Disseminated intravascular coagulation and hemorrhage in hemophilia B following elective surgery

Two patients with hemophilia B are described in whom disseminated intravascular coagulation (DIC) developed following infusion of repeated doses of Factor IX concentrate in the perioperative period. In both cases the surgery was elective, Factor IX survival studies had been done to assure proper dosing, and Factor IX levels were monitored daily. Neither patient had clinically significant liver disease. The DIC manifested itself as excessive blood loss from surgical drains without documented thrombosis and was accompanied by prolonged coagulation times, increased fibrin split products and decreased fibrinogen and platelets. In both patients the process was quickly reversed with administration of fresh frozen plasma, cryoprecipitate, and the addition of heparin to the Factor IX concentrate. These cases highlight the difficulty in managing patients with hemophilia B undergoing surgical procedures due to the potential thrombogenicity of the currently available concentrates, and the importance of differentiating the bleeding associated with DIC from underdosing with Factor IX. Furthermore, the potential complications associated with the presently available Factor IX concentrates stress the need for the development of purer, safer Factor IX concentrates.     

Mesenchymal stem cells obtained after bone marrow transplantation or peripheral blood stem cell transplantation originate from host tissue

Mesenchymal stem cells (MSC) obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Therefore, we were interested in finding out if graft MSC can be detected in leukapheresis products and in bone marrow after BMT and peripheral blood stem cell transplantation (PBSCT). Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of nine leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for origin of MSC. Almost all MSC samples exhibited a complete host profile, whereas peripheral blood cells were of donor origin. We conclude that even if trace amounts of MSC are co-transplanted during PBSCT or BMT, they do not expand significantly in the host bone marrow.     

Use of leukocyte-depleted platelets and cytomegalovirus-seronegative red blood cells for prevention of primary cytomegalovirus infection after marrow transplant.

Seventy-seven cytomegalovirus (CMV)-seronegative marrow transplant patients were randomized in a prospective controlled trial comparing the use of leukocyte-depleted platelets plus CMV-seronegative red blood cells with standard unscreened blood products for the prevention of primary CMV infection during the first 100 days after transplant. Eligible patients included CMV-seronegative patients undergoing autologous transplant or seronegative patients undergoing allogeneic transplant for aplastic anemia or non-hematologic malignancy who had seronegative marrow donors. Patients and marrow donors were serologically screened for CMV and randomized before conditioning for transplant and followed for CMV infection with weekly cultures of throat, urine, and blood and with weekly CMV serologies until day 100 after transplant. Leukocyte-depleted platelets were prepared by centrifugation, a procedure that removed greater than 99% of leukocytes. There were no CMV infections observed in 35 evaluable treatment patients compared with seven infections in 30 evaluable control patients (P = .0013). There was no statistically significant difference in the mean number of platelet concentrates in the treatment patients (164 concentrates) compared with the control patients (126 concentrates). Leukocyte-depleted platelets plus CMV-seronegative red blood cells are highly effective in preventing primary CMV infection after marrow transplant.     

Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells

Complete hematopoietic reconstitution using nonleukemic peripheral blood mononuclear cells has been achieved in animal models but not in humans. We treated two patients who had metastatic breast carcinoma involving the bone marrow and who had failed conventional therapy with high-dose chemotherapy and total body radiation. Cryopreserved autologous peripheral blood mononuclear cells (6.3-8.4 X 10(8)/kg patient weight) obtained by leukapheresis before high-dose therapy were returned to the patients intravenously. In one patient, evidence of bone marrow engraftment was present, but the patient died before full reconstitution of the peripheral blood cells occurred. Bone marrow engraftment and return of all cell lines to the peripheral blood occurred in the second patient. These findings demonstrate that human hematopoietic reconstitution can be achieved with autologous, peripheral blood, mononuclear cell transfusions following high-dose therapy. This approach may be useful to patients who have contraindications for a bone marrow harvest but who are otherwise candidates for autologous bone marrow transplantation.     

Human macrophage colony-stimulating factor induces macrophage colonies after L-phenylalanine methylester treatment of human marrow

Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L-phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment.     

