Red blood cell extended antigen typing in Omani patients with sickle cell disease to enhance daily transfusion practice.

Many Omani patients with sickle cell disease (SCD) undergo red blood cell (RBC) transfusions that are only matched for ABO and D, making RBC alloimmunization a significant concern in this population. Currently, the integration of molecular assays and hemagglutination testing helps to determine RBC phenotypes and genotypes, facilitating the provision of compatible blood and minimizing additional alloimmunization risks in patients with SCD. Based on this finding, our objective was to use molecular methods to predict the extended antigen profile of Omani patients with SCD across various blood group systems including Rh, Kell, Duffy, Kidd, Colton, Lutheran, Dombrock, Diego, Cartwright, and Scianna. This approach aims to implement RBC matching strategies and enhance daily transfusion practices for these patients. Molecular methods encompassed multiplex polymerase chain reaction for RHD, BeadChip arrays for variants of RHD and RHCE, and ID CORE XT for the primary allelic variants of RBCs. This study enrolled 38 patients with SCD, comprising 34 patients with homozygous HbSS, 1 patient with HbSC, and 3 patients with HbS Oman. The predominant ABO blood group was group O, observed in 44.7 percent of patients, followed by group A in 21.1 percent and group B in 13.2 percent. The most prevalent Rh phenotype predicted from the genotype was D+C+E-c+e+, identified in 34.2 percent of patients. All patient samples were K-, exhibiting the k+ Kp(b+) Js(b+) phenotype, with 81.6 percent demonstrating Fy(a-b-) due to the homozygous FY*02N.01 genotype and 28.9 percent displaying Jk(a+b-). RH variant alleles were detected in five patients (13.2 %), with only one type of RHD variant (RHD*DIIIa) and one type of RHCE variant (RHCE*ceVS.02.01) identified. Alloantibodies were present in 26 patients (68.4%). This study presents the initial comprehensive report of extended RBC antigen profiling in Omani patients with SCD, revealing disparities in the prevalence of RBC phenotypes compared with SCD patients from other regions and countries. Furthermore, our findings underscore a high rate of alloimmunization in these patients, emphasizing the need to implement antigen-matching programs to improve daily transfusion practices.

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.

Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Effects of prophylactic red blood cell (RBC) transfusion with extended antigen matching on alloimmunization in patients with Sickle Cell Disease (SCD).

BACKGROUND: RBC alloimmunization remains a significant problem for many patients with SCD. To reduce alloimmunization some strategies have been implemented to provide limited or extended antigen matched RBC transfusions to patients with SCD who need chronic transfusion support. The aim of this study was to evaluate the effects of prophylactic RBC transfusion with extended antigen matching on alloimmunization in patients with SCD. METHODS: This is a 20-year retrospective study of patients with SCD transfused with RBCS that were prospectively matched for D, C, c, E, e, K, Fya/Fyb, Jka/Jkb and S antigens. Our study included 95 patients, and none had antibodies documented before their first transfusion. Patients and donors were phenotyped and molecular typing was performed in all patients who had recent transfusions or a positive direct antiglobulin test to predict their antigen profile. Unexpected antibodies to the Rh system, meaning anti-Rh antibodies in patients whose serologic phenotype was Rh positive, were investigated by molecular genotyping for RH variant alleles. RESULTS: During this study-period, 12 (12.6%) were alloimmunized and 83 (87.4%) were not. Among the 12 patients who alloimmunized, 7 (58.3%) developed antibodies to Rh antigens and 5 (41.7%) produced antibodies to low prevalence antigens. All patients who developed Rh antibodies had RH variant alleles. Autoantibodies were found in 16 (16.8%) transfused patients. CONCLUSION: SCD patients benefit from receiving prophylactic RBC transfusions with extended antigen matching, as demonstrated by the reduction on the rates of alloimmunization and the lack of antibodies to K, FY, JK and S antigens, however, this strategy does not avoid alloimmunization to Rh and low-prevalence antigens.

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.

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.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

HLA-DRB1 and cytokine polymorphisms in Brazilian patients with myelodysplastic syndromes and its association with red blood cell alloimmunization.

