Technical Note: Use of a Novel Bacterial Culture Sampling Device for Pooled Whole Blood Platelets.

OBJECTIVE: Transfusion associated sepsis is a serious risk after platelet transfusion. Although platelet culture can be performed to avoid such risk, culture results are often available after transfusion due to the 4-hour shelf-life after pooling. To decrease such risk, we implemented a needleless closed system device to culture for contamination before pooling and release for transfusion. METHODS: We customized a needleless device to permit sterile sampling of whole blood platelets without retrograde or cross-contamination. Then aliquots of platelets were injected into culture media for detection of aerobic organisms and cultured for 24 hours but released for transfusion after 12 hours of negative culture. RESULTS: In a period of two years, we used this device in 5,741 whole blood derived pooled platelets and only 24 units tested positive (0.4%) but none of initial positive was later confirmed. There were 11 Staphylococcus and 9 Bacillus species identified. All but one of the positive units were discarded; there was no clinical impact in the patient who received the positive unit. CONCLUSION: This device allows for sampling of whole blood derived platelets before pooling, warranting a transfusion of a culture negative unit after 12 hours of negative culture, consequently reducing transfusion of bacterially contaminated whole blood derived pooled platelets.

A portable system for processing donated whole blood into high quality components without centrifugation.

BACKGROUND: The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of 'whole blood' into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor. METHODS AND FINDINGS: In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2-3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01). CONCLUSION: This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation-particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.

Cancer type predicts alloimmunization following RhD-incompatible RBC transfusions.

BACKGROUND: Immunosuppressed, RhD-negative oncology patients tend to have lower rates of sensitization to the D antigen when they receive transfusion with RhD-positive blood components. Clinical factors associated with alloimmunization to the D antigen in RhD-negative oncology patients when they receive transfusion with RhD-positive red blood cells (RBCs) have not been well defined. STUDY DESIGN AND METHODS: This was a 4-year, retrospective analysis identifying RhD-negative oncology patients who received RhD-positive RBCs and were not previously alloimmunized to the D antigen. Age, sex, race, ABO group, primary oncology diagnosis, and numbers of RhD-incompatible RBC transfusions were recorded. The association between antibody formation and clinical factors was studied. The incidence of alloanti-D was calculated from a subsequent antibody-detection test performed at least 28 days after receipt of the first transfusion of RhD-positive RBCs. RESULTS: In total, 545 RhD-negative oncology patients received 4295 RhD-positive RBC transfusions. Of these, 76 (14%) became alloimmunized to the D antigen. Diagnosis type was the only factor significantly associated with responder status. The logistic regression model indicated that patients who had myelodysplastic syndrome or solid malignancies were more likely to be responders than those who had acute leukemia. CONCLUSION: We measured a 14% sensitization rate to the D antigen in our RhD-negative oncology population. The rate of alloimmunization was higher in patients who had solid cancers (22.6%) or myelodysplastic syndrome (23%) compared with those who had other hematologic malignancies (7%). Knowledge of diagnoses that predispose to RhD alloimmunization enables better utilization of RhD-negative RBCs during times of shortage.

Pure Red Cell Aplasia in Major ABO-Mismatched Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with Severe Pancytopenia.

In major ABO-mismatched allogeneic hematopoietic stem cell transplantation (HSCT) persistence of antidonor isohemagglutinins leads to pure red cell aplasia (PRCA). To investigate severe pancytopenia noted in a previous study of PRCA, we analyzed all major ABO-mismatched HSCT between January 2003 and December 2012. Of 83 PRCA patients, 13 (16%) had severe pancytopenia. Severe pancytopenia was defined as an absolute neutrophil count (ANC) < 1.5 K/µL or requiring granulocyte colony-stimulating factor, platelets < 50 K/µL or transfusion dependent, and PRCA with RBC transfusion dependence at post-transplant day 90. In 6 patients (46%) severe pancytopenia resolved after PRCA resolution. Two patients (15%) received a second transplant because of persistent pancytopenia/secondary graft failure, 1 (8%) died from secondary graft failure despite a stem cell boost, 1 (8%) did not recover his platelet counts despite RBC/ANC recovery, and 3 patients (23%) died from disease relapse. We found that severe pancytopenia is frequently associated with PRCA in 16% of major ABO-incompatible HSCT with a higher incidence in males and pancytopenia resolved with resolution of PRCA in 46% of patients.

