A method for the production of CD4+ chronic myelogenous leukemia-specific allogeneic T lymphocytes.

The graft-versus-leukemia effect is critical to the maintenance of remission in patients transplanted for the treatment of chronic myelogenous leukemia (CML). A pivotal issue in transplantation for CML is whether donor lymphocytes are specific for host tumor or myeloid cells or a subset of the lymphocytes that cause graft-versus-host disease. We have enrolled seven patients in an experimental trial to evaluate the specificity of HLA-matched donor lymphocytes in vitro. We have produced 11 CD4+ cytotoxic and proliferative T-cell clones from five of the donors that only lyse or proliferate to leukemic myeloid cells. These T lymphocytes do not react with interleukin (IL)-2-stimulated blasts, natural killer-sensitive targets, donor neutrophils, or bcr-abl+ EBV-lymphoblastoid cell lines. We show that the addition of the cytokines IL-7 and IL-12 during the production of T-cell clones enhances the recovery of myeloid-specific clones in vitro. Five of the myeloid-specific clones that we produced maintained specificity over 12 weeks in culture. Adoption of this method should allow for the expansion and in vivo testing of CML-specific CD4+ T-cell clones in adoptive immunotherapy.

A paradigm for consensus. The University Hospital Consortium guidelines for the use of albumin, nonprotein colloid, and crystalloid solutions.

OBJECTIVE: To develop contemporary, comprehensive guidelines for the appropriate and efficient use of albumin, nonprotein colloid, and crystalloid solutions. DESIGN: A systematic, literature-based, consensus exercise employing a modified Delphi method. PARTICIPANTS: Thirty-one medical and allied health professionals from 26 University Hospital Consortium (Oak Brook, Ill) member institutions were initially chosen to participate. Participants were selected on the basis of their recognized research in the use of albumin, nonprotein colloid, and crystalloid solutions, and/or experience in the review of appropriateness of such use. A total of 24 participants completed the exercise. MAIN OUTCOME MEASURES: Group responses were statistically analyzed in an iterative consensus development process. Five separate questionnaire rounds were designed to establish criteria for the appropriate use of albumin, nonprotein colloid, and crystalloid solutions. RESULTS: Consensus guidelines were developed outlining the appropriate use of these products for 12 clinical indications, including hemorrhagic shock, nonhemorrhagic (maldistributive) shock, hepatic resection, thermal injury, cerebral ischemia, nutritional intervention, cardiac surgery, hyperbilirubinemia of the newborn, cirrhosis and paracentesis, nephrotic syndrome, organ transplantation, and plasmapheresis. CONCLUSIONS: The Delphi method, a systematic, literature-based consensus process, was shown to be useful in the development of complex clinical practice guidelines for the use of albumin, nonprotein colloid, and crystalloid solutions. It is anticipated that the guidelines will assist health care providers to develop local institutional policies and procedures for the appropriate and efficient use of albumin and albumin alternatives. Institutions reviewing and updating existing local guidelines may use the University Hospital Consortium guidelines as a model for comparison.

Severe hemolytic anemia due to auto anti-N.

Auto anti-N is infrequently encountered and, in most reported cases, does not cause clinical hemolysis. This case reports an auto anti-N associated with severe hemolytic anemia (Hb=2.7 g/dL) in a 6-year-old Caucasian girl with a history of vomiting, fever, and abdominal pain. Upon admission, she was found to have a metabolic acidosis, secondary to her severe anemia, with abnormal liver function tests. As in three other case reports, the autoimmune hemolytic anemia resolved, with disappearance of the auto anti-N, after corticosteroid therapy.

Relationship of hemoglobin level and duration of hospitalization after total hip arthroplasty: implications for the transfusion target.

