Clinical effectiveness of platelets in additive solution treated with two commercial pathogen-reduction technologies.

BACKGROUND: Two noninferiority, randomized, controlled trials were conducted in parallel comparing the safety and efficacy of platelets treated with Intercept or Mirasol pathogen-reduction technologies versus standard platelets. STUDY DESIGN AND METHODS: The primary endpoint was the percentage of hematology patients who developed World Health Organization Grade 2 or greater bleeding. A noninferiority margin of 11% was chosen based on expected Grade 2 or greater bleeding in 20% of controls. The study was closed for financial restrictions before reaching the planned sample size of 828 patients, and an intention-to-treat analysis was conducted on 424 evaluable patients. RESULTS: In the Intercept trial (113 treated vs. 115 control patients), the absolute risk difference in Grade 2 or greater bleeding was 6.1%, with an upper one-sided 97.5% confidence limit of 19.2%. The absolute risk difference in the Mirasol trial (99 treated vs. 97 control patients) was 4.1%, and the upper one-sided 97.5% confidence limit was 18.4%. Neither absolute risk difference was statistically significant. In both trials, posttransfusion platelet count increments were significantly lower in treated versus control patients. Mean blood component use in treated patients versus controls was 54% higher (95% confidence interval, 36%-74%; Intercept) and 34% higher (95% confidence interval, 16%-54%; Mirasol) for platelets and 23% higher (95% confidence interval, 8%-39%; Intercept) and 32% higher (95% confidence interval, 10%-57%; Mirasol) for red blood cells. Unexpected reactions and adverse events were not reported. Mortality did not differ significantly between treated and control patients. CONCLUSION: Although conclusions on noninferiority could not be drawn due to low statistical power, the study provides additional information on the safety and efficacy of pathogen-reduced platelets treated with two commercial pathogen-reduction technologies.

Ultrasound point shear wave elastography assessment of liver and spleen stiffness: effect of training on repeatability of measurements.

OBJECTIVES: To evaluate reproducibility of measurements of spleen stiffness (SS) and liver stiffness (LS) at several sites by using point shear wave elastography (pSWE) and to investigate any training effect. METHODS: Healthy volunteers were consecutively enrolled. Measurements of SS and LS were performed by an expert (observer 1) and a novice (observer 2) at three different sites of liver and spleen. To assess the effect of training the study was conducted in two periods (period 1 and period 2). Concordance correlation coefficient was used to assess intra-observer and inter-observer reproducibility. RESULTS: A total of 92 subjects (67 men and 25 women) were enrolled in the study. Both intra-observer and inter-observer agreement were higher for the liver than for the spleen. Overall, the highest intra-observer and inter-observer agreement were obtained for the assessment of LS through intercostal space, and for measurements at this site there was a significantly better performance of observer 2 after the training period. For both observers, training improved the repeatability of SS measurements at all sites. A good intra-observer agreement was obtained only for measurements at the spleen lower pole. CONCLUSIONS: The results of this study show that a learning curve in pSWE acquisition should be taken into account both for SS and LS measurements. KEY POINTS: Reproducibility of SS measurements depends on the expertise of the operator. To achieve good reproducibility between measurements a training period is required. A learning curve in pSWE acquisition should be taken into account. SS measurements are less reproducible than LS measurements.

The plasma levels of soluble HLA-G molecules correlate directly with CD34+ cell concentration and HLA-G 14bp insertion/insertion polymorphism in cord blood donors.

