Analysis of leucocyte antibodies, cytokines, lysophospholipids and cell microparticles in blood components implicated in post-transfusion reactions with dyspnoea.

BACKGROUND AND OBJECTIVES: Post-transfusion reactions with dyspnoea (PTR) are major causes of morbidity and death after blood transfusion. Transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO) are most dangerous, while transfusion-associated dyspnoea (TAD) is a milder respiratory distress. We investigated blood components for immune and non-immune factors implicated in PTR. MATERIAL AND METHODS: We analysed 464 blood components (RBCs, PLTs, L-PLTs, FFP) transfused to 271 patients with PTR. Blood components were evaluated for 1/antileucocyte antibodies, 2/cytokines: IL-1ß, IL-6, IL-8, TNF-α, sCD40L, 3/lysophosphatidylcholines (LysoPCs), 4/microparticles (MPs) shed from plateletes (PMPs), erythrocytes (EMPs) and leucocytes (LMPs). RESULTS: Anti-HLA class I/II antibodies or granulocyte-reactive anti-HLA antibodies were detected in 18.2% of blood components (RBC and FFP) transfused to TRALI and in 0.5% of FFP transfused to TAD cases. Cytokines and LysoPCs concentrations in blood components transfused to PTR patients did not exceed those in blood components transfused to patients with no PTR. Only EMPs percentage in RBCs transfused to patients with TRALI was significantly higher (P < 0.05) than in RBCs transfused to patients with no PTR. CONCLUSION: Immune character of PTR was confirmed mainly in 1/5 TRALI cases. Among non-immune factors, only MPs released from stored RBCs are suggested as potential mediators of TRALI. Our results require further observations in a more numerous and better defined group of patients.

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection.

INTRODUCTION: The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay. METHODS: We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT. RESULTS: In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression. CONCLUSION: Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Molecular and serological characterization of hepatitis B virus genotype A and D infected blood donors in Poland.

Hepatitis B virus (HBV) genotypes have distinct geographical distributions and influence severity of clinical outcome and response to antiviral therapies. HBV polymorphism in HBV surface antigen (HBsAg) positive first time blood donors from Poland was examined. HBV serological markers and HBV DNA were tested in 170 samples. Whole genome (n = 53) or specific region sequences: pre-S/S and basic core promoter/precore (BCP/PC) region (91 and 154 samples, respectively) were phylogenetically analyzed. The median age of infected donors was 21 years. Anti-HBs, anti-HBe and hepatitis B e antigen were detected in 5%, 92.4% and 10.5% of tested donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 x 10(10) IU/mL (median: 4.10 x 10(3) IU/mL). Genotypes A2 (81.2%) and D (18.8%) co-circulated. Phylogenetic analyses revealed differences between the genotypes. Viral load and level of HBsAg tended to be lower in genotype D. The median HBsAg/HBV DNA ratio expressed in IU/mL was one for both genotypes, but very low or very high ratios appeared more frequent in genotype D infections. Higher amino acid variability in the surface proteins (median: 4%vs 1.5%; P = 0.01) and in the major hydrophilic region was observed in genotype D (P = 0.01). BCP/PC region analysis revealed the double mutation 1762T/1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (P = 0.08). Mutations in PC and BCP regions correlated neither with HBsAg nor HBV DNA levels. HBV genotype A2 is dominant in HBsAg positive donors in Poland. Minority genotype D strains are significantly more substituted than genotype A2 strains potentially affecting the course of infection.

Preliminary results of fetal Rhc examination in plasma of pregnant women with anti-c.

OBJECTIVES: Anti-Rhc antibodies may be the reason for the hemolytic disease of the newborn, therefore, noninvasive Rhc determination is important for pregnancy monitoring. For this purpose, we decided to introduce real-time polymerase chain reaction (PCR) method. METHODS: Blood from 200 donors, plasma and whole-blood from 11 Rhc-negative mothers, as well as blood from fathers and newborns were examined. Rhc sensitivity and specificity were first determined by real-time PCR using genomic DNA from donors. The same Rhc genotyping method was used for fetal Rhc detection in maternal plasma. To confirm the fetal Rhc-negative result, plasma was tested with a panel of biallelic insertion/deletion polymorphisms for the presence of fetal DNA. RESULTS: The c allele assay showed full specificity. The mean Ct value for one copy of c allele diluted in C-negative DNA was determined from extrapolating the correlation curve as 39.9. Full concordance was observed between the fetal Rhc genotypes from maternal plasma and the newborn phenotypes. CONCLUSIONS: Preliminary results show that it is possible to examine fetal c allele of RHCE gene in the plasma of pregnant women with anti-c by means of a noninvasive method. The diagnostic accuracy of the procedure, however, has yet to be confirmed in a larger group.

