Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusionrelated acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcγ receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the α M (CD11b) and α L (CD11a) subunits of the leucocyte adhesion molecules (β 2 integrins). Molecular and biochemical characterization of neutrophil antigens have expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.

Comparing to another populations, Brazilians present a higher frequency of HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization. Moreover, the distribution of the HNA-4 alleles observed in Amazon Indians is quite similar to that reported among Koreans. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.

Human neutrophil reactive antibodies may cause clinical disorders such as transfusion-related acute lung injury, febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after stem cell transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test by flow cytometry, the phenotypic frequencies of the human neutrophil alloantigens (HNA)-1a, -1b, -2, -3a and -4a were determined in 100 healthy Brazilian persons. Neutrophils were separated from blood samples by sedimentation, centrifugated and incubated with HNA-specific alloantibody plus fluorescein isothiocyanate-labeled F(ab')(2) fragments of anti-human IgG. The results showed that the phenotype frequencies of HNA-1a, -1b, -2a, -3a and -4a were 65%, 83%, 97%, 95% and 94%, respectively. We detected that neutrophils from 17% of Brazilians typed positive only with anti-HNA-1a (HNA-1a/a), 35% only with anti-HNA-1b (HNA-1b/b) and 48% reacted with both antibodies (HNA-1a/b). The frequencies found for HNA-1a and -1b were quite similar to that reported among Africans and American-Africans, but different from those found in Japanese and Chinese. In addition, our data showed that the frequencies of HNA-2, -3a and -4a in Brazilians were comparable with those observed in Caucasians. The determination of HNAs frequencies among populations with distinct racial backgrounds is important not only for anthropological reasons, but also for neonatal typing in suspected cases of alloimmune neutropenia or when patients are severely neutropenic.

Granulocyte reactive antibodies have been found to cause clinical disorders such as transfusion related acute lung injury (TRALI), febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test (GIFT) by microscopic and flow cytometric analysis, the frequencies of the neutrophil antigens HNA−1a, −1b, −2a, −3a and −4a were determined among 100 random Brazilian blood donors from the Blood Center of Universidade Federal de Sao Paulo, SP, Brazil. Granulocytes were separated from mononuclear cells and red cells by sedimentation with 5% dextran, followed by centrifugation on Ficoll-paque (d = 1.077), and then incubated with anti-sera (anti-HNA−1a, −1b, −2a, −3a, and −4a obtained from American Red Cross, North Central Blood Services, St. Paul, MN) conjugated with fluorescein isothiocyanate (FITC) labeled F(ab’)2 fragments of anti-human IgG. Only cell suspensions containing ≥95% neutrophils with viability ≥90% according to the trypan-blue staining were analyzed. The frequencies of HNA−1a, −1b and −2a were 65%, 83% and 94%, respectively, and for such alloantigens exact same results were observed using either the GIFT performed by microscopy or by flow cytometry. The frequency of HNA-3a was 86% by the microscopic GIFT, and 95% by the flow cytometry analysis; while the frequency of HNA−4a was 93% by the microscopic GIFT, and 94% by the flow cytometric technique. These results indicate that: GIFT by flow cytometry is more sensitive than the GIFT by microscopy to detect HNA−3a; the phenotypic frequencies found for neutrophil antigens HNA−1a and −1b among Brazilian blood donors are quite similar to those reported among African Americans, but different from those reported for Japanese and Chinese individuals; the phenotype frequencies of the neutrophil antigens HNA−2a, −3a, and −4a in Brazilians are quite similar to those found among Caucasians (Table). (These studies were funded by FAPESP, SP, Brazil - 05/55237–9). Asians Brazilians Antigen African Americans Chineses Hindus Japaneses Caucasians M FC M, microscopy; FC, flow cytometry; nd, not done HNA-1a 46 – 68 90 44 88 52 – 54 65 65 HNA-1b 78 – 84 52 83 51 – 64 87 – 89 83 83 HNA-2a nd 99 nd 89 87 – 97 94 94 HNA-3a nd nd nd nd 99 86 95 HNA-4a nd nd nd nd 96 93 94

The HNA-4 and HNA-5 systems are located on the b2-integrin, that share a common b subunit (b 2 or CD18) noncovalently associated with four different a subunits. The HNA-4a (Mart) and HNA-5a (Ond) antigens are polymorfic variants of a M (CD11b) and a L (CD11a) subunits, respectively, and both are associated with single nucleotide polymorphisms (SNP) leading to amino-acid dimorphisms. HNA-4a has been recently linked to alloimmune neonatal neutropenia while the HNA-5a clinical significance is not clear yet. Using a PCR with sequence-specific primers (SSP) the frequency of allelic polymorphism of HNA-4a among 121 Brazilian blood donors and 114 Amazon Indians was determined. A novel PCR-restriction fragment length polymorphism (RFLP) method digesting the PCR product with the enzyme Bsp1286 I for HNA-5a genotyping was developed, and the gene frequencies of this antigen were determined among 123 Brazilian blood donors and 114 Amazon Indians. In order to validate this proposed new genotyping method, the amplified genomic DNA from 6 previously genotyped samples (2 of each genotype) was purified using a commercial kit (GFX PCR DNA and Gel Band Purification Kit, Amersham Bioscience, Piscataway, NJ, USA), sequenced using a cycle-sequencing kit (BigDye Terminator, PE Applied Biosystems, Foster City, CA, USA) and analyzed on a genetic analyzer (ABI Prism 3100, PE Applied Biosystems). The calculated HNA-4a (+/+), HNA-4a (+/−) and HNA-4a (−/−) genotype frequencies found in blood donors (0.686, 0.273 and 0.041) and Amazon Indians (1.000, 0.000 and 0.000) presented statistically significant differences (p<0.01). The frequencies of HNA-5a (+/+), HNA-5a (+/−) and HNA-5a (−/−) genotypes among blood donors (0.512, 0.399 and 0.089) and Amazon Indians (0.746, 0.219 and 0.035) also differed significantly (p<0.01). Sequencing studies demonstrated absolute concordance with PCR-RFLP genotyping results in all 6 analyzed samples. Overall, the present data indicate that comparing to other population studies, the Brazilian population presents a higher frequency of the HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization by pregnancy or blood transfusion. Moreover, the distribution of the HNA-4 system alleles observed in Amazon Indians is quite similar to that already reported among Korean individuals. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.