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation.

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

Septic transfusion reactions during blood transfusion via indwelling central venous catheters.

BACKGROUND: Transfusion of blood products requires a vascular port. Use of an indwelling central venous catheter (CVC) provides this port readily and safely in general; however, potential risks require assessment. STUDY DESIGN AND METHODS: The objective was to examine septic reactions to blood transfusions performed via CVCs owing to subclinical microbial catheter colonization. All transfusion reactions that occurred from 2007 to 2011 at The University of Texas MD Anderson Cancer Center were analyzed and correlated with microbiology culture results. Data on the reactions, including vascular access via a catheter or peripheral venipuncture, were collected prospectively. RESULTS: A total of 999 reactions were reported, with an incidence of two per 1000 transfusion events. A total of 738 reactions occurred in 642 patients during transfusion through a CVC. Among them, 606 reactions occurred in patients that had cultures of blood samples drawn within 7 days before or after reaction. Sixty of these (9.9%) had at least one significant microorganism isolated from their catheters and/or peripheral blood. The blood culture results and timing suggested that these patients likely had catheter-related bloodstream infections caused by transfusion through a CVC with subclinical microbial colonization. Fever and chills occurred in 35 of these patients (58%), which resembled febrile nonhemolytic transfusion reactions. Culture results of the transfused blood products, although not performed in all cases, were mostly negative in these CVC-related reactions. CONCLUSION: Blood transfusion through an indwelling CVC may lead to septic reaction owing to subclinical microbial colonization. This risk should be considered before transfusion and during investigation of transfusion reactions.

Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia.

BACKGROUND: The authors developed an ex vivo methodology to perform drug library screening against human leukemia. METHODS: The strategy for this screening relied on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. Several advantages were observed: 1) partial recapitulation of the leukemia microenvironment, 2) use of native hemoglobin oxygenation as a real-time sensor/reporter, 3) cost-effectiveness, 4) species specificity, and 5) a format that enables high-throughput screening. RESULTS: For a proof of concept, a chemical library (size, approximately 20,000 compounds) was screened against human leukemia cells. Seventy compounds were identified ("hit" rate, 0.35%; Z-factor = 0.71) that had activity, and 20 compounds were examined to identify 18 true-positive compounds (90%). Finally, the results demonstrated that carbonohydraxonic diamide group-containing compounds are potent antileukemia agents that induce cell death in leukemia cells and in patient-derived samples. CONCLUSIONS: The current results indicated that this unique functional assay can identify novel drug candidates and can help with the development of future applications in personalized drug selection for patients with leukemia.

Granulocyte concentrates from a single high-yield apheresis can be split to support multiple patients.

Allogeneic granulocyte transfusion has evolved into a viable therapeutic option for immunocompromised severely neutropenic leukemic patients and those with hematopoietic stem cell transplant with life-threatening bacterial and fungal infections. The collection of larger cell doses of granulocyte concentrates (GCs) has been facilitated by the stimulation of donors with granulocyte colony stimulating factor (G-CSF) and dexamethasone. The synergistic effect of G-CSF and dexamethasone has allowed the collection of larger cell doses of GCs and its use has increased steadily. This has allowed us to split the high-yield GC products and facilitated distribution of the split GC products to a second or third patient who needs GCs but lacks donors. The main objective of this article was to present our rationale for splitting GC products and how the split GC units were transfused to multiple patients. We believe that split GCs are as equally effective as unsplit GCs and that multiple patients benefit from splitting GCs.

Incidence and natural history of pure red cell aplasia in major ABO-mismatched haematopoietic cell transplantation.