BACKGROUND: Cord blood provides haematopoietic stem cells for allogeneic transplantation and, thanks to the naivety of its immune system, has several advantages over other sources of stem cells. In the transplantation setting, the presence of immunosuppressive human leucocyte antigen (HLA)-G molecules has been advocated to prevent both rejection and Graft-versus-Host disease. HLA-G is physiologically expressed throughout pregnancy and is contained in cord blood at birth. Moreover, it has recently been reported that not only cord blood mesenchymal cells, but also CD34+ cell progenies produce soluble HLA-G (sHLA-G). We tried to identify the largest producer of sHLA-G among 85 healthy cord blood donors at Pavia Cord Blood Bank, correlating the sHLA-G concentration with the HLA-G 14bp insertion/deletion (INS/DEL) genotype and CD34+ cell concentration. MATERIALS AND METHODS: We measured sHLA-G levels in 36 cord blood plasma stored at -20 °C for 2 months and 49 cord blood plasma stored at -196 °C for 4-6 years, by enzyme-linked immunosorbent assay. All cord blood donors were genotyped for the HLA-G 14bp INS/DEL polymorphism by polymerase chain reaction. For each cord blood unit, we measured the cell concentration by flow cytometry. RESULTS: We did not find differences in sHLA-G levels between cord blood plasma aliquots stored for 4-6 years at -196 °C and cord blood plasma aliquots stored for 2 months at -20 °C. We observed a higher sHLA-G concentration in cord blood plasma donors who carried the HLA-G 14bp INS/INS genotype and had higher CD34+ cell concentrations (P=0.006). DISCUSSION: This is the first report showing that the best cord blood stem cell donor is also the best sHLA-G producer, particularly if genetically characterized by the HLA-G 14bp INS/INS genotype. If the therapeutic role of sHLA-G molecules were to be finally established in the transplantation setting, our data suggest that cord blood plasma donors can provide a safe source of allogeneic sHLA-G immunosuppressive molecules ready for transfusion.

Interobserver reproducibility of the controlled attenuation parameter (CAP) for quantifying liver steatosis.

PURPOSE: This study was conducted to prospectively investigate the interobserver reproducibility of controlled attenuation parameter (CAP) measurements and the relationship among the CAP and body mass index (BMI), gender and age. METHODS: Consecutive subjects were studied using the M+ probe of the FibroScan device (Echosens, Paris, France). Measurements were performed by two raters (rater1 and rater2). Interobserver agreement was assessed by using the concordance correlation coefficient (CCC). The Pearson r coefficient was used to test correlation between two study variables, and linear regression was used for the multivariate model. RESULTS: Three hundred fifty-one subjects (227 males and 124 females) were prospectively studied. The CCC was 0.82 (95 % CI 0.78-0.85) overall, 0.80 (95 % CI 0.75-0.85) for BMI <25 kg/m(2), 0.76 (95 % CI 0.69-0.84) for BMI 25-29 kg/m(2) and 0.65 (95 % CI 0.41-0.88) for BMI ≥30 kg/m(2). The CCC was 0.44 (95 % CI 0.31-0.56) for CAP values ≤240 dB/m and 0.72 (95 % CI 0.65-0.79) for CAP values >240 dB/m. In univariate analysis, age and BMI by gender were correlated with the CAP. Multiple regression analysis confirmed the relationship of the CAP with age and BMI, but not with gender. CONCLUSIONS: The results of this study show that the interreader agreement in CAP measurement is good. In healthy volunteers, the CAP is strongly correlated with age and BMI.

Common immunogenetic profile in children with multiple autoimmune diseases: the signature of HLA-DQ pleiotropic genes.

Type 1 diabetes mellitus (T1DM), celiac disease (CD) and autoimmune thyroid disease (ATD) are autoimmune conditions relatively common in paediatric age and frequently occur in association in the same subject. This event is not by chance and requires an explanation. Here, we studied the distribution of HLA-DQ αß heterodimers in 334 Italian children with T1DM, ATD and CD alone or in association and in 224 Italian healthy controls. In particular, 164 patients had T1DM (133 alone, 20+ATD, 7+CD and 4+CD+ATD), 118 had ATD (110 alone, 8+CD) and 52 had CD (40 alone, 11+ATD and 1+T1DM). 51 patients suffered from multiple autoimmune diseases. The risk for multiple autoimmune diseases was significantly associated with the increased number of HLA-DQ markers of susceptibility for both T1DM (p = 0.003) and CD (p = 0.006). The presence of one or more diabetogenic DQ molecules significantly increased the probability of developing not only T1DM (p < 0.001) but also CD (p < 0.001) and ATD (p = 0.001). Similarly, the presence of one or more celiac HLA-DQ heterodimers significantly increased the likelihood of developing not only CD (p < 0.001), but also T1DM (p < 0.001) and ATD (p < 0.001). We confirm that the sharing of the immunogenetic background is responsible for the development of multiple autoimmune diseases although with a different risk according to the number and type of susceptible HLA-DQ heterodimers as reported in the algorithm proposed here. It is likely that combinations of DQA1 and DQB1 alleles are the real culprits of the progression towards multiple autoimmune diseases and HLA-DQ genomic typing will improve the capability to predict associated autoimmune diseases in infancy.

Best practice for peripheral blood progenitor cell mobilization and collection in adults and children: results of a Società Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO) consensus process.