We analyzed the relationship of hemoglobin level and duration of hospitalization of patients who underwent primary cemented total hip arthroplasty for degenerative joint disease at our institution. Retrospectively, we reviewed the medical records of 332 patients treated during a 16-month period (May 6, 1989, to Aug. 20, 1990). The following variables were analyzed: number of postoperative days to dismissal from the hospital, level of hemoglobin preoperatively and at dismissal, decrease in hemoglobin level from preoperatively to time of dismissal, patient age, surgeon, and blood products transfused. No correlation was found between level of hemoglobin at dismissal, preoperative hemoglobin level, or decrease in hemoglobin concentration from preoperatively to time of dismissal and number of days to dismissal from the hospital. Advanced age was associated with a longer hospital stay. A slight but statistically significant difference was noted in duration of hospitalization among patients operated on by different surgeons. Patients who received both autologous and homologous blood required more transfusion (3.8 units) and had a longer hospital stay (10.7 days) than did patients who received autologous blood only (2.4 units and 9.5 days) or homologous blood only (2.6 units and 10.2 days). We conclude that variation in hemoglobin levels among patients in our study was unrelated to duration of hospitalization. This finding suggests that transfusion of autologous or homologous blood to achieve a higher hemoglobin level (higher transfusion target) solely for shortening hospital stay is unwarranted.

Intraoperative blood loss and patient and graft survival in orthotopic liver transplantation: their relationship to clinical and laboratory data.

We reviewed the records of 83 patients who underwent 100 orthotopic liver transplantations in order to determine the following: (1) the methods to predict blood usage, (2) the consequences of an ABO-incompatible transplant, (3) the benefit of providing cytomegalovirus (CMV)-negative blood products to CMV-negative patients receiving a liver from a CMV-negative donor, (4) the association of donor anti-hepatitis B core antigens and subsequent hepatitis B, and (5) the prognostic consequences of rouleaux observed in pretransplant blood compatibility testing. Patient diagnosis, the presence of ascites, a preoperative prothrombin time greater than 15 seconds, and a multifactorial "risk category" were all predictive of intraoperative blood loss. A history of previous gastrointestinal bleeding or an operation that involved the right upper abdominal quadrant was not predictive of intraoperative blood loss. Although CMV infection is common after liver transplantation, the prophylactic use of CMV antibody-negative blood products in CMV-negative recipients receiving a liver from a CMV-negative donor in our series was not associated with postoperative CMV infection. The transplantation of a liver positive for anti-hepatitis B core antigen was associated with subsequent hepatitis B surface antigen seroconversion in two of four cases. Transplantation of an ABO-incompatible liver and the presence of rouleaux observed in pretransplant blood compatibility testing were both associated with a significantly higher mortality. A careful review of laboratory data and medical records of patients undergoing liver transplantation should enhance the ability to modify the approach to the allocation of limited blood resources and the care and management of these patients.

Blood bank support of a liver transplantation program.

Successful implementation of a liver transplantation program is dependent on extensive blood bank support. Careful planning, organization, and coordination of the blood bank and other clinical services are necessary. In our first 100 orthotopic liver transplantations, our median intraoperative erythrocyte use was 12.6 units, and 30% of the erythrocytes were provided by intraoperative cell salvage. Thus, the need for homologous blood and the number of donors to whom recipients were exposed were reduced. Use of intraoperative cell salvage and expansion of our erythrocyte inventory through the use of AS-1 preservative helped us meet the demands of the liver transplant program without compromising the availability of blood products for all other surgical and medical patients.

Chondroblastoma: an immunohistochemical study.

Five chondroblastomas were examined for the presence of monocyte/macrophage-associated antigens by an alkaline phosphatase-anti-alkaline phosphatase immunohistochemical method. The tumor cell populations were analyzed with eight antibodies reacting with separate antigens on cells of monocyte/macrophage lineage and with antibodies directed against glycoprotein IIIa and factor VIII. The "chondroblastic" tumor cells did not stain with any of the macrophage-associated antibodies. Osteoclast-like giant cells stained with the macrophage-associated antigens EBM11, KB90, leukocyte common antigen, and with the megakaryocyte/platelet-associated antigen C17. Only endothelial cells were reactive with antibody to factor VIII. Our data do not support the postulated histiocytic origin of the neoplastic cells within chondroblastomas; the osteoclast-like giant cells present within these tumors are felt to be both reactive and of monocyte/macrophage lineage.

Angiomyolipoma with regional lymph node involvement and long-term follow-up study.

