Retrospective analysis of HDFN due to ABO incompatibility in a single institution over 6 years.

OBJECTIVES: To study the rate of ABO haemolytic anaemia of fetus and newborn (HDFN) in one institution over 6 years. BACKGROUND: ABO major incompatibility between mothers and their newborns occurs in about 10% of births. So, mothers with an O blood group may form IgG-class antibodies against A and B antigens, which could pass across the placenta and lead to a variable degree of HDFN in the newborn. METHODS: At our institution, we have reviewed data regarding ABO-based HDFN in the last 6 years. RESULTS: We found that, in 28 089 deliveries, an ABO major incompatibility between mothers and newborns occurs in 11% of cases, with 72% of O/A and 28% of O/B incompatibility. In turn, 23% of these newborns had an eluate-confirmed positive direct antiglobulin test [DAT; 74% (511) were due to anti-A and 26% (179) to anti-B], with 1·0% requiring invasive treatments (exchange transfusion or intravenous immunoglobulin). Overall, 2·5% of the total newborns had a positive DAT for an anti-A or anti-B antibody, and 0·11% required invasive treatment in addition to phototherapy for their HDFN. CONCLUSIONS: Serological ABO HDFN is a relatively frequent event when an O-A/O-B incompatibility between mothers and their newborn occurs and, in most cases, translates into a self-limiting disease, with a small number of newborns requiring invasive treatments. The DAT test, although not predictive of disease severity, appears to be a useful tool to monitor babies born from O-A/O-B-incompatible pregnancies and to identify those who may require treatment.

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. METHODS: MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. RESULTS: The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. CONCLUSIONS: The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

A prospective, active haemovigilance study with combined cohort analysis of 19,175 transfusions of platelet components prepared with amotosalen-UVA photochemical treatment.

BACKGROUND AND OBJECTIVES: A photochemical treatment process (PCT) utilizing amotosalen and UVA light (INTERCEPT(™) Blood System) has been developed for inactivation of viruses, bacteria, parasites and leucocytes that can contaminate blood components intended for transfusion. The objective of this study was to further characterize the safety profile of INTERCEPT-treated platelet components (PCT-PLT) administered across a broad patient population. MATERIALS AND METHODS: This open-label, observational haemovigilance programme of PCT-PLT transfusions was conducted in 21 centres in 11 countries. All transfusions were monitored for adverse events within 24 h post-transfusion and for serious adverse events (SAEs) up to 7 days post-transfusion. All adverse events were assessed for severity (Grade 0-4), and causal relationship to PCT-PLT transfusion. RESULTS: Over the course of 7 years in the study centres, 4067 patients received 19,175 PCT-PLT transfusions. Adverse events were infrequent, and most were of Grade 1 severity. On a per-transfusion basis, 123 (0.6%) were classified an acute transfusion reaction (ATR) defined as an adverse event related to the transfusion. Among these ATRs, the most common were chills (77, 0.4%) and urticaria (41, 0.2%). Fourteen SAEs were reported, of which 2 were attributed to platelet transfusion (<0.1%). No case of transfusion-related acute lung injury, transfusion-associated graft-versus-host disease, transfusion-transmitted infection or death was attributed to the transfusion of PCT-PLT. CONCLUSION: This longitudinal haemovigilance safety programme to monitor PCT-PLT transfusions demonstrated a low rate of ATRs, and a safety profile consistent with that previously reported for conventional platelet components.

Red blood cell alloimmunization in sickle cell disease and in thalassaemia: current status, future perspectives and potential role of molecular typing.

Red blood cell (RBC) transfusions are a milestone in the treatment for sickle cell anaemia (SSA) and for thalassaemia. RBC alloimmunization remains a major challenge of chronic transfusion therapy, and it can lead to adverse life-threatening events. The alloimmunization risk could depend on multiple factors such as the number of transfusions and, most of all, the genetic background. Different ethnic groups are predisposed to immunization because of a significant degree of RBC antigenic mismatch between donor and recipient. There is no universal agreement and standards for the most appropriate selection of RBC units in chronically transfused subjects. Current practice only deals with compatibility of ABO, Rh and K antigens. Molecular RBC antigenic matching extended to other blood group systems is an innovative strategy to ensure a better quality and effectiveness of transfusion therapy.

