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Background: Accurate estimation of haematopoietic stem cell (HSC) counts by flow cytometry may be difficult in laboratories in which sophisticated equipment and staff with specific expertise are not available. Affordable flow cytometers that can perform basic functions may help to overcome these difficulties. In this study we compared HSC and leucocyte counts determined by volumetric and bead-based protocols performed with the small, low-cost Accuri(®) C6, with those obtained with two gold-standard instruments, the four-colour FACSCalibur(®) and the eight-colour FACSCantoII(®), our reference flow cytometers., Materials and Methods: With the three cytometers we tested, in parallel, 111 consecutive samples from cord blood, peripheral blood from patients with myelofibrosis and myeloproliferative syndromes, fresh and thawed HSC collected by apheresis and bone marrow products. The findings were compared with one-way ANOVA, Bland-Altman analysis and linear regression., Results: The results of HSC and leucocyte enumeration by the three devices were strongly correlated (r(2)>0.99; p<0.0001). ANOVA performed on different subgroups of samples did not reveal significant differences between HSC count determined by the C6 bead-based and reference flow cytometers in any of the subgroups. Regarding the C6 volumetric protocol, a statistically significant difference was observed only in the cord blood subgroup. Time for instrument set-up, calibration and analysis was slightly longer with Accuri(®) C6 (40 min) than with FACSCantoII(®) (30 min)., Discussion: Accuri(®) C6 is a reliable instrument for HSC enumeration in fresh samples, using both volumetric and bead-based approaches, although the volumetric protocol on cord blood samples needs to be improved. The Accuri(®) C6 is easy to use, does not require profound knowledge of flow cytometry and could be employed in an urgent setting. Its performance may be improved by more efficient calibration and shorter analysis time.

Background: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality. Antibodies against human leucocyte antigens (HLA) and human neutrophil antigens (HNA) are often detected in the implicated donors. We investigated the incidence and aetiology of TRALI in Lombardy. Moreover, we determined the rate of HLA and HNA alloimmunisation and the HNA genotype in a cohort of local blood donors., Materials and Methods: During a 2-year observational study in eight blood transfusion services, suspected TRALI cases were collected and characterised by means of HLA and HNA antibody screening of implicated donors, donor/recipient cross-matching and HLA/HNA molecular typing. In addition, 406 Italian donors were evaluated for alloimmunisation and in 102 of them HNA gene frequencies were determined., Results: Eleven cases were referred to the central laboratory, of whom three were diagnosed as having TRALI, seven as having possible TRALI and one as having transfusion-associated circulatory overload. Seven TRALI cases were immune-mediated whereas in three we did not find either alloantibodies in implicated donors or a positive reaction in the cross-match. The most frequently implicated blood component was red blood cells (in 5 males and in 1 female), whereas four cases of TRALI were associated with transfusion of fresh-frozen plasma (in 3 females and in 1 male). The frequency of reported TRALI/possible TRALI cases was 1:82,000 for red blood cells and 1:22,500 for fresh-frozen plasma. No cases were observed for platelets. Overall, the frequency of HLA or HNA alloimmunisation in blood donors was 29% for females and 7% for males. The latter could be related, at least in part, to natural antibodies. HNA gene frequencies showed that HNA-1b is more frequent than HNA-1a in our sample of donors., Discussion: The recently adopted national policy to prevent TRALI, i.e. using only plasma donated by males, would have had a positive impact in our setting.

Background: Hereditary spherocytosis is a very heterogeneous form of hemolytic anemia. The aim of this study was to relate the type of molecular defect with clinical and hematologic features and response to splenectomy using information from a large database of patients., Design and Methods: Data from 300 consecutive patients with hereditary spherocytosis, grouped according to the results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, were analyzed and the sensitivity of red cell osmotic fragility tests was compared in various subsets of patients., Results: Band 3 and spectrin deficiencies were the most common protein abnormalities (54% and 31%, respectively); 11% of cases were not classified by the electrophoretic analysis. Spectrin deficiency was more frequently diagnosed in childhood and band 3 deficiency in adulthood. Hemoglobin concentration was slightly lower, spherocyte number and hemolysis markers higher in spectrin deficiency than in band 3 deficiency. The sensitivity of the osmotic fragility tests ranged from 48% to 95%, and was independent of the type and amount of the membrane defect. The association of the acidified glycerol lysis test and the NaCl test on incubated blood reached a sensitivity of 99%. Splenectomy corrected the anemia in patients with all subtypes of hereditary spherocytosis although spectrin-deficient patients still showed increased reticulocyte numbers and levels of unconjugated bilirubin. Splenectomy allowed the identification of the membrane defect in all the previously unclassified patients, most of whom had spectrin and/or ankyrin deficiency., Conclusions: The definition of the red cell membrane defect in hereditary spherocytosis has no major clinical implications, but may be useful for a differential diagnosis from other hematologic disorders that mimic this hemolytic anemia.