OBJECTIVE(S): This study aimed investigate association of HLA-DRB1 and cytokine polymorphisms with red blood cell(RBC) alloimmunization in Brazilian Myelodysplastic syndrome(MDS) patients with prior exposure to RBC transfusion. BACKGROUND: MDS patients are at risk RBC alloimmunization due to chronic RBC transfusion. However, differences in immune response of MDS transfused patients are not completely known. METHODS/MATERIALS: A retrospective cohort of 87 polytransfused patients with MDS including 28 alloimmunized (PA) and 59 non-alloimmunized (PNA) was evaluated in three Brazilian reference hospitals. HLA-DRB1genotype was performed by polymerase chain reaction (PCR)-SSOP (Luminex platform) and cytokine polymorphisms analysed by PCR and TaqMan assays. RESULTS: While HLA-DRB1 allele frequencies did not differ between groups, IL17A 197G > A SNP and IL4 polymorphisms showed significant correlation with RBC alloimmunization. IL17A 197A allele A and AA genotype were significantly more frequent in PA than PNA(A, 46.4% versus 27.1%, p = 0.012; OR = 2.3; 95%CI = 1.1-4.9; AA, 25% versus 6.8%, p = 0.041; OR = 6.2; 95%CI 1.3-30.8). Moreover, significant association of alloimmunization to Rh antigens with IL17A 197A allele and AA genotype was also identified in PA group(A, 45% versus 27.1%, p = 0.036; OR = 2.5; 95% CI 1.1-5.7; AA, 30% versus 6.8%, p = 0.042; OR = 7.9; 95%CI 1.5-42.3). Genotype A1A2 of IL4 intron 3 was overrepresented in PA(50% versus 16.9%, p = 0.009; OR = 4.97; 95%CI 1.6-15.5). Similarly, IL4-590 CT genotype was overrepresented in PA(53.6% versus 28.8%, p = 0.049; OR = 3.3; 95%CI 1.2-9.3). CONCLUSIONS: This study showed no association regarding HLA-DRB1 alleles for RBC alloimmunization risk or protection, however the IL17A 197G>A, IL4 intron 3 and IL4 590C>T SNP was significantly associated to RBC alloimmunization risk in this cohort of Brazilian MDS patients.

Duffy phenotyping and FY*B-67T/C genotyping as screening test for benign constitutional neutropenia

ABSTRACT Objective: Low levels of neutrophils can be an intrinsic condition, with no clinical consequences or immunity impairment. This condition is the benign constitutional neutropenia (BCN), defined as an absolute neutrophils count (ANC) ≤2000 cells/mm. Diagnosis of BCN is of exclusion where patients are submitted to blood tests and possibly to invasive diagnostic search until secondary causes of neutropenia are ruled out. The natural history of the disease suggests benign evolution and Brazilian study showed an overall frequency of 2.59%. The main mechanisms include reduced neutrophil production, increased marginalization, extravasation to the tissues and immune destruction. Genetic studies showed strong association between the single nucleotide variant rs2814778 located on chromosome 1q23.2 in the promoter region of the atypical chemokine receptor 1 (Duffy blood group system) gene (ACKR1, also termed DARC) and BCN. The aim of this study is to evaluate FY phenotypes and genotypes including the analysis of the rs2814778 SNP in Brazilian patients with BCN in order to determine an effective diagnostic tool, allowing reassurance of the patient and cost reduction in their care. Methods: Case control study, with 94 individuals (18 patients and 76 controls). Phenotyping was performed by gel test and genotyping was performed by PCR-RFLP. Results: White blood cell (WBC) and absolute neutrophils (AN) counts showed lower levels in patients compared to controls. In the patient group 83.3% were genotyped as FY*B/FY*B. The SNP rs2814778 (-67T > C) was identified in 77.8% of the patients genotyped as FY*B-67C/FY*B-67C. In the control group, 72.7% were homozygous for the wild type and 23.3% were heterozygous. Conclusion: This study reinforces that FY phenotyping and genotyping can be used to detect most people with BCN, avoiding excessive diagnostic investigation. Besides, this procedure may reduce health costs and be reproductible in clinical practice.

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.

Systematic RHD genotyping in Brazilians reveals a high frequency of partial D in transfused patients serologically typed as weak D.

BACKGROUND: The discrimination between weak D types and partial D can be of clinical importance because carriers of partial D antigen may develop anti-D when transfused with D-positive red blood cell units. The aim of this study was to determine by molecular analysis the type of D variants among Brazilian patients requiring transfusions with serologic weak D phenotypes. MATERIAL AND METHODS: Samples from 87 patients (53 with sickle cell disease, 10 with thalassemia and 24 with myelodysplastic syndrome), serologic typed as weak D by manual tube indirect antiglobulin test or gel test were first RHD genotyped by using the RHD BeadChip Kit (BioArray, Immucor). Sanger sequencing was performed when necessary. RESULTS: RHD molecular analysis revealed 32 (36.8 %) variant RHD alleles encoding weak D phenotypes and 55 (63.2 %) alleles encoding partial D antigens. RHD variant alleles were present in the homozygous state or as a single RHD allele, one variant RHD allele associated with the RHDΨ allele, or two different variant RHD alleles in compound heterozygosity with each other in 70 patients, 4 patients and 13 patients, respectively. Alloanti-D was found in 9 (16.4 %) cases with RHD alleles predicting a partial D. DISCUSSION: The frequency of partial D was higher than weak D types in Brazilian patients serologically typed as weak D, showing the importance to differentiate weak D types and partial D in transfused patients to establish a transfusion policy recommendation.