Counting of leukocytes in samples from G-CSF mobilized donors, leukapheresis products, and cord blood: the performances of an analyzer with dedicated profiles.

INTRODUCTION: Accurate white blood cell counting (WBC) and differential count by blood analyzers could allow a more informative characterization of granulocyte colony-stimulating factor (G-CSF) mobilized blood (MB), leukapheresis products (LP), and cord blood (CB). However, reliable counting by a blood cell analyzer in this setting is a major challenge owing to quali-quantitative abnormalities of blood cells. METHODS: We evaluated the performances of the analyzer Pentra DX 120 by Horiba ABX working with dedicated cell-gating profiles, which generate three-part differential counts in samples obtained from donors' MB, LP, and CB. The results of the analyzer were compared to counts obtained by flow cytometry and manual counts, the latter performed for reference validation and in the case of discrepant results between study and reference counts. RESULTS: Pentra DX 120 generated highly correlated counts (R > 0.91 in all cases) to those obtained by flow cytometry in all samples (MB, LP, and CB) with high degree of count accuracy in most cases and referred to WBC absolute count and differential count including lymphocytes (LYM) %, monocytes (MON) %, and polymorphonuclear leukocytes (PMN) %. Accuracy, judged by the difference between study and reference counts and expressed as percentage of reference count, ranged from 0.8% to 8.6%, and sporadic loss of accuracy occurred for MON % only in no more than 10% of CB samples. CONCLUSION: The ABX Pentra DX 120 provided accurate WBC count and differential count during MB, LP, and CB analyses and allowed a better characterization of donors' hematologic status and graft composition.

The application of multiparameter reference intervals for pre-donation capillary blood counts: the experience of a single institution.

OBJECTIVES: To evaluate a set of reference counts applied to multiparameter pre-counts in blood donors. AIM: Analyse the impact of pre-donation counts and specific reference intervals on donors' management. BACKGROUND: Multiparameter blood counts allow an improved enrollment process of blood donors due to a prompt identification of abnormalities involving haemoglobin (Hb), white blood cells (WBC) and platelets (PLT). METHODS/MATERIALS: Multiple pre-donation capillary counts were applied in the enrollment process of 13,347 consecutive donors. The rate of specific alterations of permanent exclusion and donor readmittance to donations for temporary exclusion had been evaluated, applying a set of multiparameter reference intervals. RESULTS: Alterations involved Hb in 72.55% of cases, mean corpuscular volume (MCV) in 20.99%, total WBC in 9.39%, lymphocytes in 7.55% and PLT in 6.07%. Among donors with initial alterations (543; 4.06%), 12.70% were readmitted to donations within 15 days, 14.36% had permanent exclusion, 36.83% underwent prompt supplementation treatment and 36.09% were lost at follow-up or refused treatments. DISCUSSION: The systematic use of blood count reference intervals and pre-donation multiparameter blood counts allowed prompt identification of WBC, PLT and MCV alterations, readmittance within 15 days of 12.70% of initially excluded donors and contributed to prompt management of supplement deficiency.

Evaluation of the analytical performances of a portable, 18-parameter hemometric system using capillary blood samples for blood donor enrolment.

BACKGROUND AND OBJECTIVES: Blood donor enrolment process is frequently based on the sole capillary haemoglobin (Hb) evaluation while platelet donors by apheresis also requires platelet (Plt) count. The 'sole Hb' approach prevents a complete donor evaluation and does not allow Plt donor enrolment. To extend blood counts before donations, we evaluated the performances of a multiparametric counter using capillary blood. MATERIALS AND METHODS: The ABX Micros 60 (Micros 60) blood analyzer was employed on capillary blood and compared with venous counts by a reference counter (Coulter AcT 5diff) in a first series of 416 donors and in a second series of 136, after a 3-month period of routine use of this study counter. An average of 50 microl of capillary blood was collected whose 10 microl had been aspirated by Micros 60. RESULTS: High correlations were found between capillary counts using Micros 60 and venous counts using the reference counter. Mean Plt counts differed of 37 x 10(9)/l less for capillary approach in the first series of comparisons, but decreased to 10 x 10(9)/l less in the second series due to a greater expertise of operators in capillary sampling. All other parameters were accurate and never reached clinical relevance albeit they showed statistically significant differences. CONCLUSION: Data on Micros 60 demonstrated that capillary predonation counts may represent a feasible and effective approach to realize an accurate enrolment process of blood and Plt donors.

