The strategies to reduce iron deficiency in blood donors randomized trial: design, enrolment and early retention.

BACKGROUND AND OBJECTIVES: Repeated blood donation produces iron deficiency. Changes in dietary iron intake do not prevent donation-induced iron deficiency. Prolonging the interdonation interval or using oral iron supplements can mitigate donation-induced iron deficiency. The most effective operational methods for reducing iron deficiency in donors are unknown. MATERIALS AND METHODS: 'Strategies To Reduce Iron Deficiency' (STRIDE) was a two-year, randomized, placebo-controlled study in blood donors. 692 donors were randomized into one of two educational groups or one of three interventional groups. Donors randomized to educational groups either received letters thanking them for donating, or, suggesting iron supplements or delayed donation if they had low ferritin. Donors randomized to interventional groups either received placebo, 19-mg or 38-mg iron pills. RESULTS: Iron deficient erythropoiesis was present in 52·7% of males and 74·6% of females at enrolment. Adverse events within 60 days of enrolment were primarily mild gastrointestinal symptoms (64%). The incidence of de-enrolment within 60 days was more common in the interventional groups than in the educational groups (P = 0·002), but not more common in those receiving iron than placebo (P = 0·68). CONCLUSION: The prevalence of iron deficient erythropoiesis in donors enrolled in the STRIDE study is comparable to previously described cohorts of regular blood donors. De-enrolment within 60 days was higher for donors receiving tablets, although no more common in donors receiving iron than placebo.

Plasma filtration on mediators of thrombotic microangiopathy: an in vitro study.

OBJECTIVES: Sepsis-induced thrombotic microangiopathy is successfully treated by plasma exchange therapy. However, certain putative mediators of thrombotic microangiopathy may not be removed by plasma filtration. METHODS: We conducted an in vitro study to determine whether plasma filtration can remove ultralarge von Willebrand factor (ULvWF) multimers and other mediators. In separate experiments, human umbilical venous endothelial cell (HUVEC) supernatant enriched with ULvWF or human whole blood was passed through a therapeutic plasma exchange (TPE 2000, PRISMA) filter and samples were taken for measurement of ULvWF, vWF ristocetin cofactor, vWF antigen and PAI-1. RESULTS: The sieving coefficients for vWF and PAI-1 were above 0.9. The ULvWF was gradually eliminated, and nearly disappeared after four circulations. CONCLUSION: The TPE 2000 filter can directly remove potential mediators of sepsis-induced thrombotic microangiopathy.

Viability of cryopreserved BM progenitor cells stored for more than a decade.

BACKGROUND: PBPC or BM is increasingly being harvested in remission for possible use in the event of relapse. Although the value of this approach has not been demonstrated, the long-term storage of progenitor cell components has become commonplace in many facilities. METHODS: We used multi-parameter flow cytometry to determine the viability of 11 long-term cryopreserved BM components (mean = 11.8 years) in liquid phase nitrogen. The components, prepared for autotransplantation but deaccessioned after confirming patient death, were carefully thawed, washed, and assayed immediately. The flow cytometry assay was performed according to the ISHAGE protocol, modified by the addition of 7AAD for analysis of progenitor viability (CD45+ CD34+ 7AAD-) and total leukocyte viability (CD45+ 7AAD-). In addition, total viability was assessed by fluorescence microscopy using acridine orange dye exclusion; granulocyte-monocyte colony-forming units (CFU-GM) were measured after 14 days culture. RESULTS: Leukocyte viability by flow cytometry and fluorescence microscopy agreed well (r2 = 0.55, slope = 0.83, P < 0.0005 by linear regression). CFU-GM did not correlate with CD34% or any of the viability parameters. Compared with short-term stored (mean = 33 days) PBPC assayed at infusion, long-term stored BM had a comparable percentage of CD34+ cells, comparable CFU-GM activity, increased CD34 viability, but decreased total cell viability, the latter most likely due to an increased proportion of differentiated myeloid cells. DISCUSSION: The results indicate that BM products can be cryopreserved for more than a decade without apparent loss of progenitor activity, as measured by these laboratory surrogates. This agrees with clinical anecdotes describing successful engraftment with long-term stored BM, and argues that expiration dates cannot be set for cryopreserved hematopoietic stem-cell components stored in liquid phase nitrogen.

Effective removal of copper by plasma exchange in fulminant Wilson's disease.