Large scale purification of human blood CD34+ cells from cryopreserved peripheral blood stem cells, using a nylon-fiber syringe system and immunomagnetic microspheres

Isolation of large numbers of human peripheral blood CD34+ cells could lead to therapeutic applications, including purging of malignant cells from blood cell transplantations, purging of T cells from allogeneic bone marrow, and even blood cell transplantation. This procedure has limitations if there are not sufficient numbers of progenitor cells in the leukapheresis concentrates available for selection after detection of tumor cells in apheresis products. Use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples would make feasible pooling of two or even more stem cell harvests collected at different time points and the total number of CD34+ progenitor cells available would increase. We established an efficient method for purification of CD34+ cells from cryopreserved apheresis products, using a nylon-fiber syringe system and immunomagnetic microspheres. We compared purity, recovery rate and clonogenicity of CD34+ cells purified from fresh (n = 22) and cryopreserved apheresis products (n = 14), using a nylon-fiber syringe system and immunomagnetic microspheres. The purity of CD34+ cells from cryopreserved products was less than that from fresh products (85.9 +/- 14.4% vs 94.6 +/- 10.0%), but the recovery rate of CD34+ cells and colony-forming cells was comparable between fresh and cryopreserved products. One patient underwent grafting with peripheral blood CD34+ cells selected after freezing, with good success. Therefore, these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy. Bone Marrow Transplantation (2000) 26, 787-793.     

Correlation of quantitative bone marrow and blood cultures in AIDS patients with disseminated Mycobacterium avium complex infection.

The relationship between Mycobacterium avium complex (MAC) infection of blood and bone marrow was studied in human immunodeficiency virus-infected patients before and during treatment. Quantitative cultures were obtained at baseline from 17 persons with newly detected MAC bacteremia. Serial blood cultures were obtained, and a second bone marrow sample was obtained at 4 or 8 weeks. At baseline, the median MAC load in bone marrow core samples was 3 log10 higher than in blood. Bone marrow MAC loads ranged widely (866-847,315 cfu/g), and no significant correlation was found between MAC load in blood and that in bone marrow core samples. MAC loads in bilateral bone marrow biopsy samples from 7 subjects were highly correlated. MAC loads declined in blood and bone marrow at similar rates during therapy, but blood was sterilized before bone marrow. Length of survival was inversely associated with initial bone marrow core MAC load but not with blood MAC load. Initiation of treatment when tissue MAC load is low may increase the likelihood of favorable clinical outcome.     

Evidence for secretion of human eosinophil granule major basic protein and Charcot-Leyden crystal protein during eosinophil maturation

Prior electron microscopic studies have suggested that immature eosinophils degranulate during normal maturation in human bone marrow. This hypothesis was tested by measuring levels of eosinophil granule major basic protein (MBP) and Charcot-Leyden crystal (CLC) protein in bone marrow sinusoidal blood. MBP and CLC protein levels were elevated initially in bone marrow sinusoidal blood from normal volunteers when compared with peripheral blood, and levels of both proteins decreased during serial sampling; CLC protein levels remained significantly elevated, while MBP levels declined to equal those of peripheral blood. To investigate whether MBP and CLC protein were secreted during eosinophil maturation, bone marrow cells were cultured in soft agar; MBP and CLC protein levels were measured in culture supernatants by RIA. There was a significant positive correlation between eosinophil colony growth and levels of each protein. Electron micrographs of cells in soft agar colonies provided ultrastructural evidence for secretion of granule products by immature eosinophils. These results support prior observations that eosinophil promyelocytes degranulate during maturation.     

Myeloma interacts with the bone marrow microenvironment to induce osteoclastogenesis and is dependent on osteoclast activity

Myeloma tumour growth, except in the most advanced stages of the disease, is restricted to the bone marrow. We used the severe combined immunodeficient-human (SCID-hu) host system, in which primary human myeloma cells grow in, disseminate to and interact with a human microenvironment, to study the interactions between myeloma cells and cells in the bone marrow microenvironment. We used inhibitors of osteoclast activity to determine the role of osteoclasts and their products in supporting myeloma cell growth. Treatment of myelomatous SCID-hu hosts with an inhibitor of osteoclast activity (pamidronate or zoledronate) or with a specific inhibitor of the receptor activator of NF-kappaB ligand (RANKL) halted myeloma-induced bone resorption, when present, and resulted in inhibition of myeloma cell growth and survival. In contrast, myeloma cells from patients with extramedullary disease had a different growth pattern in the SCID-hu hosts and were not inhibited by these interventions, indicating that, while still dependent on a human microenvironment, these cells no longer required the bone marrow microenvironment for survival. This study demonstrates the dependence of myeloma cells on osteoclast activity and their products, and highlights the importance of the myeloma-osteoclast-myeloma loop for sustaining the disease process. Breaking this loop may help control myeloma.     