The first long-term follow-up study of a patient who had renal angiomyolipoma with regional lymph node involvement is reported. The absence of recurrence after 15 years favors the view that regional lymph node involvement represents a multicentric hamartomatous change and is not a metastasis from a malignant renal tumor.

Long-term engraftment failure after marrow ablation and autologous hematopoietic reconstitution: differences between peripheral blood stem cell and bone marrow recipients.

We infused peripheral blood stem cells (PBSC) into 51 patients with various malignant disorders, after myeloablative conditioning. Twenty-four patients also received autologous bone marrow (PBSC + BM). In a multivariate analysis, the only statistically significant predictors of neutrophil engraftment were log-dose CFU-GM (P < 0.001) and the number of prior chemotherapy regimens (P = 0.004). The factors predicting RBC and platelet engraftment were log-dose CFU-GM (P = 0.002), PBSC + BM infusion (P = 0.007) and the absence of neoplastic bone marrow involvement (P = 0.009). Seven patients remained platelet and/or red cell transfusion-dependent for 100 days or more post-transplant after good neutrophil recovery. Six of these seven long-term engraftment failures, as well as five additional patients, received < 10(5) CFU-GM/kg. Of the 11 patients who received < 10(5) CFU-GM/kg (low-dose patients), seven were PBSC recipients, of whom six were long-term engraftment failures. In contrast, there were no long-term engraftment failures among the four low-dose autologous marrow recipients. This difference in long-term engraftment failure rate was significant (P = 0.015). The low-dose PBSC patients all had a diagnosis of lymphoma with bone marrow involvement. The low-dose PBSC + BM group was more heterogeneous, but no patient had malignant involvement of the marrow. The low-dose PBSC patients had also received significantly more prior chemotherapy regimens than the low-dose PBSC + BM patients and a significantly higher proportion received total body irradiation (TBI) as part of their conditioning regimen. We conclude that marrow damage resulting from a combination of neoplastic infiltration, chemotherapy and TBI may result not only in low PBSC yields but also in an impaired capacity of the marrow microenvironment to support transplanted stem cells.

Absence of mononuclear phagocyte antigens in malignant fibrous histiocytoma.

Nine malignant fibrous histiocytomas (MFHs) were tested for the presence of macrophage-associated antigens by an alkaline phosphatase-anti-alkaline phosphatase immunohistochemical method. One myxoid and eight pleomorphic-storiform tumors were analyzed with eight antibodies reacting with separate antigens previously found on cells of mononuclear phagocyte lineage. Malignant cells did not react with any of the antibodies, while benign histiocytes within tumor were consistently labeled with all of the antibodies tested. While the malignant cells of MFH previously have been found to share some functional and morphologic properties with histiocytes, they do not possess the surface and cytoplasmic antigens that have been found on various macrophage populations. The malignant cells of MFH are fibroblastic or poorly differentiated mesenchymal cells rather than neoplastic histiocytes.

Lack of efficacy for conventional gamma irradiation of platelet concentrates to abrogate bacterial growth.

The maximum storage time for platelet concentrates is 5 days, owing to the higher risk bacterial contamination with longer storage. The expiration date could potentially be extended if a rapid system to detect microbial contamination or a safe sterilization technique could be developed and easily implemented. Gamma irradiation has decreased bacterial contamination in food products. Conventional doses of gamma irradiation were tested for their efficacy in decreasing bacterial growth during the 5-day platelet shelf life. An initial pilot study determined that bacteria suspended in normal saline at concentrations of 1 to 2 x 10(7) colony-forming units per milliliter showed a dose-related susceptibility to gamma irradiation. Subsequently, four platelet concentrates were pooled, inoculated with a known concentration of Staphylococcus autreus or Serratia marcescens, and divided. The concentrates were exposed to varying amounts of gamma irradiation, ie, no irradiation (control), 25, 50, and 75 Gy, and subjected to typical blood bank storage conditions. The platelet concentrates were sampled daily for 7 consecutive days to monitor bacterial growth by quantitative cultures. An inverse linear dose-related extinction of bacteria was evident in the pilot study with an extrapolated total kill in the 100 to 150 Gy range. There is no difference in bacterial growth with S aureus using irradiation levels from 0 to 75 Gy. A 1-day delay in bacterial growth at 75 Gy was found with S marcescens compared with units irradiated with 0 through 50 Gy. Exposure of bacteria-contaminated platelet concentrates on storage day zero to gamma irradiation at levels up to 75 Gy is ineffective at sterilizing the platelet concentrates. Higher levels of irradiation may be effective in sterilizing platelet concentrates. Function, survival, and sterility after higher than conventional levels of irradiation need further study.