Hepatitis B virus blood screening: impact of nucleic amplification technology testing implementation on identifying hepatitis B surface antigen non-reactive window period and chronic infections.

BACKGROUND: Despite improvements in hepatitis B surface antigen (HBsAg) test sensitivity, post-transfusion hepatitis B virus (HBV) infection still occurs because HBsAg is undetectable during the early window phase (WP) of the infection, in the convalescence core window phase of the infection, or in serologically silent chronic hepatitis or in mutant forms of HBV. HBV-DNA screening using high sensitivity nucleic amplification technology (NAT) assays has recently been introduced to reduce the residual risk of transmission of HBV by transfusion of blood components. MATERIALS: Over 1 year 75 063 donations were individually screened for HBV-DNA by the Ultrio Procleix assay on the Tigris platform. The donations were collected in the Latium region, an area of the central Italy, and they accounted for the 40% of the total blood units collected in this area per year. The initial reactive samples were re-tested and confirmed by the discriminatory HBV assay. Additional HBV serological markers were also performed. Suspected WP infections were followed-up to monitor the development of the immune response. All HBV-DNA-positive donors were called back to check up their infectious status. RESULTS: The results of testing the 75 063 donations are: 33 donations HBsAg positive, 31 out of them HBV-DNA-positive and two HBV-DNA negative; 22 donations HBsAg-negative but HBV-DNA positive with low viral load. Six of the 22 were found to be consistently HBV-DNA reactive whereas the remaining 16 donations showed inconsistent results on multiple NAT retesting. One WP infection was confirmed by the follow-up of the donor for 3 months following the index blood donation. CONCLUSIONS: In the donor population of the Latium region, NAT screening has revealed a higher than expected number of donors who were HBsAg non-reactive but HBV-DNA-positive with three donors showing HBV-DNA as the only marker of infection. The adoption of genome screening has increased the safety of the blood supply and has also contributed to the protection of donor health by identifying either WP or clinically silent infections.

Expression of CD133-1 and CD133-2 in ovarian cancer.

Cancer stem cells have been isolated from several solid tumors including prostate, colon, liver, breast, and ovarian cancer. Stem cells isolated from nervous system and prostate express CD133 antigen, which is widely used to isolate hematopoietic stem and progenitor cells. The aims of this study were to investigate the expression of the CD133-1 and CD133-2 epitopes in primary ovarian tumors and to biologically characterize CD133(+) ovarian cancer cells, also according to clinicopathologic parameters. Tissue specimens were obtained at primary surgery from 41 ovarian carcinomas; eight normal ovaries and five benign ovarian tumors were also collected. Flow cytometry with monoclonal antibodies against CD133-1 and CD133-2 epitopes was employed. FACS (fluorescence activated cell sorting) analysis enabled the selection of CD133(+) cells, whose epithelial origin was confirmed by immunofluorescence analysis with monoclonal anti-cytokeratin 7. CD133(+) cells gave rise to a 4.7 +/- 0.9-fold larger number of colonies than that documented in CD133(-) population (P < 0.001). Moreover, CD133(+) cells showed an enhanced proliferative potential compared to CD133(-) cells. The percentages of CD133-1- and CD133-2-expressing cells were significantly lower in normal ovaries/benign tumors with respect to those in ovarian carcinoma. Both the percentages of CD133-1- and CD133-2-expressing cells were significantly lower in omental metastases than in primary ovarian cancer (P = 0.009 and 0.007 for CD133-1- and CD133-2-expressing cells, respectively). There seems not to be any difference in the distribution of the percentage of CD133-1- and CD133-2-expressing cells according to clinicopathologic parameters and response to primary chemotherapy. CD133-1 and CD133-2 may be useful in order to select and enrich the population of CD133(+) ovarian tumor cells, which are characterized by a higher clonogenic efficiency and proliferative potential.

Accurate prediction of autologous stem cell apheresis yields using a double variable-dependent method assures systematic efficiency control of continuous flow collection procedures.