Reduced serum haptoglobin values in hemophiliacs receiving monoclonally purified factor VIII concentrates

Hemophiliacs often have mild anemia, and hemolysis has been suggested as the likely mechanism on the basis of the reduced serum haptoglobin values frequently observed in these patients. It has been suggested that hypohaptoglobinemia results from isohemagglutinins or other contaminating proteins in the infused factor concentrates. The advent and increased utilization of Factor VIII concentrates that are highly purified by use of monoclonal antibodies have provided the opportunity to study whether proteins other than Factor VIII contained in the concentrate induce hemolysis. Of 49 consecutively studied Factor VIII-deficient hemophiliacs, 19 (39%) had a reduced serum haptoglobin level (less than 27 mg/dl). In particular, 16 of 35 (46%) of patients receiving only monoclonally purified Factor VIII products (Monoclate or Hemofil-M) had a reduced serum haptoglobin value. Haptoglobin measurements were variable on repeat measurement in 8 patients. Haptoglobin levels did not correlate with type or severity of hemophilia, hemoglobin value, or alterations in liver function. Low serum haptoglobin values were also observed in children with leukemia, without apparent hemolysis, who had extensive cutaneous hemorrhage associated with thrombocytopenia. We propose that reduced serum haptoglobin values in hemophiliacs do not result from immune-mediated hemolysis due to contaminating proteins in the concentrate. Moreover, hypohaptoglobinemia may not be due to hemolysis at all but may instead result from dissolution of hematomas and other foci of internal hemorrhage.     

Characterization of erythroid and granulocyte monocyte progenitors in human cord blood

Some characteristics of both erythroid and granulocyte monocyte progenitors in human cord blood were compared to those in adult blood and bone marrow. The number of progenitors in cord blood was higher than that in adult blood and bone marrow. Most colonies in cord blood culture were monocyte-macrophage, whereas those from adult blood were largely eosinophilic. Cord blood progenitors had a slower sedimentation velocity than that reported for marrow, but sedimented faster than that for adult blood. A significant proportion of progenitors in cord blood as well as adult marrow was found to be in the DNA synthetic phase of the cell cycle whereas progenitors in adult blood were not. Cord blood BFU-E were more resistant than adult blood BFU-E but cord blood CFU-GM were not different from adult blood CFU-GM with regard to radiation sensitivity. Cord blood CFU-GM appeared to be more radio-resistant than adult marrow GFU-GM. From these results is seems clear that progenitors in cord blood differ in some aspects from those in adult blood and bone marrow.     

Bacteremia is as unpredictable as clinical manifestations in human brucellosis.

OBJECTIVES: Because of the suboptimal recovery rate of brucellae from blood, it has been proposed that cultures of bone marrow, liver tissue, and lymph nodes may improve the recovery rate of the organism. Data in support of these recommendations are limited and not clearly convincing, especially that of bone marrow culture. The main purpose of this work was to evaluate the roles of blood, bone marrow, liver, and lymph node cultures in the diagnosis of human brucellosis. METHODS: Blood and bone marrow cultures were evaluated in parallel in 103 cases of human brucellosis using Castaneda's biphasic technique. Simultaneous cultures of blood, bone marrow, liver, and lymph node aspirates were also carried out for 13 of these 103 cases. RESULTS: Blood culture identified 47 (45.6%) cases and bone marrow culture identified 85 (82.5%) cases. Faster recovery of Brucella spp was accomplished with the bone marrow culture (2.8+/-0.7 days, p<0.05). When the results of cultures of blood and bone marrow were compared with each other in the 13 cases, it was found that bone marrow specimens could be sterile (six cases (46%)) when bacteremia was present, but Brucella melitensis was detected in liver aspirate in all these six bacteremic cases. CONCLUSIONS: Our data indicate that it is worthwhile practicing bone marrow culture by conventional biphasic technique for the definitive and rapid diagnosis of brucellosis; this is particularly the case in developing countries where diagnostic facilities by advanced technologies such as automated culture systems with PCR are not available. Bone marrow culturing would be a better gold standard in areas where antibiotic pretreatment is common. Also, adopting the practice of culturing liver/lymph node fluids may enhance bacterial isolation and aid in the establishment of a diagnosis of brucellosis in cases for whom blood and bone marrow cultures are negative.     

