Fecal blood loss: A quantitative method of evaluating hemostasis in patients with thrombocytopenia.

BACKGROUND: The purpose of our studies was to determine if fecal blood loss can provide a quantitative measure of bleeding at platelet counts of 20 000/µL or less in patients with hypoproliferative thrombocytopenia and to document the effects of different prophylactic platelet transfusion triggers on fecal blood loss. METHODS AND MATERIALS: Patients had an aliquot of their autologous red blood cells (RBCs) labeled with 51 Cr. Following reinjection of their radiolabeled RBCs, all feces and a daily blood sample were collected to determine fecal blood loss per day. Three different studies were performed in patients with thrombocytopenia: The first was in patients with thrombocytopenia with aplastic anemia who were not receiving platelet transfusions, and the other two trials involved thrombocytopenic patients with cancer who were receiving prophylactic platelet transfusions at platelet transfusion triggers of 5000/µL, 10 000/µL, or 20 000/µL. RESULTS: In patients with thrombocytopenia not receiving platelet transfusions, fecal blood loss does not increase substantially until platelet counts are 5000/µL or less. When platelet transfusions are given prophylactically to patients with cancer with chemotherapy-induced thrombocytopenia at platelet counts of 5000/µL or less, fecal blood loss and red cell transfusion requirements are the same as those for patients transfused prophylactically at higher transfusion triggers of 10 000 platelets/µL or 20 000 platelets/µL. However, the total number of platelet transfusions needed increases significantly, and the duration of the patient's thrombocytopenia tends to be longer at the higher platelet transfusion thresholds. CONCLUSION: A prophylactic platelet transfusion threshold of 5000/µL or greater is sufficient to maintain hemostasis in patients with thrombocytopenia.

Transfusion-related immunomodulation: gamma irradiation alters the effects of leukoreduction on alloimmunization.

BACKGROUND: Adverse events following blood transfusion include allosensitization and generalized immunosuppression, collectively referred to as transfusion-related immune modulation. We evaluated the immunological effects of red blood cell (RBC) and platelet transfusions on alloantibody responses and on immunoregulatory cells in nonimmunosuppressed patients undergoing cardiovascular surgery. STUDY DESIGN AND METHODS: Patients were randomized to receive standard unmodified (STD), leukoreduced (LR), or leukoreduced and γ-irradiated (LRγ) RBCs. Patients received only apheresis platelets that were in-process LR and were γ-irradiated for the third arm. Nontransfused patients served as controls for the effects of surgery itself on immunologic changes. Antibodies to HLA were assessed with use of solid-phase assays. The effects of transfusion on adaptive and innate immunity were evaluated by assessing T regulatory cells (Tregs) and invariant natural killer T (iNKT) cells. RESULTS: LR of blood products reduced the development of human leukocyte antigen (HLA) alloantibodies, but only in patients without preexisting HLA antibodies. However, if LR blood products were γ-irradiated, HLA antibody production was not reduced. Compared to nontransfused patients, recipients of STD or LR transfusions showed a significant increase in CD4+CD25hi T cells expressing FoxP3 or CTLA4 and also of iNKT cells producing interleukin-4. In contrast, recipients of LRγ blood products showed markedly lower increases in all three cellular assays. CONCLUSION: LR decreased HLA alloantibody production in naïve recipients, but did not reduce the immunosuppressive effects of transfusion. LRγ reduced immunosuppression and was not associated with decreased HLA alloantibody production.

Leukofiltration plus pathogen reduction prevents alloimmune platelet refractoriness in a dog transfusion model.