BACKGROUND: A large heterogeneity in current mobilization and collection practices is perceived. Moreover, recent evidence introduced novel issues into some specific topics. Optimization of the clinical practice, through the adoption of clinical practice guidelines, previously proved to reduce health care resource use. STUDY DESIGN AND METHODS: Two Italian scientific societies, Società Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO), perceived the need of hematologists and transfusionists to share a common paradigm in the setting of hematopoietic stem cell transplantation (SCT). The aim of the current position paper is to provide common definitions and criteria for mobilization and collection of peripheral blood stem cells both in autologous and in the allogeneic setting. Current international and national standards (i.e., International Society of Hematotherapy and Graft Engineering) and recommendations (i.e., European Group for Blood and Marrow Transplantation) were harmonized with the Panel recommendations. RESULTS: The Expert Panel consisted of nine members (five transfusionists and four hematologists with both clinical and scientific experience of SCT in both pediatric and adult setting) and one methodologist and first convened on April 19, 2010: they in turn agreed on the questions to be answered by the project. Available literature was reviewed by one expert and the methodologist and presented to the other members. Statements were then formulated. SIDEM and GITMO planned an informal meeting of the Panel every 2 years to discuss relevant updates and possible changes to the recommendations. CONCLUSION: The efforts of the expert panel members allowed to set up and share a common approach to the mobilization, enumeration, and collection issues in the field of both autologous and allogeneic peripheral blood SCT.

Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma: the Pavia experience.

BACKGROUND: The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). STUDY DESIGN AND METHODS: Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the "2010 Workshop on Molecular Blood Group Genotyping"; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the "1(st) Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype". To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). RESULTS: The results for the 31 investigated samples showed 100% concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. DISCUSSION: These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.

Excess of activating killer cell immunoglobulin­like receptors and lack of HLA-Bw4 ligands: a two­edged weapon in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is an inflammatory disease of unknown aetiology. Researchers have proposed infectious, neurological and immunological causes of this syndrome. Recently, the xenotropic murine leukemia virus-related virus was detected in 67% of patients with CFS in a US study. This observation is in agreement with one ascertained aspect of the disease: a decreased efficiency in NK cell lytic activity in CFS patients. Here, we analyzed the genomic polymorphism of killer cell immunoglobulin-like receptors (KIRs) and their HLA class I cognate ligands in patients with certified CFS. An excess of KIR3DS1 was found in CFS patients with respect to controls, as well as an increased frequency of the genotype missing KIR2DS5. Forty-four CFS patients and 50 controls also underwent genomic typing for the HLA-ligands. In the patients, a great proportion of KIR3DL1 and KIR3DS1 receptors were found to be missing their HLA-Bw4Ile80 binding motif. We hypothesize that an excess of KIR3DS1, combined with an excess of ligand-free KIR3DL1 and KIR3DS1 receptors, may hamper the clearance of a pathogen via NK cells, thus favouring the chronicity of the infection.

Extracorporeal photochemotherapy in graft-versus-host disease: a longitudinal study on factors influencing the response and survival in pediatric patients.

BACKGROUND: Extracorporeal photochemotherapy (ECP) is a valid therapeutic option in the treatment of acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively). No standard clinical and laboratory criteria of response to ECP treatment are available at the moment. STUDY DESIGN AND METHODS: Clinical and laboratory variables on 73 pediatric patients with aGVHD (n = 50) and cGVHD (n = 23) were correlated with response to ECP and survival. RESULTS: An overall response (OR) was obtained in 34 of 50 (68%) aGVHD and in 16 of 23 (69.5%) cGVHD patients. Steroid tapering within 30 days of 1.3 mg/kg in OR (p = 0.004) was the sole highly significant correlation with response found in aGVHD while no correlation emerged for cGVHD (p = 0.28). Among aGVHD patients, response to ECP was inversely associated with death: among OR, deaths were 13 of 34 (38.2%), while among nonresponders, deaths were 15 of 16 (93.8%; p < 0.001). On the other hand, decrease of steroid dose at 30 days was associated with survival: for each 1 mg/kg reduction, the hazard ratio was 2.2, and the 95% confidence interval was 1.5 to 3.2 (p < 0.001). No other clinical or laboratory variables statistically associated with survival were found. CONCLUSIONS: Our results demonstrate that steroid tapering within the first 30 days of ECP treatment in aGVHD and response to ECP in acute and chronic GVHD are the only variables influencing response and survival, respectively.