Major ABO mismatching is not considered a contraindication to allogeneic haematopoietic stem cell transplantation (HSCT). Modern reduced-intensity conditioning and reduced-toxicity regimens cause much less myeloablation than conventional myeloablative regimens, such as cyclophosphamide with busulfan or total body irradiation, which may affect the incidence of pure red cell aplasia (PRCA). We estimated the incidence and described the natural history of PRCA in patients with major ABO-mismatched donor stem cells. Between 2007 and 2008, 161 (27% of all patients undergoing HSCT) underwent allogeneic HSCT with major ABO-mismatched stem cells and 12 (7·5%) of these patients developed PRCA. Thirty and ninety day T-cell and myeloid cell chimerism and neutrophil and platelet engraftment did not differ between patients who developed PRCA and those who did not. The only risk factor associated with PRCA was the use of a fludarabine/busulfan conditioning regimen. All patients with PRCA needed red cell transfusion for several months after HSCT resulting in significant iron overload. Pure red cell aplasia resolved spontaneously in the majority (seven patients) but only resolved after stopping tacrolimus in three patients. Hence, after major ABO-mismatched HSCT, the incidence of PRCA was 7·5% and it resolved spontaneously or after withdrawal of immunosuppression in the majority of patients.

Targeting neuropilin-1 in human leukemia and lymphoma.

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/Tyr-Any-Leu-Arg-Ser (F(F)/(Y)XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence (D)(KLAKLAK)2. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for ligand-directed therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-(D)(KLAKLAK)2 is a promising drug candidate in this setting.

Results of HLA antibody testing using ELISA vs the fluorescent bead method and retrospective review of data for recipients of packed RBCs and platelets from male HLA-immunized donors.

We reviewed HLA antibody testing results using an enzyme-linked immunosorbent assay (ELISA) for all male blood donors at our institution during a 3.5-month period to look for HLA immunization. Confirmatory testing of 33 blood samples positive for HLA class I and/or II antibodies was performed using the fluorescent bead method. A retrospective review of recipients of packed RBCs and platelets processed from these 33 HLA-immunized male donors were conducted to identify transfusion-related acute lung injury and cognate antigens. The agreement rates between the methods for HLA class I and II antibodies were 21% (7/33) and 6% (2/33), respectively. We noted HLA antibodies in the male donors corresponding to cognate antigens in 2 recipients of packed RBCs and in 3 recipients of platelets. Of 8 donors positive for HLA antibodies, 5 did not have a history of blood transfusion. We conclude that ELISA was too sensitive and had a high false-positive rate for the detection of HLA class II antibodies.

Impact of safety concerns and regulatory changes on the usage of erythropoiesis-stimulating agents and RBC transfusions.

PURPOSE: Safety concerns raised in the recent oncology trials with erythropoiesis-stimulating agents (ESAs) have led to regulatory restrictions on their use. We wished to determine the impact of these changes on the use of ESAs and RBC transfusions. METHODS: In a retrospective observational study of patients treated at a comprehensive cancer center in 2006-2008, data on all ESA doses dispensed, RBCs transfused, and hemoglobin levels on the days of transfusions and ESA initiations were analyzed. RESULTS: Compared with 2006, the total patients treated was 14% higher (28,339 versus 24,806) in 2007 and 22% higher (30,254) in 2008. Patients receiving ESAs decreased by 26% and 61%, and ESA units dispensed decreased by 29% (from 30,206 units to 21,409 units) and 80% (6,102 units) in 2007 and 2008, respectively. However, RBC transfusions increased by only 2% (from 38,218 units to 38,948 units) in 2007 and by 8% (41,438) in 2008. The mean hemoglobin on the day of transfusion was the same for each year (8.4 g/dl); however, an increasing proportion of patients initiated ESAs at lower hemoglobin (< 10 g/dl) levels. After adjusting for demographics and diagnostic variables for 3 years (n = 83,399), a multivariate logistic regression showed a significant decline in ESA use (p < .0001) without an increase in RBC transfusions. CONCLUSIONS: Recent ESA safety concerns and regulatory restrictions have significantly decreased ESA use. The lack of a significant impact on transfusions may be related to a lower hemoglobin threshold used to initiate ESAs or treatment of patients less likely to respond.

Impact on patient outcome following transfusion of bacterially contaminated platelets: the M.D. Anderson Cancer Center experience.