Human lymphocyte antigen alloimmunization to filter leukoreduced (F-LR) platelets occurs in about 18% of immunosuppressed thrombocytopenic hematology/oncology patients and represents a significant challenge for effective chemotherapy. In a dog platelet transfusion model, we have evaluated other methods of preventing alloimmune platelet refractoriness and demonstrated that successful methods in our dog model are transferable to man. In the present study, donor/recipient pairs were dog lymphocyte antigen DR-B incompatible (88% of the pairs), and recipient dogs received up to 8 weekly treated transfusions from a single donor (a highly immunogenic stimulus), or until platelet refractoriness. Continued acceptance of F-LR platelets occurred in 6 of 13 recipients (46%), but neither γ-irradiation (γ-I; 0 of 5) nor Mirasol pathogen reduction (MPR; 1 of 7) treatment of donor platelets prevented alloimmune platelet refractoriness. Combining γ-I with F-LR was associated with only 2 of 10 (20%) recipients accepting the transfused platelets. Surprisingly, F-LR platelets that then underwent MPR were accepted by 21 of 22 (95%) recipients (P < .001 vs F-LR + γ-I recipients). Furthermore, 7 of 21 (33%) of these accepting recipients demonstrated specific tolerance to 8 more weekly donor transfusions that had not been treated. In addition, platelet concentrates prepared from F-LR + MPR whole blood were also nonimmunogenic; that is, 10 of 10 (100%) recipients accepted donor platelets. Overall, 31 of 32 (97%) recipients accepted F-LR + MPR platelets; none developed antibodies to donor lymphocytes. These data are the highest rate of acceptance for platelet transfusions reported in either animals or man. This approach to platelet transfusion may be particularly important when supporting patients with intact immune systems, such as in myelodysplastic syndromes.

Ultraviolet B irradiation in the prevention of alloimmunization in a dog platelet transfusion model.

BACKGROUND: Alloimmune platelet (PLT) refractoriness remains a significant problem for chronically transfused patients with thrombocytopenia. STUDY DESIGN AND METHODS: In a dog PLT transfusion model, we evaluated ultraviolet B irradiation (UV-B) of donor PLTs-either alone or in combination with centrifuge leukoreduction (C-LR) or filtration leukoreduction (F-LR)-to prevent refractoriness to donor PLTs and to induce tolerance to standard (STD) PLTs from the same donor or to tertiary donors. RESULTS: Recipient acceptance rates for C-LR donor PLT transfusions were 14%, F-LR were 33%, and UV-B irradiated were 45% with no significant differences among the treatments given to the donor's PLTs. Adding UV-B irradiation to C-LR or F-LR PLTs increased acceptance rates to 50 and 68% (p = 0.02 and p = 0.05), respectively, comparing single treatments to the combined treatments. After a recipient had accepted any type of UV-B-treated donor PLTs, specific tolerance to subsequent transfusions of the same donor's STD PLTs averaged 65%. Nonspecific tolerance to third-party donor's STD PLTs averaged 36% if they had accepted their initial donor's treated PLTs but was only 4% (p < 0.001) if they had rejected these PLTs. CONCLUSION: Combining UV-B irradiation with a method of leukoreduction produces additive effects on prevention of alloimmune PLT refractoriness.

Quantitative Influence of ABO Blood Groups on Factor VIII and Its Ratio to von Willebrand Factor, Novel Observations from an ARIC Study of 11,673 Subjects.

ABO blood groups are known to influence the plasma level of von Willebrand factor (VWF), but little is known about the relationship between ABO and coagulation factor VIII (FVIII). We analyzed the influence of ABO genotypes on VWF antigen, FVIII activity, and their quantitative relationship in 11,673 participants in the Atherosclerosis Risk in Communities (ARIC) study. VWF, FVIII, and FVIII/VWF levels varied significantly among O, A (A1 and A2), B and AB subjects, and the extent of which varied between Americans of European (EA) and African (AA) descent. We validated a strong influence of ABO blood type on VWF levels (15.2%), but also detected a direct ABO influence on FVIII activity (0.6%) and FVIII/VWF ratio (3.8%) after adjustment for VWF. We determined that FVIII activity changed 0.54% for every 1% change in VWF antigen level. This VWF-FVIII relationship differed between subjects with O and B blood types in EA, AA, and in male, but not female subjects. Variations in FVIII activity were primarily detected at low VWF levels. These new quantitative influences on VWF, FVIII and the FVIII/VWF ratio help understand how ABO genotypes differentially influence VWF, FVIII and their ratio, particularly in racial and gender specific manners.

Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets.

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at -65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events.

Extended storage of buffy coat platelet concentrates in plasma or a platelet additive solution.

BACKGROUND: Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22 °C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria. STUDY DESIGN AND METHODS: Normal subjects donated whole blood that was stored at 22 °C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays. RESULTS: BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals. CONCLUSION: BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.

Exploratory studies of extended storage of apheresis platelets in a platelet additive solution (PAS).

To evaluate the poststorage viability of apheresis platelets stored for up to 18 days in 80% platelet additive solution (PAS)/20% plasma, 117 healthy subjects donated platelets using the Haemonetics MCS+, COBE Spectra (Spectra), or Trima Accel (Trima) systems. Control platelets from the same subjects were compared with their stored test PAS platelets by radiolabeling their stored and control platelets with either (51)chromium or (111)indium. Trima platelets met Food and Drug Administration poststorage platelet viability criteria for only 7 days vs almost 13 days for Haemonetics platelets; ie, platelet recoveries after these storage times averaged 44 ± 3% vs 49 ± 3% and survivals were 5.4 ± 0.3 vs 4.6 ± 0.3 days, respectively. The differences in storage duration are likely related to both the collection system and the storage bag. The Spectra and Trima platelets were hyperconcentrated during collection, and PAS was added, whereas the Haemonetics platelets were elutriated with PAS, which may have resulted in less collection injury. When Spectra and Trima platelets were stored in Haemonetics' bags, poststorage viability was significantly improved. Platelet viability is better maintained in vitro than in vivo, allowing substantial increases in platelet storage times. However, implementation will require resolution of potential bacterial overgrowth during storage.

Signal transducer and activator of transcription 3 (STAT3) regulates collagen-induced platelet aggregation independently of its transcription factor activity.

BACKGROUND: Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. METHODS AND RESULTS: The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide-induced but not thrombin receptor-activating peptide- or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. CONCLUSIONS: These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.

Platelet concentrates prepared after a 20- to 24-hour hold of the whole blood at 22°C.

BACKGROUND: The Food and Drug Administration (FDA) requires that red blood cells must be refrigerated within 8 hours of whole blood collection. Longer storage of whole blood at 22°C before component preparation would have many advantages. STUDY DESIGN AND METHODS: Two methods of holding whole blood for 20 to 24 hours at room temperature were evaluated, refrigerated plates or a 23°C incubator. After extended whole blood storage, platelet (PLT) concentrates were prepared from PLT-rich plasma on Day 1 postdonation, and the PLTs were stored for 6 more days. On Day 7 of PLT storage, blood was drawn from each subject to prepare fresh PLTs. The stored and fresh PLTs were radiolabeled and transfused into their donor. RESULTS: Eleven subjects' whole blood was stored using refrigerated butanediol plates (Compocool, Fresenius), and 10 using an incubator. Poststorage PLT recoveries averaged 47 ± 13% versus 53 ± 11% and survivals averaged 4.6 ± 1.7 days versus 4.7 ± 0.9 days for Compocool versus incubator storage, respectively (p = NS). With all results, poststorage PLT recoveries averaged 75 ± 10% of fresh and survivals 57 ± 13% of fresh; PLT recoveries met FDA guidelines for poststorage PLT viability but not survivals. CONCLUSION: Seven-day poststorage PLT viability is comparable when whole blood is stored for 22 ± 2 hours at 22°C using either refrigerated plates or an incubator to maintain temperature before preparing PLT concentrates.

Suppression of the immune response to FVIII in hemophilia A mice by transgene modified tolerogenic dendritic cells.

