Fleur M. Aung

Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplantation.

Anti-HLA donor-specific Abs (DSAs) have been reported to be associated with graft failure in mismatched hematopoietic stem cell transplantation; however, their role in the development of graft failure in matched unrelated donor (MUD) transplantation remains unclear. We hypothesize that DSAs against a mismatched HLA-DPB1 locus is associated with graft failure in this setting. The presence of anti-HLA Abs before transplantation was determined prospectively in 592 MUD transplantation recipients using mixed-screen beads in a solid-phase fluorescent assay. DSA identification was performed using single-Ag beads containing the corresponding donors HLA-mismatched Ags. Anti-HLA Abs were detected in 116 patients (19.6%), including 20 patients (3.4%) with anti-DPB1 Abs. Overall, graft failure occurred in 19 of 592 patients (3.2%), including 16 of 584 (2.7%) patients without anti-HLA Abs compared with 3 of 8 (37.5%) patients with DSA (P = .0014). In multivariate analysis, DSAs were the only factor highly associated with graft failure (P = .0001; odds ratio = 21.3). Anti-HLA allosensitization was higher overall in women than in men (30.8% vs 12.1%; P < .0001) and higher in women with 1 (P = .008) and 2 or more pregnancies (P = .0003) than in men. We conclude that the presence of anti-DPB1 DSAs is associated with graft failure in MUD hematopoietic stem cell transplantation.

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.

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.

Outcomes of Patients with Myeloid Malignancies Treated with Allogeneic Hematopoietic Stem Cell Transplantation from Matched Unrelated Donors Compared with One Human Leukocyte Antigen Mismatched Related Donors Using HLA Typing at 10 Loci

Most candidates for hematopoietic stem cell transplantation (HSCT) lack a human leukocyte antigen (HLA)-identical sibling donor. Some patients may have a related donor with whom they are mismatched at 1 antigen/allele. It is not known whether such a match is preferable to a matched unrelated donor (MUD). We evaluated the outcomes (survival, relapse, nonrelapse mortality [NRM]) of all 28 patients with a single HLA antigen/allele mismatch identified through high-resolution HLA typing at HLA-A, -B, -C, -DRB1, and -DQB1, and all 318 patients with myeloid malignancies who received transplants from a 10/10 MUD treated during the same period of time at a single institution. Overall, outcomes for patients treated from a 1-antigen/allele mismatch related donor were significantly worse than from a MUD, primarily because of increased NRM. Overall survival (OS) rates at 3 years for 1-antigen/allele mismatched related donor and MUD transplant recipients were 19% and 45% (P = .007), and NRM rates were 40% and 26% (P = .05), respectively. Patients with class I mismatches appeared to have poorer OS than did patients with class II mismatches. A higher incidence of graft rejection was identified in the mismatched related donor group (P = .02). These results indicate that transplant outcomes are better with a MUD than with a 1 antigen/allele-mismatched related donor.

Analysis of the Leprosy Agents Mycobacterium leprae and Mycobacterium lepromatosis in Four Countries

OBJECTIVES To differentiate the leprosy agents Mycobacterium leprae and Mycobacterium lepromatosis and correlate them with geographic distribution and clinicopathologic features. METHODS Species-specific polymerase chain reactions were used to detect each bacillus in archived skin biopsy specimens from patients with leprosy from Brazil (n = 52), Malaysia (n = 31), Myanmar (n = 9), and Uganda (n = 4). Findings were correlated with clinical and pathologic data. RESULTS Etiologic species was detected in 46 of the 52 Brazilian patients, including 36 patients with M leprae, seven with M lepromatosis, and three with both bacilli. The seven patients with sole M lepromatosis all had tuberculoid leprosy, whereas only nine of the 36 patients infected with M leprae exhibited this type, and the rest were lepromatous (P < .001). All patients with dual infections had lepromatous leprosy. Of the nine patients from Myanmar, six were test positive: four with M leprae and two with M lepromatosis. Of the Malaysian and Ugandan patients, only M leprae was detected in 27 of the 31 Malaysians and two of the four Ugandans. CONCLUSIONS The leprosy agents vary in geographic distribution. Finding M lepromatosis in Brazil and Myanmar suggests wide existence of this newly discovered species. The leprosy manifestations likely vary with the etiologic agents.

Lipid profile of platelets and platelet-derived microparticles in ovarian cancer

Background Ovarian cancer patients have a high risk of developing venous thrombosis. The membrane lipid bilayer of platelets and platelet-derived microparticles (PMP) provides a platform for assembly of coagulation proteins and generation of blood clots. Methods We compared the lipid composition of platelets and PMPs in patients with ovarian cancer to those in healthy subjects. We used shotgun lipidomics to quantify 12 classes and 177 species of lipids. Results We found a significant change in 2 classes of lipids in platelets and PMPs isolated from ovarian cancer patients: higher phosphatidylinositol and lower lyso-phosphatidylcholine. The level of 28 species of lipids was also significantly altered in the direction of an increase in the pro-coagulant and a reduction in the anticoagulant lipids. We found that cancer platelets expressed less lipid phosphate phosphatase 1 (LPP1), a key enzyme in phospholipid biosynthesis pathways, than normal platelets. The reduction in LPP1 might contribute to the changes in the lipid profile of cancer platelets. Conclusion Our results support a procoagulant lipid profile of platelets in ovarian cancer patients that can play a role in the increased risk of venous thrombosis in these patients. General significance As far as we are aware, our study is the first study on platelet lipidomics in ovarian cancer. The importance of our findings for the future studies are: 1) a similar change in lipid profile of platelets and PMP may be responsible for hypercoagulability in other cancers, and 2) plasma level of high-risk lipids for venous thrombosis may be useful biomarkers.

Are changes in HLA Ags responsible for leukemia relapse after HLA-matched allogeneic hematopoietic SCT?

Loss of heterozygosity (LOH) has been shown to be associated with leukemia relapse after haploidentical transplantation. Whether such changes are an important cause of relapse after HLA-matched transplantation remains unclear. We retrospectively HLA-typed leukemic blasts for 71 patients with AML/myelodysplastic syndrome obtained from stored samples, and the results were compared with those obtained at diagnosis and/or before the transplant. No LOH or any other changes in HLA Ag were found in any of the samples tested post transplant as compared with pretransplant specimens. One patient had LOH in HLA class I Ag (HLA-A,-B and -C); however, these changes were present in the pretransplant sample indicating that they occurred before the transplant. We concluded that, in contrast with haploidentical transplantation, HLA loss does not have a major role as a mechanism of relapse after allogeneic transplantation with a closely HLA-matched donor.

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.

A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets

Abstract Cyclooxygenase (COX) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids, and has two isoforms, COX1 and COX2, which share ~65% amino acid homology. COX1 is universally expressed in many cell types including platelets; however, expression of COX2 is known to be more limited. We examined expression of COX2 mRNA and protein in platelets and platelet-derived microparticles (MPs); using quantitative RT-PCR, immunostaining, and Western blotting. We have detected a significant amount of COX2 in platelets, both at mRNA and protein levels. We found that COX1/COX2 mRNA and protein ratios in platelets were 370:1 and 17:1, respectively. Expression level of COX2 in platelets was less than COX1, but comparable to the expression of COX2 in malignant epithelial cells. Considering the important role of COX2 in tumorigenesis and thrombosis, and the large number of circulating platelets, we propose that platelet COX2 may play an important role in physiologic and pathologic conditions.

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.