This study examined the clinical outcome of every patient who received a bacterially contaminated unit of platelets at The University of Texas M.D. Anderson Cancer Center, Houston, during 2007. Samples of platelets were aerobically cultured and read for 1 day at 35 degrees C. Positive bottles were subcultured in the appropriate media. The effect of independent variables in the clinical outcome of patients infused with bacterially contaminated platelet units was analyzed. A total of 23,199 platelet units were transfused, 71 of which were bacterially contaminated units; 8 were apheresis platelets and 63 were whole blood platelets. Of the 71 units, 70 were contaminated with gram-positive bacteria and 1 with gram-negative bacteria. Only 1 patient developed fever, and coagulase-negative staphylococci were isolated from the transfused unit. Transfusion of fresh units and antibiotic therapy possibly explain the lack of clinical consequences in our patients.

Two-surgeon technique of parenchymal transection contributes to reduced transfusion rate in patients undergoing major hepatectomy: analysis of 1,557 consecutive liver resections.

BACKGROUND: Blood transfusions are an independent risk factor for adverse outcomes after hepatectomy. In-hospital transfusions are still reported in one third of patients in major series. Data on factors affecting blood transfusions in large series of liver resection are limited. The aim of this study was to evaluate factors predictive of blood transfusion in hepatectomies performed at a tertiary referral center. METHODS: Records of 1,477 patients who underwent 1,557 liver resections between 1998 and 2007 were reviewed. Multivariate analysis of risk factors for red cell transfusion was performed. RESULTS: Median intra-operative blood loss was 250 cc, and 30-day peri-operative red cell transfusion rate was 27%. On multivariate analysis, factors that significantly predicted increased red cell transfusion rates were female sex, pre-operative hematocrit<30%, platelet count<100,000/mm3, simultaneous resection of other organs, major hepatic resection, use of the Pringle maneuver, and tumors>10 cm. Parenchymal transection technique was an independent risk factor for perioperative red cell transfusion; the usage of the 2-surgeon technique (combined saline-linked cautery and ultrasonic dissection) was associated with a lower transfusion rate than other techniques, including ultrasonic dissection alone, finger fracture, and stapling (P<.001). CONCLUSION: Although most factors that affect the red cell transfusion rate for liver resection are patient- or tumor-related, the parenchymal transection technique is under the surgeon's control. The decrease in transfusion rate associated with the use of the 2-surgeon technique emphasizes the important role of the hepatobiliary surgeon in determining outcomes after liver resection.

In vivo efficacy of shipped HLA-matched platelets.

BACKGROUND: Platelet (PLT) concentrates are currently stored in an incubator at 20 to 24 degrees C with continuous gentle agitation. PLTs are routinely shipped for transfusion to thrombocytopenic patients, however. There is a concern that PLT concentrates may be adversely affected during the shipping process. CASE REPORT: A 40-year-old woman with severe aplastic anemia and immune refractory to unselected PLT transfusions was transferred to a distant medical center for a hematopoietic peripheral blood progenitor cell transplant where she continued to receive HLA-matched PLTs from her dedicated donors. Sixteen such components were collected and air-shipped in insulated boxes to the transplant center. Thirty-seven plateletpheresis components from the same dedicated donors had been transfused to the patient before transfer. Corrected count increments (CCIs) at the two sites were compared, with assessment of the role of HLA-match grades. The mean interruption time of controlled agitation during shipment was approximately 10.5 hours. The mean CCI of all distant transfusions was 14,450 +/- 9700 PLTs per microL x m2 per 10(11) and that of local transfusions was 10730 +/- 4870. The mean donor-paired difference between CCIs at the two sites was 1140 +/- 9940. At the remote location no clinically significant bleeding occurred and one posttransfusion febrile reaction was noted. CONCLUSION: Despite the study limitations, the effectiveness, in a single patient, of leukoreduced, irradiated apheresis PLTs shipped by lengthy combined surface and airline transport is reported, as measured by posttransfusion CCIs.

Recombinant interferon gamma1b immune enhancement in 20 patients with hematologic malignancies and systemic opportunistic infections treated with donor granulocyte transfusions.

