Guidelines for the collection, processing and storage of human bone marrow and peripheral stem cells for transplantation

SUMMARY. There are no current U.K. or international guidelines or regulations covering the production, processing and storage of haemopoietic cells such as to allow their engraftment following myeloablative therapy. This paper seeks to provide such guidelines. It enumerates how quality control and assurance can be applied to this area of transfusion medicine; procedural steps relating to bone marrow harvest on peripheral blood stem cell collection are outlined and recommended doses of nucleated cells suggested for both procedures. General specifications for identification, storage and transportation of bone marrow and peripheral blood stem cells are included and specific laboratory procedures related to the provision of haemopoietic cells for engraftment are outlined. Umbilical cord blood transplants and long-term bone marrow culture are alluded to but these are still in a research phase.     

Second-messenger pathways involved in the regulation of survival in germinal-centre B cells and in Burkitt lymphoma lines.

Spontaneous apoptosis in germinal-centre (GC) B cells can be prevented by treatment with anti-immunoglobulin (Ig). By contrast, susceptible group-I Burkitt lymphoma (BL) cells can be driven to apoptosis by anti-Ig. The second-messenger pathways involved in the regulation of apoptosis in GC B lymphocytes and in BL cell lines were studied using pharmacological agonists or inhibitors of intracellular calcium ([Ca2+]i) and protein kinase C (PKC). Anti-Ig was found to mobilize Ca2+ in group-I cells. Pre-incubation with the Ca2+ chelator EGTA partially reduced apoptosis induced by anti-Ig or by Ca2+ ionophore in group-I BL cells. Activation of PKC with phorbol ester reduced such Ca(2+)-driven programmed cell death (PCD) to control levels of apoptosis. Apoptosis in group-I BL cell lines could also be triggered by the kinase inhibitors staurosporine and Ro-31-8220 at concentrations selective for PKC activity. Expression of the bcl-2 protein in BL group-I cells following gene transfer affords protection from apoptosis induced by ionomycin or anti-Ig. In the present study, bcl-2 was additionally found to protect from apoptosis driven by staurosporine. The high levels of spontaneous apoptosis exhibited by normal GC B cells were reduced, but not abrogated, by co-culture with phorbol ester. These results indicate that, in group-I BL cells, imbalance in the phosphoinositide pathway of signalling, in favour of [Ca2+]i and away from PKC, results in apoptosis: constitutive phosphorylation of key proteins by PKC may therefore suppress apoptosis in BL as well as in GC B cells.     

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.     

Primary acquired sideroblastic anaemia and myeloproliferative disease: a report on three cases

Summary Three patients who presented with primary acquired sideroblastic anaemia (PASA) later developed myelofibrosis (MF). As both are clonal disorders a second mutation is suggested.     

Thrombelastograph (TEG) analysis of platelet gel formed with different thrombin concentrations

Autologous blood transfusion is the safest and most successful way to decrease transfusion-related risks such as postoperative infections, allo-immunization, and short- and long-term immunosuppression. In addition, these fibrin sealants are known to provide coagulation support at the surgical site and act as an adjunct to the control of postoperative bleeding. The physical formation of autologous platelet fibrin gel clot is dependent on both the common pathway of the coagulation cascade and platelet activation. Platelet gel can help provide control of intraoperative and postoperative bleeding. The Thrombelastograph Hemostasis Analyzer (TEG) measures the viscoelastic properties of a clot as it forms. Based on the information that the TEG provides, it promises to be a good choice for point of care measurement of the integrity of thrombus formed by platelet gels. Bovine blood from a single donor was sequestered into platelet-rich plasma and was made into platelet gel using calcium and three different concentrations of thrombin. The platelet gel samples were then analyzed with the TEG analyzer. The results for MA, tMA, CI, and angle were recorded and statistical analysis was performed to accept or reject the null hypothesis, which is: There is no difference between TEG parameters when analyzing platelet gels formed with calcium chloride, platelet-rich plasma and three different concentrations of thrombin A one-way analysis of variance test was performed between thrombin concentrations for MA (p = 0.19), tMA (p = 0.443), CI (p = 0.257), and angle (p = 0.323). The results showed that thrombin concentration did not affect the MA, tMA, CI, or angle as measured by the TEG analyzer. The null hypothesis was accepted. Based on a one-way analysis of variance test for MA, tMA, CI, and angle there was no significant statistical difference for the TEG samples in this experiment as reported with a 95% confidence interval.     

Site‐Dependent Reference Point Microindentation Complements Clinical Measures for Improved Fracture Risk Assessment at the Human Femoral Neck

In contrast to traditional approaches to fracture risk assessment using clinical risk factors and bone mineral density (BMD), a new technique, reference point microindentation (RPI), permits direct assessment of bone quality; in vivo tibial RPI measurements appear to discriminate patients with a fragility fracture from controls. However, it is unclear how this relates to the site of the most clinically devastating fracture, the femoral neck, and whether RPI provides information complementary to that from existing assessments. Femoral neck samples were collected at surgery after low‐trauma hip fracture (n = 46; 17 male; aged 83 [interquartile range 77–87] years) and compared, using RPI (Biodent Hfc), with 16 cadaveric control samples, free from bone disease (7 male; aged 65 [IQR 61–74] years). A subset of fracture patients returned for dual‐energy X‐ray absorptiometry (DXA) assessment (Hologic Discovery) and, for the controls, a micro‐computed tomography setup (HMX, Nikon) was used to replicate DXA scans. The indentation depth was greater in femoral neck samples from osteoporotic fracture patients than controls (p < 0.001), which persisted with adjustment for age, sex, body mass index (BMI), and height (p < 0.001) but was site‐dependent, being less pronounced in the inferomedial region. RPI demonstrated good discrimination between fracture and controls using receiver‐operating characteristic (ROC) analyses (area under the curve [AUC] = 0.79 to 0.89), and a model combining RPI to clinical risk factors or BMD performed better than the individual components (AUC = 0.88 to 0.99). In conclusion, RPI at the femoral neck discriminated fracture cases from controls independent of BMD and traditional risk factors but dependent on location. The clinical RPI device may, therefore, supplement risk assessment and requires testing in prospective cohorts and comparison between the clinically accessible tibia and the femoral neck. © 2015 American Society for Bone and Mineral Research.     

Meta‐Analysis of Risk Factors for Secondary Traumatic Stress in Therapeutic Work With Trauma Victims

Revisions to the posttraumatic stress disorder (PTSD) diagnostic criteria in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM‐5; American Psychiatric Association, 2013) clarify that secondary exposure can lead to the development of impairing symptoms requiring treatment. Historically known as secondary traumatic stress (STS), this reaction occurs through repeatedly hearing the details of traumatic events experienced by others. Professionals who work therapeutically with trauma victims may be at particular risk for this exposure. This meta‐analysis of 38 published studies examines 17 risk factors for STS among professionals indirectly exposed to trauma through their therapeutic work with trauma victims. Small significant effect sizes were found for trauma caseload volume (r = .16), caseload frequency (r = .12), caseload ratio (r = .19), and having a personal trauma history (r = .19). Small negative effect sizes were found for work support (r = ?.17) and social support (r = ?.26). Demographic variables appear to be less implicated although more work is needed that examines the role of gender in the context of particular personal traumas. Caseload frequency and personal trauma effect sizes were moderated by year of publication. Future work should examine the measurement of STS and associated impairment, understudied risk factors, and effective interventions.