BACKGROUND AND OBJECTIVES: Stem cell collection is a standard procedure for the procurement of autologous grafts to rescue myelosuppression induced by high-dose treatments. Accurate prediction of collection yields may contribute to optimize planning and quality control of collection. MATERIALS AND METHODS: Data of 313 autologous haematopoietic stem cell (AHSC) evaluable collections performed in 208 patients with haematologic and non-haematologic neoplasms from seven centres were prospectively analysed to test the accuracy of yield predictions generated by a formula that required the input of peripheral blood (PB) CD34+ cell precount and desired PB volume to be processed. Data were matched in a standard linear regression, in a zero-point regression analysis and tested for prediction accuracy. Further 165 AHSC collections were analysed on a single-centre basis, using yield predictions as reference standards. RESULTS: Analysis showed high levels of correlation between measured collection yields (my) and predictions (py) (R = 0.85; P = 0.000000) as well as high degree of prediction accuracy (my vs. py at paired t-test: P = 0.114781; median my/py ratio = 1.23). Analysis of additional 165 AHSC collections on a single-centre basis showed that the analysed centres had 70% or more measured yields comprising the 0.6-1.8 interval of the my/py ratio. The observance of the 'efficiency' my/py interval assured collection quality control in these centres confirming the reliability of the method. CONCLUSIONS: This prediction method generates accurate and immediate yield predictions allowing collection planning and rapid efficiency control. As a consequence of our study, four centres out of seven use the described method to plan both leukapheresis number and single-procedure blood processing volume while the remaining three centres plan leukapheresis number on the basis of our predictions, maintaining a fixed single-procedure 200 ml/kg blood volume processing, according to their centre AHSC collection policy.

Stem cell-based treatments for gynecological solid tumors.

BACKGROUND AND OBJECTIVE: We have recently assisted to an increasing scientific interest and a new research effort in the field of stem cell-based therapy. Since the late 1980s hematopoietic stem cells (HSC) have been used to set up therapeutic strategies for the treatment of solid tumors such as gynecological cancers. In this context, different approaches have been suggested and clinically investigated. STATE OF THE ART: In the autologous setting we can describe the well-known use of HSC as hematologic support to high-dose chemotherapy regimens, and the use of HSC as a source of dendritic cells for cancer vaccination protocols. In our institution a long-term experience has been developed in high-dose chemotherapy with autologous HSC transplantation as first-line treatment of advanced ovarian cancer, and in the use of cytokines both for HSC collection and for post-transplantation hematopoietic recovery and immune reconstitution. An alternative approach consists of allogenic HSC transplantation following either myeloablative/standard or non-myeloablative/reduced conditioning regimens, which have been proposed as new adoptive immunotherapeutic treatments for different non-hematologic malignancies. PERSPECTIVES: Future strategies in the use of HSC in oncology comprise the possibility of HSC ex-vivo expansion, the use of umbilical cord blood HSC, and the development of HSC-based gene-therapy programs. Further investigations are expected in the new field of cancer stem cells.

A human umbilical cord stem cell rescue therapy in a murine model of toxic liver injury.

BACKGROUND: Several studies have demonstrated that bone marrow contains a subpopulation of stem cells capable of participating in the hepatic regenerative process, even if some reports indicate quite a low level of liver repopulation by human stem cells in the normal and transiently injured liver. AIMS: In order to overcome the low engraftment levels seen in previous models, we tried the direct intraperitoneal administration of human cord blood stem cells, using a model of hepatic damage induced by allyl alcohol in NOD/SCID mice. METHODS: We designed a protocol based on stem cell infusion following liver damage in the absence of irradiation. Flow cytometry, histology, immunohistochemistry and RT-PCR for human hepatic markers were performed to monitor human cell engraftment. RESULTS: Human stem cells were able to transdifferentiate into hepatocytes, to improve liver regeneration after damage and to reduce the mortality rate both in both protocols, even if with qualitative and quantitative differences in the transdifferentiation process. CONCLUSIONS: We demonstrated for the first time that the intraperitoneal administration of stem cells can guarantee a rapid liver engraftment. Moreover, the new protocol based on stem cell infusion following liver damage in the absence of irradiation may represent a step forward for the clinical application of stem cell transplantation.