Preliminary results of HBV DNA testing of Polish haemophilia patients--lack of occult HBV infection.

Identification of hepatitis B virus (HBV) infection in the absence of surface antigen (HbsAg) became possible with the introduction of HBV DNA detection methods. Such occult HBV infection was diagnosed recently in about half of the Japanese HBsAg-negative haemophilia patients. The aim of our study was to assess the prevalence of occult HBV infection in Polish severe haemophilia population on the sample of 115 haemophilia A and B patients (mean age 34.9 +/- 10.9) treated with non-virus inactivated clotting factor preparations before 1995. HBV DNA was detected in nine HBsAg-positive patients (7.8%). The mean HBV DNA load was 72,800 IU mL(-1) (250-400,000 IU mL(-1)). Hepatitis C virus (HCV) RNA was found in six out of nine HBV-positive patients. In conclusion, HBV DNA was identified only in HBsAg-positive patients. Unlike in Japan, the frequency of occult HBV infection in Polish haemophilia population seems extremely rare or absent.

Noninvasive determination of fetal RHD status by examination of cell-free DNA in maternal plasma.

BACKGROUND: Cell-free fetal DNA in maternal plasma opens the way for routine risk-free diagnosis of fetal D status of D- mothers. The focus was on accuracy of RHD typing and confirmation of fetal DNA in maternal plasma while RHD was not detected. STUDY DESIGN AND METHODS: Plasma DNA was extracted (by manual and/or automatic method) from 255 D- pregnant women and amplified in exons 7 and 10 and intron 4 of RHD gene with real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY and, when negative, by one of 11 different polymorphisms found in the father but not in the mother. The results were compared with the D status of the newborns. RESULTS: After exclusion of 25 cases (10%) because of material shortage, in 230 cases (90%) available for complete study, the predictive value of the procedure of fetal RHD testing (RHD genotyping plus confirmation of fetal DNA) was 99.6 percent. SRY detection confirmed fetal DNA presence in maternal plasma in all boys, whereas the detection of various polymorphisms in all girls but one. CONCLUSIONS: Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).

The hepatitis C virus genotype and subtype frequency in hepatitis C virus RNA-positive, hepatitis C virus antibody-negative blood donors identified in the nucleic acid test screening program in Poland.

BACKGROUND: Since 2002, blood donors in Poland have been tested not only for hepatitis C virus antibodies (anti-HCV) but also for HCV RNA or HCV core antigen. This screening program identifies asymptomatic, recently infected individuals with no anti-HCV (in the "window period"). The aim of this study was to compare HCV genotype and subtype distribution in window-period (wp) donors, anti-HCV-positive donors, and chronic hepatitis C (CHC) patients. STUDY DESIGN AND METHODS: A total of 2.37 million donors were investigated for HCV RNA, and 340,000 for HCV core antigen. HCV genotypes and subtypes were investigated in 50 HCV RNA-positive, anti-HCV-negative donors; in 70 anti-HCV-positive donors; and in 170 CHC patients. Re-questioning of wp donors for probable risk factors was introduced. RESULTS: HCV RNA was detected in 50 donors of 2.71 million (1:54,200) anti-HCV-negative blood donations. Of these 50 donors, 36 percent exhibited Subtype 1b, whereas Subtypes 3a and 4c/d were identified in 40 and 14 percent, respectively. In anti-HCV-positive donors and CHC patients, the frequency of Subtype 1b was significantly higher (75.7 and 85.3%, respectively); in both groups the lower frequency of Subtypes 3a (14.3 and 10.6%, respectively) and 4c/d (4.3 and 1.2%, respectively) was found. The probable source of infection was identified in 9 wp donors. CONCLUSIONS: The frequency of wp donors is 18.5 per 1 million. The unexpected high frequency of Genotype 4 and Subtype 3a and the low frequency of Subtype 1b was observed in wp donors compared to anti-HCV-positive individuals. Additional epidemiologic questioning introduced after HCV RNA detection may help to identify infection source.

Polymorphism of the TT virus and its frequency in Polish blood donors.