Time course of oxidant markers and antioxidant defenses in subgroups of amyotrophic lateral sclerosis patients.

Oxidative stress markers have been found in nervous and peripheral tissues of familial and sporadic amyotrophic lateral sclerosis patients. Here, we evaluated the activity of some antioxidant enzymes glutathione peroxidase, glutathione reductase and Cu-Zn superoxide dismutase in erythrocyte, the marker of non-enzymatic antioxidant response (total antioxidant status), as well as plasma reactive oxygen species, at the enrolment and during disease progression in 88 patients affected by the sporadic form of amyotrophic lateral sclerosis. Our study has been performed along 72 months by grouping the patients according to the ALS functional rating score or rate of disease progression. Our results showed a significant impairment of erythrocytes glutathione peroxidase activity in all groups of patients that remained low during disease time course. SOD1 activity significantly decreased along disease course in subjects with a more impaired functional status. A decreasing activity of all assayed enzymes was found in patients who have a faster disease progression rate. By this work we have the evidence that different ALS phenotypes present with different profile of enzymatic and non-enzymatic antioxidant response.

The HLA alleles DRB1*13 and DQB1*06 are associated to Whipple's disease.

BACKGROUND & AIMS: Whipple's disease is a systemic, chronic, relapsing disorder caused by a combination of environmental (Tropheryma whipplei) and unknown host factors. Because it is a rare disease, the association between HLA type and Whipple's disease has been studied in only small numbers of patients; these studies have led to conflicting results. We aimed to investigate whether disease phenotype and outcome are associated with HLA type in 122 patients with Whipple's disease. METHODS: Genomic DNA was collected from 103 German, 11 Italian, and 8 Austrian patients with Whipple's disease, along with 62 healthy Austrian workers exposed to T whipplei (14 stool samples contained the bacterium). HLA class I and II alleles were identified by polymerase chain reaction analysis. Patient genotypes were compared with those of healthy German and Austrian populations; data for Italian controls were obtained from the Pavia HLA bone marrow donors' bank. RESULTS: HLA-DRB1*13 and DQB1*06 alleles occurred significantly more frequently in patients with Whipple's disease but not in healthy individuals who had been exposed to T Whipplei. The cumulative odds ratios for disease were 2.23 for the DRB1*13 allele (P < .0001) and 2.25 for the DQB1*06 allele (P < .0001). CONCLUSIONS: DRB1*13 and DQB1*06 alleles were found to be risk factors in the largest HLA study ever performed in patients with Whipple's disease.

Strategies for the transfusion of subjects with complex red cell immunisation: the Bank of rare blood donors of the Region of Lombardy.

INTRODUCTION: Selecting units of rare blood for transfusion to patients with complex immunisation is one of the most critical processes of a Transfusion Centre. In January 2005 the 'Rare Blood Components Bank - Reference Centre of the Region of Lombardy' w as established with the following goals: 1) identifying regional rare blood donors; 2) creating a regional registry of rare donors; 3) organising a regional bank of liquid and frozen rare blood units; 4) setting up a regional Immunohaematology Reference Laboratory (IRL) to type donors and resolve complex cases. METHODS: The key elements in establishing the Bank were periodic meetings organised by the directors and representatives of the regional Departments of Transfusion Medicine and Haematology (DTMH) and the institution of three working groups (informatics, regulations, finance). RESULTS: The regional IRL was set up, the relevant operating procedures were distributed region-wide, software features were defined and later validated upon activation, and the funds assigned were allocated to various cost items. The number and characteristics of the donors to be typed were identified and 14 regional DTMHs started to send samples. Overall, 20,714 donors were typed, for a total of 258,003 typings, and 2,880 rare donors were identified. Of these, 97% were rare donors because of combinations of antigens (2,139 negative for the S antigen and 659 negative for the s antigen) and 3% (n=82) because they were negative for high-frequency antigens. In the first 2 years of activity, the IRL carried out investigations of 140 complex cases referred from other Centres and distributed 2,024 units with rare phenotypes to 142 patients. CONCLUSIONS: The main goal achieved in the first 24 months from the start of the project was to set up a regional network able to meet the transfusion needs of patients with complex immunisation.

Clinical impact of a new automated system employed for peripheral blood stem cell collection.