BACKGROUND: The response to antifungal therapy alone often is suboptimal in patients with cancer who have therapy-refractory neutropenia, and even donor-derived granulocyte transfusions (GTX) are not always successful. The authors evaluated the safety and efficacy of immune enhancement using recombinant interferon gamma1b (rIFN-gamma1b) in patients with cancer who received GTX for refractory, systemic, opportunistic infections. METHODS: Twenty recipients of high-dose donor GTX ( approximately 5.5 x 10(10) neutrophils per transfusion) who had received concurrent rIFN-gamma1b between October 2001 and December 2004 were evaluated retrospectively. RESULTS: The median age (+/- standard deviation [SD]) was 45 +/- 17 years. Ten patients (50%) were men, 17 patients (85%) had leukemia, and 3 patients (15%) had myelodysplastic syndrome. The median +/- SD Acute Physiology and Chronic Health Evaluation II score was 15 +/- 4 (range, 7-22). Most patients (n = 18 patients; 90%) had recurrent or refractory cancer. In 6 patients (30%) who received allogeneic hematopoietic stem cell transplantation, GTX plus rIFN-gamma1b was given a median +/- SD of 26 +/- 100 days (range, 12-372 days) after transplantation. Seventeen patients (85%) had neutropenia during GTX therapy. Five patients (25%) had possible invasive fungal infection, 3 patients (15%) had probable invasive fungal infection, and 11 patients (55%) had proven invasive fungal infection. One patient (5%) had refractory Pseudomonas aeruginosa sepsis. Eight patients (40%) received corticosteroids during GTX plus rIFN-gamma1b therapy. Patients received a median +/- SD of 8 +/- 7 GTX doses (range, 4-28 doses) and 9 +/- 7 rIFN-gamma1b doses (range, 1-28 doses), for a mean +/- SD cumulative dose (CD) of 400 +/- 2621 microg. Other concomitant cytokines were granulocyte-colony stimulating factor (12 +/- 3 doses; CD, 6720 +/- 4721 microg) in 15 patients (75%) and granulocyte-macrophage-colony stimulating factor (12 +/- 9 doses; CD, 4750 +/- 4410 microg) in 14 patients (70%). Four patients (20%) developed fever, and 2 patients (10%) developed skin rashes. Reversible liver dysfunction (n = 3 patients; 15%) and tachycardia (n = 1 patients; 5%) were considered rIFN-gamma1b-associated adverse reactions; whereas, in 1 patient (5%), transient dyspnea was attributed to GTX. Four weeks after therapy started, 9 patients (45%) had complete or partial resolution of infection; and, in another 3 patients (15%), the invasive fungal infection had become stable. CONCLUSIONS: The current results indicated that no serious adverse events were associated with rIFN-gamma1b immune enhancement in patients with systemic opportunistic infections who received donor GTX therapy.

Thirty-day typing and screening for patients undergoing elective surgery: experience at a large cancer center.

BACKGROUND: Preoperative testing in patients scheduled to undergo surgery often includes determining the ABO group and Rh type and screening for atypical alloantibodies in blood samples. AABB recommends obtaining blood samples within 3 days of transfusion. This was extended to 30 days to minimize the number of phlebotomies, avoid delays in providing blood during surgery, and decrease the laboratory workload. This study was conducted to show that extending the expiration date of the preoperative blood sample for blood typing and screening to 30 days will serve our purpose and provide better patient care. STUDY DESIGN AND METHODS: Data were collected for all patients undergoing elective surgery with perioperative blood samples submitted to our blood bank over 31 months. Each patient completed a questionnaire to determine whether his or her samples qualified for a 30-day preoperative clot. The questionnaires were validated upon preoperative screening. A transfusion medicine physician made the final determination regarding whether the samples qualified for 30-day typing and screening. These blood samples were used for cross-matching to find compatible blood during surgery. RESULTS: A total of 12,310 preoperative blood samples were received with a request for typing and screening, 4,370 (35.5%) of which qualified for a 30-day expiration date. No significant problems were encountered with these blood samples. CONCLUSION: Extension of the preoperative clot expiration date from 3 to 30 days has improved service to our patients and their physicians and indirectly reduced the laboratory workload.