BACKGROUND: Patients who present with fulminant hepatic failure due to Wilson's disease may develop hemolytic anemia and renal insufficiency. In this entity, acute hepatocellular necrosis triggers the release of copper ions into the circulation, which leads to toxic effects on red cell metabolic pathways and hemolysis. STUDY DESIGN AND METHODS: The utility of therapeutic plasma exchange to rapidly remove copper and reduce toxic serum copper levels was studied in two patients with fulminant Wilson's disease. RESULTS: Intensive plasma exchange using fresh-frozen plasma replacement removed substantial amounts of copper from the hypercupremic patients, resulting in a rapid reduction in serum copper levels and decreased hemolysis. The net copper removal was proportional to the serum level, ranging from 7,000 to 11,800 micrograms per procedure in one patient and from 3,700 to 6,800 micrograms in the other. CONCLUSION: Plasma exchange allows a rapid reduction in elevated serum copper levels in patients with fulminant Wilson's disease. This leads to an amelioration of hemolytic anemia and provides clinical stabilization until liver transplantation can be performed.

Mobilization of peripheral blood progenitor cells using 16 versus 10 mg/kg/d G-CSF in children with malignancies.

Administration of hematopoietic growth factors, with or without chemotherapy, can augment progenitor cell numbers available for collection. The dose of granulocyte colony stimulating factor (G-CSF) used for mobilization of peripheral blood progenitor cells (PBPC) is controversial, and doses between 5 and 32 microg/kg/d have been reported in adults. In order to determine the dose-response effect for G-CSF in mobilizing PBPC in children, we randomized 30 children with malignancies to receive either 16 or 10 microg/kg/d subcutaneously starting on the day after the disease-oriented chemotherapy regimen and continuing until the completion of leukapheresis. Leukapheresis commenced after threshold WBC > 1 x 10(9)/L was achieved and continued until 10 x 10(6) CD34+ cells/kg were obtained or for 6 procedures. Both treatment groups achieved an adequate yield of CD34+ cells with an average of 4 leukapheresis procedures. The numbers of CD34+ cells/kg were 8.3 x 10(6) and 11.7 x 10(6) in patients receiving 16 and 10 microg/kg/d doses of G-CSF, respectively, or 2.1 x 10(6) and 3.7 x 10(6) cells/kg per leukapheresis. The levels of CD34+ cells in peripheral blood had a wide interindividual variation, and were not significantly different after 16 or 10 microg/kg doses of daily G-CSF. We conclude that there is no advantage to using 16 microg/kg/d of G-CSF post-chemotherapy for PBPC mobilization in children.

Immunohematologic disorders.

Immunohematology encompasses a broad array of clinical disorders in which immune reactions are involved in the pathogenesis of hematologic diseases. Immune reactions can involve the formed elements of the blood, producing hemolytic anemia, thrombocytopenia, or neutropenia. Autoimmune phenomena and drug-induced reactions are the most common mechanisms. In newborns, maternal antibodies can cross the placenta and destroy red blood cells, platelets, or neutrophils. Immune reactions can also occur during transfusion of blood products, leading to hemolysis, febrile reactions, allergic reactions, and lung injury. The role of leukocytes and cytokines released during blood component storage in mediating febrile transfusion reactions has prompted the increased use of leukocyte-reduced components. Immune reactions can occur to soluble clotting factors and can produce bleeding or thrombosis. Finally, immunohematologic features of B-cell disorders are considered.

Relationship of CD34+ cell dose to early and late hematopoiesis following autologous peripheral blood stem cell transplantation.

We evaluated early and late hematopoietic reconstitution in 27 patients with advanced lymphoma, Hodgkin's disease, and breast or ovarian cancer after treatment using high-dose/myeloablative conditioning regimens and autologous peripheral blood stem cell PBSC) transplantation. Eighteen patients (67%) received G-CSF 5 micrograms/kg/day following chemotherapy and nine (33%) were mobilized using G-CSF alone. Each patient had 7 x 10(8) mononuclear cells (MNC) per kg collected. G-CSF was administered post-PBSC infusion. While all patients showed prompt granulocyte recovery by day 14, platelet recovery failed to occur in our (15%) heavily pretreated patients with non-Hodgkin's lymphoma. Retrospective analysis in 17 patients revealed that the infused number of CD34 surface antigen-positive cells correlated with time to granulocyte (r = 0.59, P = 0.012) and platelet (r = 0.58, P = 0.021) recovery. Patients receiving the higher numbers of CD34+ cells had consistently better hematologic parameters at 11 times examined. At 180 days post-transplant, the median Hb level was 124 g/l vs 88 g/l (P = 0.004); platelet count was 202 x 10(9)/l vs 25 x 10(9)/l (P = 0.004); and neutrophil count was 3100 x 10(6)/l vs 1400 x 10(6)/l (P = 0.15). Hemoglobin strongly correlated with the CD34+ cell dose at 360 days (r = 0.90, P = 0.01). We conclude that graft CD34+ cell content appears to be an indicator of the quality of late as well as early hematopoietic function.