Accurate counting of low numbers of leukocytes. Use of flow cytometry and a manual low-count chamber.

The leukocyte depletion that current filters make possible in erythrocyte and platelet preparations can result in leukocyte concentrations too low to be counted accurately by automated and standard manual methods. A recently described method for counting low numbers (1 to 10 x 10(6) per L) of leukocytes by flow cytometry and the use of a manual low-count chamber on 25 venous samples serially diluted to 1:1,000 were evaluated. The results show that both methods for counting leukocytes can reliably determine counts of 1 to 10 x 10(6) leukocytes/liter (a three-order of magnitude reduction from venous blood) in terms of order of magnitude but lack accuracy for specific measurement. The flow cytometric method is more expensive, less readily available, and suffers from greater sample variability. Use of a low-count chamber is a superior technique to evaluate and maintain quality control of methods for leukocyte depletion, resulting in a final leukocyte concentration of 1 to 10 cells per microliter.

Rejuvenation of erythrocytes preserved with AS-1 and AS-3.

Rejuvenation of erythrocytes (biochemical modification of adenosine triphosphate and 2,3-diphosphoglycerate) preserved with currently available nutrient additive solutions has not been licensed or adequately studied. The ability of the commercially available solution PIPA to modify erythrocytes preserved with AS-1 or AS-3 for 35 and 42 days was studied. Use of PIPA produced greater-than-normal levels of adenosine triphosphate in the preserved erythrocytes. Restoration of 2,3-diphosphoglycerate was suboptimal at both periods. (PIPA treatment produces normal levels of 2,3-diphosphoglycerate in erythrocytes preserved for 35 days with citrate-phosphate-dextrose-adenine-1.) In erythrocytes preserved with AS-1 or AS-3 for 42 days, 2,3-diphosphoglycerate returned to normal levels after a second PIPA rejuvenation. This study demonstrates that biochemical modification is possible in erythrocytes preserved with AS-1 or AS-3 for 42 days.

MUC1 expression in hematopoietic tissues.

The MUC1 gene encodes the core protein of episialin, which is recognized by several antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) detection of MUC1 transcripts has been proposed for the detection of micrometastases from breast cancers. MUC1 expression in hematopoietic tissues has been reported but not confirmed. Our preliminary RT-PCR studies confirmed MUC1 expression by MDA-231 breast cancer cells. Western blots of MDA-231 proteins stained with anti-MUC1 core gave one 68-kd (core protein) band, with an additional high molecular weight (HMW) band in blots stained with anti-epithelial membrane antigen (EMA). MUC1 expression was detectable by RT-PCR in 4 samples each of peripheral blood, bone marrow, and lymph node. MUC1 expression was detectable by Western blot analysis using anti-MUC1 core and anti-EMA in 2 peripheral blood samples and all bone marrow samples. Western blots from all lymph node samples stained positively with anti-EMA for the HMW product, but the 68-kd product was less prominent. Separated peripheral blood lymphocytes and granulocytes showed similar levels of MUC1 expression. RT-PCR studies demonstrated MUC1 expression in various hematopoietic cell lines. Western blots showed the 68-kd and HMW products in a granulopoietic line, with only the 68-kd product in 3 lymphoblastoid lines. MUC1 is expressed ubiquitously in hematopoietic tissues and is unsuitable for use as a marker for epithelial micrometastases.

Delayed hemolysis resulting from anti-A1 after liver transplantation.

