent blood donation is dependent upon characteristics of individual donors and also varies in different regions of Brazil. However, social capital was not associated with the likelihood of return behaviour. A better understanding of altruistic categories and appeals may help to improve donor recruitment and retention.

SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

A blockage monoclonal antibody protocol as an alternative strategy to avoid anti-CD38 interference in immunohematological testing.

BACKGROUND: As CD38 is expressed on red blood cells (RBCs), the plasma of patients on daratumumab (DARA) reacts with the panel cells of pretransfusion tests, masking underlying alloantibodies. The treatment of RBCs with dithiothreitol (DTT) is the most disseminated method to overcome DARA effect on immunohematological tests, but it hampers the identification of potentially harmful antibodies. Our goal was to validate a new strategy, the blockage monoclonal antibody protocol (BMAP), to mitigate the DARA interference on RBCs using anti-CD38 and antihuman globulin. METHODS: Samples of patients receiving DARA were included in the study. Sera were tested using both DTT- and BMAP-treated RBCs, which comprised three steps: 1) titration of monoclonal anti-CD38, 2) treatment of RBCs obtained from donors with anti-CD38, and 3) blockage of anti-CD38-adsorbed RBCs with antihuman globulin. RESULTS: Twenty patients were included in the study. Donor RBCs were treated with anti-CD38 and successfully blocked with antihuman globulin. In 19 patients, DARA-mediated agglutination was eliminated using both DTT- and BMAP-treated RBCs. In one patient, agglutination persisted when tested against the BMAP-treated RBCs, and alloantibodies were identified. Patient samples were mixed with commercial anti-D, -C, -e, -K, -Jka, -Kpb and tested against antigen-positive BMAP-treated RBCs, resulting in detection of these antibodies. CONCLUSION: This study validated a new strategy to minimize the interference of DARA on immunohematological tests. The protocol preserves the integrity of RBC antigens, permitting the detection of antibodies from all blood group systems. The BMAP has potential use in other situations where specific antibodies may interfere with pretransfusion screening.

Prevalence and laboratorial determinants of the clinical relevance of antibodies of undetermined specificity.

BACKGROUND AND OBJECTIVES: Antibodies of unknown specificity (AUS) are frequently identified in the pre-transfusion testing. These antibodies can be insignificant or potentially cause post-transfusion haemolysis. Information about the prevalence of clinically relevant AUS is still lacking. Our aim was to predict the potential clinical relevance of AUS using the monocyte monolayer assay (MMA) and to identify the clinical and laboratorial determinants of AUS' significance. MATERIALS AND METHODS: Antibodies of unknown specificity identified at a single institution from 2015-2017 were evaluated through MMA. A monocyte index (MI) of more than 5% was predictive of potential post-transfusion haemolysis. RESULTS: Thirty-two patients with AUS were included in the study. Of the studied AUS, 37·5% (12/32) presented with a monocyte index (MI) more than 5%. In the group of significant AUS, 41·7% of the patients presented with sickle cell disease (SCD) and the AUS were associated with Rh antibodies in 75% of the cases. In the group of insignificant AUS, only 10% of the patients had SCD and the association with Rh antibodies was detected in 20% of the cases. The presence of Rh antibodies was independently associated with the AUS clinical relevance (P = 0·012). CONCLUSION: More than one-third of the AUS are potentially clinically relevant, and the association with Rh antibodies is predictive of AUS relevance. Services must honour AUS in the pre-transfusion process in order to ensure transfusion safety.

Evaluation of the applicability and effectiveness of a molecular strategy for identifying weak D and DEL phenotype among D- blood donors of mixed origin exhibiting high frequency of RHD*Ψ.

BACKGROUND: Molecular tests designed to detect the presence of active RHD gene among D- donors have been successfully applied in people of European ancestry, but not in admixed populations with a considerable frequency of RHD*Ψ. Our goal was to evaluate the performance of a molecular screening tool for identifying active RHD alleles among Brazilian blood donors classified as D- C+ and/or E+. STUDY DESIGN AND METHODS: Pools of five DNA samples of serologically D- C+ and/or E+ donors were checked by a RHD polymerase chain reaction (PCR) assay specific for RHD Intron 4 and Exon 7. When a pool result was positive, samples were genotyped individually for RHD Intron 4 and Exon 7, RHD*Ψ, RHCE*Cc, and RHD zygosity. Donors suspected of active RHD gene were further evaluated by whole-coding region and flanking intron direct sequencing. RESULTS: A total of 405 donors were included. Two percent exhibited active RHD gene, codifying D-weak (38 and 45) or DEL phenotype. The most prevalent DEL allele was RHD*DEL1 (c.1227G>A), which is proven to be immunogenic. A high frequency of RHD*Ψ was detected in the donors with nondeleted RHD alleles (31%), far superior to the frequency of RHD variant alleles (15.5%). The proposed approach presented sensitivity of 100% and specificity of 85.7% for identifying active RHD gene. CONCLUSION: The strategy of checking D- donors with RHD PCR followed by exclusion of RHD*Ψ allele has proved efficient in identifying weak-D and DEL phenotype in the Brazilian population.

Defining the clinical relevance of red blood cell autoantibodies by Monocyte Monolayer Assay.

BACKGROUND: The Monocyte Monolayer Assay (MMA) is an in vitro simulation of red blood cell (RBC) alloantibody behavior. It has been classically applied to predict the risks of post-transfusion hemolytic reactions when transfusing incompatible RBC units. Quantifying erythrophagocytosis by MMA may be an interesting option for situations where there is doubt whether a RBC autoantibody is mediating significant hemolysis. Here, we present three situations involving RBC autoantibodies in which the MMA was decisive for clarifying the diagnosis and choosing the best clinical treatment. CASE REPORT: Case 1. Pregnant patient with severely anemic fetus exhibited warm autoantibody without signs of hemolysis. MMA revealed 30% of monocyte index (MI) highlighting that fetal hemolysis was caused by maternal autoantibody. Prednisone was prescribed with fetal clinical improvement. Cases 2 and 3. Two patients with the diagnosis of mixed auto-immune hemolytic anemia and poor response to corticosteroids were evaluated using MMA. The resulting MI was less than 10% in both cases, suggesting that the cold-agglutinin rather than the warm auto-IgG was responsible for overt hemolysis. Treatment with rituximab was begun, with good clinical response. CONCLUSION: MMA can be used to evaluate the ability of RBC autoantibodies to mediate overt hemolysis. It can be especially useful to determine the role played by cold and warm auto-antibodies in mixed auto-immune hemolytic disease, helping to define the best treatment option.