Are job characteristics related to fibrinogen levels in middle-aged women?

This study examined whether employment status or job characteristics thought to be stressful were related to fibrinogen level in a sample of 161 healthy middle-aged women. Employed women had higher fibrinogen levels than did nonemployed women. Moreover, among employed women, those who perceived high levels of job stress or low support from their bosses had elevated fibrinogen, independent of menopausal status. Perception of low support from one's boss was related to higher fibrinogen levels only among premenopausal women or postmenopausal women who were not using hormone replacement therapy. These results are consistent with the notion that stress associated with some job characteristics influences levels of fibrinogen in women.

Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy.

The CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC-labeled anti-CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE-labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < 6 microns in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG-1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+lin- cells. Duplicate samples varied by < 10%. Initial studies indicate that this procedure can be reliably used to measure CD34+lin- cells in blood, pheresis products, and bone marrow harvests. This CD34 enumeration procedure should result in increased consistency in enumerating this stem cell population.

Hemostatic factors according to menopausal status and use of hormone replacement therapy.

The rise in cardiovascular disease (CVD) risk after menopause may be reduced by hormone replacement therapy (HRT) although the mechanism is unclear. Because little is known about the potential role of hemostatic factors, fibrinogen level and other coagulation parameters were measured in a study on the change in CVD risk factors through the climacteric (the Healthy Women Study). Of 239 subjects measured to date, 32 taking aspirin or other medications thought to alter coagulation were excluded from analyses. Results (adjusted for age and obesity) showed that women taking HRT had lower plasma concentrations of fibrinogen and higher levels of plasminogen and factor VIIc than did postmenopausal subjects not taking HRT. Pre- as compared with postmenopausal women had lower plasma levels of fibrinogen, factor VIIc, and antithrombin III. Adjusting for cigarette smoking did not change the findings. Thus, among women aged 49 to 55, selected hemostatic measures varied (within normal ranges) by menopausal status and were altered by HRT. These findings generally support a hypothesis of hemostatic change contributing to the increase of CVD after menopause. The fact that subjects taking HRT showed no increase in fibrinogen relative to premenopausal women is consistent with an observed decreased risk of CVD among women taking HRT, while the implication of an elevation in factor VIIc among these women is uncertain.

Intravenous gamma globulin decreases platelet-associated IgG and improves transfusion responses in platelet refractory states.

In an attempt to improve platelet transfusion responses, intravenous immunoglobulin (IV-IgG) was administered to 19 patients who were refractory to random and best available HLA-matched platelets. A response to IV-IgG was defined as two or more successive transfusions of HLA-matched products that provided recoveries greater than 30%. Thirteen of 19 (68%) patients responded to therapy at a median time of 7 days after initiation of IV-IgG (range = 2-17). Baseline platelet associated IgG levels (PalgG) were elevated in both the responders (61.6 +/- 76.2) (mean +/- SD) and the non-responders (47.0 +/- 46.3 fg/plt). Post-therapy, PalgG levels remained unchanged in the nonresponders but were decreased significantly (p = 0.05) to 11.1 +/- 6.2 fg/plt in the responders. The latter levels were similar to those (11.6 +/- 8.2 fg/plt) measured in a series of 36 transfusion responsive patients. This apparent decline in PalgG was not explained by differences in lymphocytotoxic antibodies (LCT-Ab) after therapy. Moreover, a high degree of alloimmunization was associated with a poorer response to IgG. Only two of eight patients with LCT panel-reactive antibody (PRA) of greater than 85% were responders. By contrast, improved transfusion outcomes were seen uniformly in patients with PRA greater than or equal to 85%. Improved recoveries were obtained using LCT-Ab compatible but not incompatible platelets. The median increment (% predicted) with compatible platelets before therapy was 6.0 +/- 9.9 (SD). Post-IgG, median recoveries were 37.0 +/- 31.2 percent, P less than 0.001. These findings suggest that IV-IgG may alter destructive mechanisms that affect the survival of compatible platelets in refractory patients.

Hemorrhagic sequelae of immune thrombocytopenic purpura in human immunodeficiency virus-infected hemophiliacs.