The authors report a case of a delayed hemolytic transfusion reaction in a type A2 recipient of a type O liver allograft. An anti-A1 antibody reactive at 37 degrees C in the indirect antiglobulin test was identified both in the patient's serum and in an eluate. Hemolysis secondary to the production of anti-A1 has been reported to be extremely rare. The English-language literature has six cases before 1979. During the 1980s, an additional seven cases were reported; six involved transplantation of a solid organ, and in each such case the donor was type O and the recipient was type A1 or A2. Recommendations regarding transfusion practice in such cases are made. An association among the transplantation of a type O organ to a type A recipient, the use of cyclosporine, and an apparent increase in the occurrence of clinically significant anti-A1 is suggested.

Blood volume determination as a function of hematocrit and mass in three preservative solutions and saline.

Accurate blood volume determination is useful both clinically and in research. In many instances, however, direct measurement of blood volume is impractical due to the risk of bacterial contamination. For this reason, mass is often used to estimate volume. The relationship between mass and volume (density) varies with different suspension solutions and hematocrits. In this paper, equations are derived to calculate volume as a function of hematocrit and mass for pooled red cells suspended in four solutions: CPD plasma (whole blood), additive solutions 1 and 3 (AS-1 and AS-3), and saline. To validate this approach, the actual versus predicted blood volumes in 10 individual blood samples suspended in either AS-1 or saline are compared. The equations predict the volume of blood to within 0.5% and 1.0% in samples with low/normal and high hematocrits (15% to 85%, respectively). Use of these equations allow for accurate and rapid conversion of mass to volume for these blood products.

Correction of bone marrow nucleated cell counts for the presence of fat particles.

Although the use of bone marrow transplantation has increased greatly in recent years, the quality control procedures used in bone marrow processing laboratories remain less than ideal. Accurate marrow total nucleated cell (TNC) counts are essential for effective monitoring of bone marrow collection and processing. Aspirated marrow is variably contaminated by fat particles, resulting in overestimation of marrow TNC by automated analyzers. A recently-marketed hematological analyzer (Cobas-Helios; Roche Diagnostic Systems, Branchburg, NJ) offers the potential to correct marrow TNC counts for fat particles using available software. The authors investigated the accuracy of corrected TNC counts on 21 marrow samples, using a visual chamber count as the reference method. The correction methods studied were software correction, using the Cobas-Helios differential system, and replacement of the sample plasma with saline. Uncorrected automated marrow TNC counts (mean, 28.4 x 10(9)/L) were significantly higher than the visual reference counts (mean, 23.1 x 10(9)/L). Neither the mean corrected automated count (24.3 x 10(9)/L) nor the mean saline replaced count (24.6 x 10(9)/L) differed significantly from the mean visual reference count. For both the corrected automated and saline replaced counts, 20 of the 21 data points (95%) fell within a 95% confidence interval computed for the reference method. The authors conclude that both the corrected automated method, using the Cobas-Helios, and the saline replacement method are acceptable alternatives to the visual chamber count.

Transfusion-transmitted bacterial infection.

Transfusion-transmitted bacterial infection is a persistent but often underemphasized problem facing transfusion medicine today. The present status of bacterial contamination of red cells, platelet products and plasma, frequency of contamination, and types of organisms implicated is reviewed. Current methods of prevention and detection are discussed, as well as typical clinical presentations and therapy.

Apheresis. New opportunities.

Recent advances in apheresis have emerged in a variety of clinical settings. Improvements in granulocyte and peripheral blood stem cell collection and mobilization, the changing role of intravenous gamma globulin, the importance of patient assessment and selection of replacement fluids, and the progress in both affinity apheresis and photopheresis are presented. These current trends, their limitation, and their promise in treating a diversity of diseases inspire the future of this technology.

Autologous blood transfusion.

Autologous blood transfusion is an endorsed blood conservation strategy that has become widely practiced in elective surgical procedures. We review ten years' experience in this arena, along with emerging strategies designed to continue to minimize allogeneic blood exposure but reduce the costs associated with autologous blood procurement. We conclude that point-of-care autologous blood procurement (acute normovolemic hemodilution and intraoperative autologous blood salvage) can replace the predonation of autologous blood in surgical patients when transfusion medicine specialists, anesthesiologists, and surgeons develop a prospective, comprehensive approach to blood conservation.