Reemphasis on Leukocyte Transfusions: Induction of Myeloid Marrow Recovery in Critically ill Neutropenic Children with Cancer

Neutropenia is the major factor predisposing to sepsis in cancer patients, and its duration is important for survival. We administered leukocyte transfusions (LT) to 10 children suffering from documented life-threatening infections during profound, prolonged neutropenia. Nine had acute leukemia, and one had aplastic anemia; three were bone marrow transplant recipients. These 10 were the only patients in our unit who received LT during the past 7 years. The median leukocyte dose was 0.6×10⁹/kg in total. Instead of leukapheresis products, we used pooled buffy coats from random donors, with a high relative content of lymphocytes and especially T lymphocytes. The leukocyte concentrates were irradiated with 15 Gy. In all 10 patients, we observed prompt and sustained myeloid marrow recovery following LT. Such an effect of LT has never been described before. We hypothesize that in the internal milieu of these aplastic patients the transfused leukocytes were stimulated to secrete cytokines, the result being a cascade-like phenomenon and stimulation and proliferation of the patient's own bone marrow cells. The bone marrow-stimulating effect of LT merits further study.     

Prevention of cytomegalovirus infection following bone marrow transplantation: a randomized trial of blood product screening.

From 1983 to 1987, cytomegalovirus seronegative allogeneic bone marrow recipients were randomized to receive screened cytomegalovirus (CMV) seronegative or unscreened blood products and 125 patients were available for analysis. CMV infection occurred in 18% of patients in the screened versus 38% in the unscreened blood product group. However, only two of 64 patients in the screened group and seven of 61 in the unscreened group developed culture or biopsy-proven CMV infections. Bone marrow donor CMV seropositivity was associated with an increased risk of developing CMV infection (21% with seronegative and 46% with seropositive donor), and CMV infection was not prevented by blood product screening if the bone marrow donor was sero = positive (62% for screened, 42% for unscreened group, p = 0.80). One year survival censored for relapse was 52% in the screened group versus 68% in the unscreened group (p = 0.08). Gram negative bacteremia complicated bone marrow transplantation (BMT) in 35% of patients receiving screened and 15% of those receiving unscreened blood products (p = 0.02). Relapse did not differ in the screened and unscreened groups. By multivariate analysis, high risk disease (p = 0.0002), CMV infection (p = 0.004), screened blood products group (p = 0.011), recipient age greater than 17 (p = 0.027), chronic graft-versus-host disease (p = 0.014) and gram negative bacteremia (p = 0.004) independently had a negative influence on survival. We conclude that blood product screening was effective in preventing CMV infections following BMT if both the recipient and bone marrow donor were CMV seronegative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody Production to the Factor in Human Urine Stimulating Colony Formation In Vitro by Bone Marrow Cells

Rabbits immunized with human urine concentrates from leukaemia patients developed antibody which neutralized the mouse bone marrow colony stimulating factor in human urine and human serum. The undiluted antibody partially neutralized the colony stimulating factor in mouse urine and mouse serum and culture medium conditioned by mouse embryo cells.     

Colony Formation by Human Haemopoietic Precursor Cells Cultured in Semi-solid Agar in Diffusion Chambers

A technique which allows colony growth of haematologically normal human bone marrow cells is described. The cells are supported by semi-solid agar-medium inside modified Millipore diffusion chambers implanted in the peritoneal cavity of irradiated mice. After 9 days incubation colonies containing up to 1000 cells are found in these Agar Diffusion Chambers. All haematologically normal patients studied so far produced colonies, the majority with between 10 and 40 colonies per 2 × 10⁵ bone marrow cells inoculated.
This culture system therefore provides a convenient and reliable clonal assay for human bone marrow cells which, in contrast to the agar colony assay in vitro , does not require a source of Colony Stimulating Factor (CSF).     

Adherent Cell Dependent Colony-Stimulating Activity in Human Serum: A Granulopoietic Regulator?

Bacterial infections and trauma which increase production of granulocytes and monocytes by the bone marrow, may do so through factors in serum capable of stimulating growth of granulocyte-macrophage cells in vitro. Human serum possesses two types of colony stimulating activity (CSA), one which stimulates granulopoietic progenitor cells directly, and another which results from the interaction of serum and bone marrow adherent cells (monocyte-macrophages) or peripheral blood leucocytes: adherent cell dependent CSA. These activities are due to different factors which may be separated by gel filtration. Sera of 7 patients undergoing hystectomy who developed post-operative infection showed post-operative elevation of the adherent cell dependent activity in all cases but no change in direct acting CSA. These results suggest that the direct acting CSA in human serum does not represent the principal humoral ‘message’ to the bone marrow from sites of trauma and infection in the tissues and that granulopoiesis may be controlled indirectly by the action of a different humoral factor which increases production of CSA by marrow monocyte-macrophages. Preliminary experiments suggest that lymphocytes stimulated by bacterial products may be one source of this factor.     