Clinical bleeding tendency and tests of immune function were studied prospectively in 11 human immunodeficiency virus (HIV)-infected hemophiliacs with immune thrombocytopenic purpura (ITP) and a platelet count less than 50,000/microL. These 11 patients represented 13% of a well-characterized cohort of 87 HIV + hemophiliacs. ITP developed a mean 3.5 years after seroconversion, mean platelet count at presentation was 36,000/microL (range 15,000 to 49,000/microL), and the mean age at seroconversion was 37.1 years. Nine patients (82%) suffered bleeding complications, including four with intracranial hemorrhage, which was fatal in three. At the onset of ITP, five had AIDS and six were asymptomatic. Mean T4 lymphocyte count at onset of ITP was 126 +/- 32/microL (range 5 to 267/microL). Sustained treatment responses occurred with intravenous gammaglobulin (2 of 2), one of whom spontaneously remitted, and with zidovudine (1 of 2), but not with steroids (0 of 6) or danazol (0 of 3). In conclusion, 13% of a cohort of HIV + hemophiliacs has developed ITP with platelets less than 50,000/microL, a significant proportion of whom (82%) have experienced bleeding complications. It is recommended that treatment for ITP in HIV + hemophiliacs be instituted once the platelet count falls below 50,000/microL in order to avoid serious hemorrhagic sequelae.

Suitability of liquid-stored donor platelets in platelet compatibility testing.

Current platelet crossmatch procedures to select compatible donors for alloimmunized thrombocytopenic patients are hampered by the lack of a convenient platelet storage method. This study examined the feasibility of using washed apheresis donor platelets stored for up to 1 year in a modified Hank's buffer solution at 4 degrees C as crossmatch reagents in an indirect IgG-enzyme immunoassay. Pooled and monospecific HLA and PlA1 antisera were used to determine the antigenic reactivity of donor platelets in relation to duration of storage. There were no significant differences between mean HLA and PlA1 antigen expression in fresh and stored platelets. HLA reactivity was detected on 12 of 13 donor platelet samples stored for 3 to 9 months and on 14 of 17 platelets stored for 12 to 14 months. PlA1 reactivity was maintained at 12 to 14 months for all 12 donor platelet samples tested. In addition, incompatibility remained in 23 of 24 paired fresh and stored platelet crossmatches using individual alloimmunized patient plasmas. These data indicate that both HLA and platelet-specific PlA1 antigen reactivity can be maintained adequately in liquid storage at 4 degrees C for up to 1 year. The availability of a convenient platelet storage method should facilitate the general application of platelet crossmatching procedures for alloimmunized patients.

Liver transplantation: intraoperative changes in coagulation factors in 100 first transplants.

Six intraoperative blood samples were obtained at intervals from each of 100 individuals undergoing their first liver transplants. The patients fell into the following diagnostic categories: postnecrotic cirrhosis 28, primary biliary cirrhosis 20, sclerosing cholangitis 19, miscellaneous diseases 14, carcinoma/neoplasia 12 and fulminant hepatitis 7. Coagulation factor values in the initial (baseline) blood samples varied by patient diagnosis. In general, all factor levels were reduced except factor VIII:C, which was increased to almost twice normal. The slight intraoperative changes in factors II, VII, IX, X, XI and XII suggested that a steady-state relationship existed between depletion (consumption/bleeding) and repletion (transfusion, transit from extra- to intravascular space), even in the anhepatic state. In contrast, there were rapid and very significant falls in factor VIII and fibrinogen and a less pronounced decrease in factor V, all reaching their nadirs in early to mid-Stage III. The cause of these coagulation changes appears to be activation of the fibrinolytic system.

Blood use in liver transplantation.

During the first 5 years (1981-1985) of the liver transplantation program in Pittsburgh, a total (preoperative, intraoperative, and postoperative) of 18,668 packed red cell units, 23,627 fresh-frozen plasma units, 20,590 platelet units, and 4241 cryoprecipitate units was transfused for the procedures. This represents 3 to 9 percent of the total of blood products supplied by the Central Blood Bank to its 32 member hospitals. Six hundred thirty-six (636) transplants were performed on 485 patients in two hospitals: the Presbyterian University Hospital (564 beds) and Children's Hospital of Pittsburgh (236 beds). All of the blood components used in the operations were procured and released by the Central Blood Bank. This report describes some of these findings.

Danazol therapy in myelodysplasia.

Platelet-associated IgG was markedly elevated in three patients with myelodysplasia and severe thrombocytopenia that had become refractory to platelet transfusions. The patients were treated with danazol because of its efficacy in treating immune thrombocytopenic purpura where platelet destruction is primarily mediated by IgG autoantibodies. After danazol therapy, the platelet counts of each patient rose and clinical bleeding stopped, and a decline in hemolysis was seen in two patients. Danazol probably impeded the peripheral clearance of cells by macrophages; however, a beneficial effect of danazol on hematopoiesis cannot be excluded.