Current methods for eradicating clinically significant inhibitory antibodies to human factor VIII (hFVIII) in patients with hemophilia A rely on repeated delivery of high doses of factor concentrates for a minimum of many months. We hypothesize that tolerance can be induced more efficiently and reliably through hFVIII antigen presentation by tolerogenic dendritic cells (tDCs). In this study, we generated tDCs from hemophilia A mice and modified them with a foamy virus vector expressing a bioengineered hFVIII transgene. Naive and preimmunized mice infused with hFVIII expressing tDCs showed suppression of the T cell and inhibitor responses to recombinant hFVIII (rhFVIII). Treatment with hFVIII expressing tDCs was also associated with a higher percentage of splenocytes demonstrating a regulatory T cell phenotype in immunized mice. Furthermore, CD4(+) T cells harvested from recipients of hFVIII expression vector-modified tDCs were able to mediate antigen-specific immune suppression in naive secondary recipients. We also demonstrated a trend for improved suppression of inhibitor formation by coexpressing interleukin-10 (IL-10) and hFVIII from a bicistronic vector. These preclinical results demonstrate the potential for employing vector modified ex vivo generated tDCs to treat high titer inhibitors in patients with hemophilia A.

Extended storage of platelet-rich plasma-prepared platelet concentrates in plasma or Plasmalyte.

BACKGROUND: Using bacterial detection or pathogen reduction, extended platelet (PLT) storage may be licensed if PLT viability is maintained. The Food and Drug Administration (FDA)'s poststorage PLT acceptance guidelines are that autologous stored PLT recoveries and survivals should be 66 and 58% or greater, respectively, of each donor's fresh PLT data. STUDY DESIGN AND METHODS: Nonleukoreduced PLT concentrates were prepared from whole blood donations. Autologous PLT concentrates from 62 subjects were stored in 100% plasma (n=44) or 20% plasma/80% Plasmalyte (n=18), an acetate-based, non-glucose-containing crystalloid solution previously used for PLT storage. Fresh PLTs were obtained on the day the donor's stored PLTs were to be transfused. The fresh and stored PLTs were alternately radiolabeled with either (51) chromium or (111) indium, and in vitro measurements were performed on the stored PLTs. RESULTS: The FDA's PLT recovery criteria were met for 7 days of plasma storage, but PLT survivals maintained viability for only 6 days. Plasmalyte-stored PLTs did not meet either acceptance criteria after 6 days of storage. After 7 days of storage, PLT recoveries averaged 43±4 and 30±4% and survivals 4.1±0.4 and 2.0±0.2 days for plasma- and Plasmalyte-stored PLTs, respectively (p=0.03 for recoveries and p<0.001 for survivals). Poststorage PLT recoveries correlated with the commonly used in vitro PLT quality measurements of hypotonic shock response and annexin V binding, while survivals correlated with extent of shape change, morphology score, and pH. CONCLUSION: There is a progressive decrease in recoveries and survivals of plasma-stored PLTs over time. PLT viability is better maintained in plasma than Plasmalyte.

Suppression of FVIII inhibitor formation in hemophilic mice by delivery of transgene modified apoptotic fibroblasts.

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20-40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.

Effects of pretransfusion warming of platelets to 35 degrees C on posttransfusion platelet viability.

