Morelati F et al.Table III - Clinical <strong>and</strong> laboratory characteristics of patients with DAT-negative <strong>and</strong> MS-DAT positive AIHAN. S e x Hb g/dL Total bilirubin Reticulocytes Haptoglobin LDH Steorid DAT MS-DAT(indirect) µmol/L % mg/L U/L therapy (*) IgG ng/mL1 F 13.3 8 (5) 1.4 1,200 420 Yes neg 2792 F 12.4 13.7 (12) 0.8 2,290 296 Yes neg 3213 F 12.3 10.3 (7) 0.7 1,830 377 No neg 4334 M 13.1 10 (9) 1.8 1,200 472 No neg 3025 M 12 10 (8) 0.9 690 303 Yes neg 2566 M 15 13.7 (8) 0.2 870 291 No neg 3227 M 12 27 (22) 3.7 200 480 No neg 8138 F 10.9 28 (22) 4.4 200 420 No neg 4339 M 11.1 20 (18) 1.1 200 550 No neg 1,23010 F 5.3 20 (19) 6.9 200 232 No neg 1,66011 M 8.8 n.d. 4.3 200 520 Yes neg 51612 M 10.8 40 (27) 15.9 200 470 No neg 85613 F 11.1 9 (8) 0.9 500 338 No neg 314Normal Women 0-17 (0-12)
New immunohaematological technologies- the risk of haemolytic disease of the newborn,- the risk of neonatal alloimmune thrombocytopenia,- RHD zygosity;c) large scale typing of blood donors for red cell <strong>and</strong>platelet antigens, when typing antisera are not availableor difficult to obtain.Molecular characterisation of antigens is essential intransfused immunised patients, in order to select compatibleunits of red blood cells <strong>and</strong> in pregnant women, in order todecide whether to administer RhD prophylaxis.Over the course of about 2 years, molecular techniqueswere used to study 28 blood donors (5 suspected ABOvariants, 5 discrepancies in Rh determination, 3discrepancies in the typing of other red cell antigens <strong>and</strong>15 donors negative for high incidence antigens) <strong>and</strong> 40patients sent for immunohaematological investigations (6with ABO discrepancies <strong>and</strong> 4 with Rh discrepancies in theagglutination techniques, 11 transfused subjects for bonemarrow transplantation or thalassaemia or malignancy, 10DAT-positive transfused subjects, 3 immunised pregnantwomen <strong>and</strong> their partners, whose offspring were suspectedto be affected by foetal or neonatal alloimmunethrombocytopenia, 1 foetus with suspected haemolyticdisease, 4 subjects with haemolytic <strong>transfusion</strong> reactions<strong>and</strong> 1 with platelet-specific alloimmunisation). Moleculartyping was carried out using DNA, at a concentration of 5-10 µg, extracted by from peripheral blood in EDTA, usingthe salting out method 22 . The commercial kits used werebased on the Polymerase Chain Reaction Sequence-Specific Primers (PCR-SSP) method, <strong>and</strong> prepared accordingto international knowledge in the relevant field 23-42 . Thefindings were 3 ABO variants (A el), 4 D variants (1 DFR, 1Rh33, 1 Dweak type 1 <strong>and</strong> 1 Dweak type 5), 14 cases ofabsence of a high incidence antigen (3 k, 2 Lu b , 1 Co a , 7 Fynull, 1 HPA-1a); in another 6 cases (5 donors <strong>and</strong> 1 patient)genomic typing revealed the presence of antigens that theserological techniques had not detected (3 Fy b weak, 2 Lu b<strong>and</strong> 1 antigen of the KEL system).A second study was carried out to identify donors ofplatelets negative for human platelet antigens (HPA).The anti-HPA alloantibodies <strong>and</strong> relative antigens wereinvolved in cases of post-<strong>transfusion</strong> purpura, inimmunological refractoriness to st<strong>and</strong>ard platelet<strong>transfusion</strong>s <strong>and</strong> in cases of foeto-neonatal alloimmunethrombocytopenia. The availability of donors of knownplatelet type is essential in order to ensure effective<strong>transfusion</strong>s of platelet concentrates in these subjects.However, it is difficult to identify such subjects because ofthe scarcity of specific typing antisera to use in the classicalmethods (in ELISA, in flow cytometry or in solid phase).Genomic DNA from 149 Caucasian, group O blood donorswas analysed in order to determine the genotype of theHPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-5a <strong>and</strong> HPA-5b antigens. The PCR-SSP method <strong>and</strong> acommercial kit were used. The allelic, phenotypic <strong>and</strong>genotypic frequencies observed in this population of blooddonors were compatible with those reported in otherEuropean studies 43-48 (Table IV).ConclusionsIn this article we have presented our recent experiencewith some of the new technologies internationally appliedto the field of red cell <strong>and</strong> platelet immunohaematology 49-54to reduce the risk of incompatibility between donors <strong>and</strong>recipients <strong>and</strong>, therefore, to improve <strong>transfusion</strong> safety.The use of automation, in particular, seems to be a validapproach for reducing the risk related to human error <strong>and</strong>guaranteeing the traceability of all the operative phases inall critical processes involving large numbers, such asextended red cell typing, identification of antibodies inmultiply immunised subjects <strong>and</strong> in the <strong>transfusion</strong> therapyof patients affected by immunological refractoriness tost<strong>and</strong>ard platelet concentrates.The use of molecular techniques has becomeindispensable, in combination with agglutination methods,for defining the red cell antigen profile in recently transfused<strong>and</strong> immunised subjects or in cases in which discrepanciescannot be resolved with traditional serological techniques<strong>and</strong> in the consequent selection of compatible red bloodcell units. These techniques represent the only methodavailable for characterising the platelet antigen profile,which cannot be determined otherwise because of the lackof suitable typing reagents, <strong>and</strong> in antenatal investigationsto evaluate the risk of haemolytic disease of the newborn,the risk of alloimmune thrombocytopenia <strong>and</strong> the antigenprofile of the foetus. The mitogen stimulation test is aclinically important assay in the management of cases ofsuspected AIHA, that gives negative results in thetraditional test.Finally, it should be emphasised that <strong>transfusion</strong> safetydepends on a series of processes which must be improved<strong>and</strong> monitored over time <strong>and</strong> that the use of newtechnologies is only one element in the <strong>transfusion</strong>procedure.It is, therefore, essential to use new technologies withina carefully defined process including all the phases betweenselection of the donor, the <strong>transfusion</strong> of bloodcomponents <strong>and</strong> the follow-up of the patients.<strong>Blood</strong> Transfus 2007; 5: 58-65 DOI 10.2450/2007.0006-0763058-65_Morelati.p65 6309/07/2007, 9.20
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