Background: Neonatal alloimmune thrombocytopenia or neutropenia (FNAIT, NAIN) may result from passive transfusion of maternal antibodies against platelet (HPA) or neutrophil antigens (HNA). The simultaneous occurrence of these cytopenias in the newborn is rare (0.03%), however a new agent has been identified as a potential cause of FNAIT and/or NAIN: antibodies against HLA class I antigens, that may increase the incidence of these syndromes. In the last years, many case reports of suspected FNAIT and/or NAIN with only HLA class I antibodies detected in the mother were reported in the literature. Aim: To investigate the specificity and intensity of HLA class I antibodies detected in the serum of mothers of newborns who had simultaneous thrombocytopenia and neutropenia at birth. Methods: From an initial cohort of 10,000 consecutive newborns, 20 cases (0.2%) of concurrent neonatal thrombocytopenia (platelet count <150 x 109/L) and neutropenia (neutrophil count <2 x 109/L) were selected for investigation. For the evaluation of fetal-maternal incompatibility, HPA (HPA-1 to 11, -15), HNA (HNA-1 to 5) and HLA class I genotyping was performed on the samples of mothers and neonates by PCR-SSP, PCR-RFPL and bead-based technology. Specific alloantibodies were investigated in maternal serum by ELISA, MAIPA, bead-based assays, granulocyte immunofluorescence and granulocyte agglutination test. Results: Considering the HNA, HPA and HLA genotyping, 18/20 (90%) cases had feto-maternal incompatibility. Antibody screening identified 4/20 (20%) cases with the involvement of HPA antibodies (anti-HPA-5b, -5a and -9bw), 4/20 (20%) cases with HNA antibodies (anti-HNA-2 and -3b) and 6/20 (30%) cases with specific HLA class I antibodies only. The prevalence of simultaneous FNAIT and NAIN caused by HNA and HPA antibodies was 0.03% (3/10,000). When HLA class I specific antibodies were included, such prevalence raised to 0.09% in our cohort. Four out of six cases with HLA antibodies alone, showed a remarkably high mean fluorescence intensity (MFI), ranging from 15,700 to 21,600 and were specific to HLA A2, A3, B44 and Cw7. Doing a parallel with transplants recipients, these MFI values are considered highly unacceptable for donor specific antibodies. The HLA B44 specificity was associated with the lowest platelet count (14 x 109/L) and HLA A2 with the lowest neutrophil count (0.66 x 109/L). Discussion: An immune etiology was identified in 45% of the cases of neutropenia and thrombocytopenia at birth, however, few HNA and HPA antibodies were detected simultaneously. Although maternal alloimmunization against HLA class I is common, a considerable rate (20%) of specific and high intensity HLA antibodies observed in the studied cases has led us to reinforce the idea that they should be considered as a potential cause of NAIN and FNAIT. Antibodies directed against HLA-A and HLA-B alleles suggests platelets or other nucleated cells that have crossed into maternal circulation as an immunising agent rather than syncytiotrophoblasts, which only express HLA-C,E,G. Possibly the individual expression of HLA class I antigen on platelets and neutrophils could also contribute to the variation in outcome. Conclusion: High intensity antibodies specific to HLA A2, A3, B44 and Cw7 were related to low platelet and neutrophil counts and should be considered as a potential cause of FNAIT and NAIN.