At the moment, PBSC collections can be performed using semi-automated or automated cell separator devices. The automated methods offer the advantages of a decreased working load for dedicated personnel and high standardization of the collection procedure. Herein we report our single institutional experience in 80 PBSC collections employing the new automated COM.TEC Fresenius autoMNC program that provides the ability to predict the total number of CD34(+) cells collected, based on the pre-leukapheresis CD34(+) cell count in peripheral blood. Fourty-eight patients and 21 healthy donors were mobilized with chemotherapy + G-CSF or G-CSF alone, respectively. Eighty leukapheresis collections were performed starting with a CD34(+) cell count in peripheral blood at least of 20/microL. Collection parameters and related side effects were evaluated. The mean CD34(+) cell collection efficiency in patients and donors was 81.8% (sd 27.6) and 95.1% (sd 15.6), respectively. The mean difference between real and predicted CD34(+) cells was +30.2% (sd 92.9) for patients and +4.6% (sd 30.3) for donors. The mean leukapheresis bag volume was 240.7 ml (sd 67.3) and 310.3 ml (sd 86.8) with a mean HCT of 10.9% (sd 34.4) and 9.2% (sd 3.9) for patients and donors, respectively. The automated PBSC collection with the new program COM.TEC Fresenius autoMNC demonstrated a very high CD34(+) cell collection efficiency. Moreover, the ability to predict the CD34(+) cell yield permits improved management of the leukapheresis collection, with the only disadvantage of larger leukapheresis volume and higher hematocrit.

Daily plasma-exchange for life-threatening class I HELLP syndrome with prevalent pulmonary involvement.

HELLP syndrome (Hemolysis, Elevated Liver enzymes, Low Platelets) is a thrombotic microangiopathy affecting a minority of women with pre-eclampsia and usually resolves after delivery. The role of plasma exchange in HELLP syndrome has to be defined. Herein is reported a case of a primipara with a class I HELLP syndrome with prevalent pulmonary involvement successfully treated with 8 consecutive plasma exchange plus corticosteroids. In class I HELLP syndrome with cardiopulmonary complications early plasma exchange could be considered a therapeutic option.

Peripheral blood progenitor cell mobilization and collection in 42 patients with primary systemic amyloidosis.

BACKGROUND: High-dose chemotherapy followed by an inoculum of autologous peripheral blood progenitor cells (PBPCs) can improve survival in patients affected with primary systemic amyloidosis (AL). It has been documented, however, that the morbidity and mortality of PBPC mobilization and collection in this setting are higher than in patients with other diseases. To minimize the mobilization and collection-related risks, we developed a multidisciplinary approach involving different specialists to manage AL patients with predominant heart and renal involvement. STUDY DESIGN AND METHODS: We report our experience in 42 patients (23 men, 19 women; median age, 51.2 years; range, 28-68 years) with AL who underwent PBPC mobilization and collection. Twenty of the 42 patients (47.6%) had cardiac involvement and 35 of 42 (83.3%) renal involvement. Thirty-three patients (78.5%) were mobilized with granulocyte-colony-stimulating factor (G-CSF) alone (10 microg/kg) and 9 (21.4%) with cyclophosphamide (CTX) (3 g/m(2)) plus G-CSF (10 microg/kg). RESULTS: The median number of collections per patient after either G-CSF or CTX plus G-CSF was 1.8 (range, 1-3). The median number of CD34+ cells collected in patients mobilized with G-CSF alone was 8.2 x 10(6) per kg (range, 1.35 x 10(6)-21.3 x 10(6)/kg) and in patients mobilized with CTX plus G-CSF it was 8.9 x 10(6) per kg (range, 5.5 x 10(6)-14.9 x 10(6)/kg). Forty of the 42 (95.2%) patients produced the minimum required CD34+ cell target dose (4 x 10(6)/kg). The overall rate of morbidity during the collections was 50 percent (21/42 patients): 18 patients (42.8%) had asymptomatic hypotension, 1 (2.4%) had symptomatic hypotension with nausea and vomiting, and 2 (4.7%) experienced a life-threatening hypotensive episode. There were no procedure-related deaths. CONCLUSION: Our multidisciplinary approach was effective in limiting the serious side effects related to PBPC mobilization and collection in AL patients.

Immunomagnetic cell selection performed for HLA haploidentical transplants with the CliniMACS device: effect of additional platelet removal on CD34+ cell recovery.

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein-Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.