BACKGROUND: Three of four prior studies suggested that warming platelets (PLTs) to 37 degrees C before transfusion into patients with thrombocytopenia gave improved corrected PLT count increments. STUDY DESIGN AND METHODS: Eighteen normal subjects had apheresis PLTs collected that were stored at 22 degrees C for 5 days in two storage bags. One bag of PLTs was warmed to 35 degrees C before infusion, and one remained at 22 degrees C. Three different methods of warming the donor's autologous PLTs before reinfusion were evaluated: warming PLTs to 35 degrees C for 10 or 60 minutes followed by radiolabeling or radiolabeling the PLTs followed by warming to 35 degrees C for 60 minutes. In the first two methods, the warmed PLTs would have returned to 22 degrees C before infusion, and in the third, the PLTs would still be warm when injected. The paired test and control PLTs were radiolabeled with either (111)In or (51)Cr to determine posttransfusion PLT recoveries and survivals. PLT morphology score, pH, hypotonic shock response, extent of shape change, and annexin V binding were determined just before transfusion. RESULTS: There were no differences in posttransfusion autologous radiolabeled PLT recoveries and survivals or in the in vitro measurements for the PLTs maintained at 22 degrees C versus those warmed to 35 degrees C using any of the three methods of PLT warming before infusion. CONCLUSION: Based on these 5-day-stored autologous radiolabeled PLT recovery and survival measurements, there is no evidence that warming PLTs to 35 degrees C before infusion improves postinfusion PLT viability.

Viability and function of 8-day-stored apheresis platelets.

BACKGROUND: Methods of bacterial detection and pathogen inactivation of platelets (PLTs) may allow extended storage of PLTs as long as PLT quality is maintained. STUDY DESIGN AND METHODS: Twenty normal volunteers had their PLTs collected with an apheresis machine (Haemonetics Corp.). A variety of in vitro PLT function and metabolic assays were performed both on Day 0 and after 8 days of storage. On Day 8, a small blood sample was drawn from each donor to obtain fresh PLTs. The fresh and stored autologous PLTs were labeled with either (51)Cr or (111)In, and the radiolabeled PLTs were transfused. Posttransfusion serial blood samples were drawn to determine the relative posttransfusion recoveries and survivals of the fresh versus the stored PLTs. RESULTS: Although the in vitro assays showed some differences between the two trial sites, the results were generally within the ranges expected for fresh and stored PLTs. Overall, PLT recoveries averaged 66 +/- 16 percent versus 53 +/- 20 percent and survivals averaged 8.5 +/- 1.6 days versus 5.6 +/- 1.6 days, respectively, for fresh compared to 8-day-stored PLTs. There were no significant differences in the in vivo PLT data between the trial sites or based on the radiolabel used for the measurements. CONCLUSION: After 8 days of storage, the in vivo posttransfusion recovery and survival of autologous Haemonetics apheresis PLTs meet the proposed standards for poststorage PLT quality.

Evaluation of different methods of leukoreduction of donor platelets to prevent alloimmune platelet refractoriness and induce tolerance in a canine transfusion model.

The effectiveness of different methods of leukoreduction in preventing alloimmune platelet refractoriness was evaluated in a canine model. Platelets from a random donor dog were administered for up to 8 weeks or until platelet refractoriness. Standard (STD; unmodified) platelets were accepted by 14% of recipients (n = 7) compared with 14% for centrifuge leukoreduced (C-LR) platelets (n = 21) and 31% for filter leukoreduced (F-LR) platelets (n = 13; no significant differences). Surprisingly, using both F-LR and C-LR platelets was highly effective (87% acceptance, n = 15). Transfusing F-LR/C-LR red blood cells (n = 4) or F-LR/C-LR plasma (n = 4), along with F-LR/C-LR platelets, did not affect platelet acceptance (100% acceptance). Overall acceptance of F-LR/C-LR platelets was 91% (n = 23; P < or = .05 versus STD, C-LR, or F-LR platelets). F-LR/C-LR transfusions also induced tolerance to subsequent STD platelet transfusions from the same donor (82% acceptance, n = 19) as well as to donor skin grafts without recipient immunosuppression (57% acceptance, n = 7). To evaluate mechanisms of tolerance induction, F-LR/C-LR platelets were gamma-irradiated. Although the gamma-irradiated F-LR/C-LR platelets were uniformly accepted (n = 6), tolerance to STD platelets was lost. These data suggest that some allostimulatory white cells are filter adherent, whereas others escape filtration but can be removed by centrifugation and tolerance requires a residual functioning white cell.