Bone marrow cavity: A supportive environment for islet engraftment

An important goal in advancing islet transplantation for the treatment for type 1 diabetes, is to discover transplantation sites that promote long-term islet engraftment. Here, we investigate the bone marrow cavity in rats as a potential site for islet transplantation. Dark agouti streptozotocin diabetic recipients received DA islets to one of three sites: to the renal subcapsular, intrahepatic or bone marrow cavity site. Assessment of graft function was made by measuring blood glucose concentrations using a wireless continuous glucose monitoring system (CGM), performing a glucose tolerance test (GTT), and histological analysis. To determine if bone tissue secretes factors supportive to islet function and survival, human islets were cultured in the presence of osteoblast conditioned medium. Gene expression, insulin secretion and content were assessed in islets after culture. All transplant recipients with islets transplanted to the bone marrow cavity site had reversal of hyperglycemia and remained diabetes free until the end of the experiment at four months. Mean blood glucose concentrations, glucose variability and GTT, using CGM in recipients, yielded similar results between all transplantation sites. Histological assessments at four months after transplantation showed viable islets within the bone marrow space. Incubation of human islets in the presence of osteoblast conditioned medium resulted in positive changes in gene expression, insulin secretion and content. These positive changes were mediated by osteocalcin which was present in the conditioned medium. In summary, islets transplanted to the bone marrow cavity in diabetic rats showed good engraftment. In addition, the bone marrow cavity may provide an environment that is protective against post-transplant cellular stress thus increasing the chances of long-term islet function and survival.     

Bone marrow cavity: a supportive environment for islet engraftment

An important goal in advancing islet transplantation for the treatment for type 1 diabetes, is to discover transplantation sites that promote long-term islet engraftment. Here, we investigate the bone marrow cavity in rats as a potential site for islet transplantation. Dark agouti streptozotocin diabetic recipients received DA islets to one of three sites: to the renal subcapsular, intrahepatic or bone marrow cavity site. Assessment of graft function was made by measuring blood glucose concentrations using a wireless continuous glucose monitoring system (CGM), performing a glucose tolerance test (GTT), and histological analysis. To determine if bone tissue secretes factors supportive to islet function and survival, human islets were cultured in the presence of osteoblast conditioned medium. Gene expression, insulin secretion and content were assessed in islets after culture. All transplant recipients with islets transplanted to the bone marrow cavity site had reversal of hyperglycemia and remained diabetes free until the end of the experiment at four months. Mean blood glucose concentrations, glucose variability and GTT, using CGM in recipients, yielded similar results between all transplantation sites. Histological assessments at four months after transplantation showed viable islets within the bone marrow space. Incubation of human islets in the presence of osteoblast conditioned medium resulted in positive changes in gene expression, insulin secretion and content. These positive changes were mediated by osteocalcin which was present in the conditioned medium. In summary, islets transplanted to the bone marrow cavity in diabetic rats showed good engraftment. In addition, the bone marrow cavity may provide an environment that is protective against post-transplant cellular stress thus increasing the chances of long-term islet function and survival.     

Human leucocyte antigen alloimmunization after bone marrow transplantation: an association with chronic myelogenous leukaemia

Platelet refractoriness due to human leucocyte antigen (HLA) alloimmunization is a significant risk to allogeneic bone marrow transplant recipients. To identify factors contributing to this risk, we reviewed the records of 317 consecutive, paediatric, allogeneic bone marrow transplant recipients at a single institution. The 6-year estimated cumulative incidence of platelet refractoriness due to HLA alloimmunization was 2.6% +/- 0.9%. The incidence among patients with chronic myelogenous leukaemia (CML) 12.5% +/- 5.3% was significantly greater than that of other patients (1.1% +/- 0.6%, P < 0.001). Graft rejection (P = 0.003) and the number of platelet transfusions during the first 90 d after bone marrow transplantation (BMT) (P = 0.0025) were also significantly associated with alloimmunization. The association with CML and with graft rejection was not seen among patients alloimmunized before transplantation. Eight patients developed alloimmunization, of whom three had mismatched grafts and four had unrelated grafts. Alloantibody specificities, identified in seven patients, were unrelated to host or graft major histocompatibility complex (MHC). Host recognition of alloantigens in transfused blood products, not graft-host recognition, therefore seems predominantly responsible for the alloimmunization. These results show that paediatric CML patients have a significantly increased risk of platelet refractoriness due to HLA alloimmunization after BMT. Identifying the mechanism for the increased alloimmunization risk may assist in the development of therapies to prevent platelet refractoriness.