OBJECTIVE: To determine, in Polish blood donors, the frequency of TT virus (TTV) using different primers and the sequence diversity of TTV genotypes. MATERIALS AND METHODS: Two-hundred blood donors were studied. TTV DNA was detected by the polymerase chain reaction (PCR) using primers for the coding (ORF1) and non-coding (NC) regions. Twenty isolates were genotyped by sequencing the ORF1 fragment. RESULTS: TTV DNA was detected in 78% of donors using NC primers and in 10% using ORF1 primers. The frequency of TTV DNA detection by NC primers was observed to increase with donor age, whereas the frequency of detection by ORF primers did not differ between various age-groups. The nucleotide sequence homology of Polish TTV isolates ranged from 59 to 99%. Three genotypes (1b, 2b and 2c) were identified. CONCLUSIONS: The frequency of TTV detection depends on the primers used for the PCR. Using the NC primers the virus is detected in the majority of donors, whereas the ORF1 primers strongly underestimate the prevalence of TTV. The frequency of TTV DNA increases with age. Polish TTV isolates are highly polymorphic and are classified as 1b, 2b and 2c.

The risk of antibody formation against HNA1a and HNA1b granulocyte antigens during pregnancy and its relation to neonatal neutropenia.

The evaluation of immunization by the HNA1a and 1b antigens during pregnancy was based on (i) their genotyping in 1038 unselected mothers and newborns of homozygous mothers, (ii) granulocyte counting in all born infants and (iii) examination of granulocyte antibodies in maternal sera if an HNA1 incompatibile child was born. A total of 548 (52.8%) mothers were heterozygous--thus further examinations were not done. Four hundred and ninety (47.2%) were homozygous, of whom 203 (41.3%) delivered an incompatible child, i.e. 19.6% of all the infants. Among available sera from 195 mothers with feto-maternal incompatibility, the granulocyte-specific antibodies were found in nine (4.5%); six of these (3%) were HNA1 (four anti-1a, two anti-1b), and in three others the specificity was not determined. In the remaining 28 sera, the only antibodies detected were HLA. Hence, six out of 1000 pregnant women can be expected to develop anti-HNA1. In none of the newborns was the cord neutrophil count below 1.5 x 109 L-1 and signs of infection found, thus the incidence of NAIN seems to be lower than 1 per 1000 infants. A comparison with our previous, unpublished data suggests that the incidence of severe NAIN is roughly 1 per 6000 (four cases among 24101 newborns).

Factors influencing natural history of chronic hepatitis C.

The aim of the study was to asses influence of selected epidemiologic and virusologic factors on the course of chronic hepatitis C (CHC). Data obtained from 550 CHC patients was analyzed (F/M: 241/309; age: 14-87, average age: 44.9 +/- 15.6). HbsAg and HIV-positive, as well as patients taking drugs were excluded from the study. Progression of the liver disease was assessed by the maximal ALT activity, presence of clinical or histopathological symptoms of hepatic cirrhosis, and 363 liver biopsy results. Clinical and histological data was analyzed depending on: patients sex, age (= 40, and > 40 years old), portal of infection (history data on transfusion or another source of infection), history of HBV infection (presence or absence of anti-HBc antibodies), and HCV genotype (1b or no-1b group). HCV genotype was determined in 170 patients by the use of commercial InnoLipa kit (Innogenetics). Statistical analysis was based on t-Student test and chi-squared test with or without Yates correction. It was proved that in patients over 40 years old or with history of transfusion inflammatory activity and liver fibrosis activity are significantly higher than in the rest of patients. More advanced age, transfusion and history of HBV infection are risk factors for hepatic cirrhosis development in CHC patients. Neither patient's sex nor HCV genotype were found to have significant influence on the course of CHC.

DRB1 alleles in relation to severity of liver disease in patients with chronic hepatitis C.

The aim of our study was analysis of relation between HLA class II antigens and the liver disease severity in chronic hepatitis C (CHC) patients. The subject of analysis was data obtained from 134 CHC patients with disease confirmed by histopathologic test (F/M: 62/72; age 16-74; average age 41.4 +/- 12.7 yrs), HCV RNA-positive, HbsAg- and HIV-negative with no coexistence of any other liver diseases. Liver biopsy specimens were estimated according to Ishak's criterions (grading 0-18; staging 0-6). HLA DRB1 alleles were determined by a commercial method INNOLiPA DRB (Innogenetics, Belgium). Statistical analysis considered alleles occurring with frequency higher than 10%. The necroinflammatory activity (average grading score) was compared in groups of patients with- and without particular allele. The frequency of each allele's occurrence was analyzed according to patients sex, age and staging score of liver fibrosis. In statistical analysis t-Student test and chi-squared test with or without Yates' correction were applied. Statistically significant correlation was found between occurrence of DRB1*13 and DRB1*07 alleles and necroinflammatory activity intensification, and between occurrence of DRB1*13 allele and progression of liver disease. Mild liver damage, instead, expresses statistically significant relation with DRB1*11 allele.