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.

Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations.

BACKGROUND: The correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.' STUDY DESIGN AND METHODS: The molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil. RESULTS: Atypical serologic D typing occurred in 0.79 % of blood donors. The majority of RHD variant alleles (88 %) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47 %), followed by RHD*weak D type 3 (29.9 %), RHD*weak D type 2 (3.9 %) and RHD*weak D type 1 (3.1 %). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22 %), RHD*DAR3 (weak 4.0.1) (2.4 %), RHD*DAR3.1 (weak 4.0) (22 %) and RHD*DAR4 (weak 4.1) (0.8 %). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6 %), RHD*DAU-5 (2.4 %), RHD*DAU-6 (1.6 %), RHD* DIII type 8 (1.6 %), RHD*DVII (3.9 %) and RHD* DMH (0.8 %) were also observed. CONCLUSION: The prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.

Duffy phenotyping and FY*B-67T/C genotyping as screening test for benign constitutional neutropenia.

OBJECTIVE: Low levels of neutrophils can be an intrinsic condition, with no clinical consequences or immunity impairment. This condition is the benign constitutional neutropenia (BCN), defined as an absolute neutrophils count (ANC) ≤2000 cells/mm. Diagnosis of BCN is of exclusion where patients are submitted to blood tests and possibly to invasive diagnostic search until secondary causes of neutropenia are ruled out. The natural history of the disease suggests benign evolution and Brazilian study showed an overall frequency of 2.59%. The main mechanisms include reduced neutrophil production, increased marginalization, extravasation to the tissues and immune destruction. Genetic studies showed strong association between the single nucleotide variant rs2814778 located on chromosome 1q23.2 in the promoter region of the atypical chemokine receptor 1 (Duffy blood group system) gene (ACKR1, also termed DARC) and BCN. The aim of this study is to evaluate FY phenotypes and genotypes including the analysis of the rs2814778 SNP in Brazilian patients with BCN in order to determine an effective diagnostic tool, allowing reassurance of the patient and cost reduction in their care. METHODS: Case control study, with 94 individuals (18 patients and 76 controls). Phenotyping was performed by gel test and genotyping was performed by PCR-RFLP. RESULTS: White blood cell (WBC) and absolute neutrophils (AN) counts showed lower levels in patients compared to controls. In the patient group 83.3% were genotyped as FY*B/FY*B. The SNP rs2814778 (-67T > C) was identified in 77.8% of the patients genotyped as FY*B-67C/FY*B-67C. In the control group, 72.7% were homozygous for the wild type and 23.3% were heterozygous. CONCLUSION: This study reinforces that FY phenotyping and genotyping can be used to detect most people with BCN, avoiding excessive diagnostic investigation. Besides, this procedure may reduce health costs and be reproductible in clinical practice.

Association of positive direct antiglobulin test (DAT) with nonreactive eluate and drug-induced immune hemolytic anemia (DIHA).

BACKGROUND AND OBJECTIVES: Drug-induced immune hemolytic anemia is a rare condition that occurs primarily because of drug-induced antibodies, either dependent or independent and positive direct antiglobulin test. Our aim was to evaluate the association of positive DAT with nonreactive eluate and DIHA. MATERIALS AND METHODS: From 2014-2018, we evaluated 159 patients who presented positive DAT with a nonreactive eluate. Laboratory and clinical analyses were performed including HIV, HBV and HCV testing. All patients were exposed to the following drugs: Dipyrone in 63.5 %, Furosemide in 28.9 %, Metoclopramide in 34.6 % and Ondansetron in 41.5 %. RESULTS: Results of DAT showed IgG in 125 (78.4 %) patients and C3d in 24 (15.1 %) with reactions varying from 1+ to 4+. HIV test was positive in 10 (16.1 %) patients, HBV was positive in 3 (4.7 %) and HCV was positive in, 1 (1.5 %). There was no clinical significance when the parameters of hemoglobin, hematocrit, reticulocytes and LDH were evaluated, only a slight increase in bilirubin, especially, in patients with positive DAT reacting 3+/4+ due to IgG and C3d sensitization. Clinical evaluations showed that all patients were asymptomatic. CONCLUSIONS: The association of drugs with positive DAT can be a challenge to transfusion services and immunohematology reference laboratories. There was no evidence of any case of severe hemolysis with clinical repercussion through the clinical and laboratory findings analyzed with the drugs associated with positive DAT. Dipyrone and Furosemide have already been associated with DIHA but there are no studies reporting the association of Metoclopramide and Ondansetron with DIHA.