Commentary on 50 years of research at the NBRL, Boston, Massachusetts.

Research at the NBRL, Boston, MA over the past 50 years assessed the survival and function of RBC and platelets and the function of plasma clotting proteins. Liquid preserved RBC can be stored at 4 °C for only 2 weeks to maintain a 24-hour posttransfusion survival value of 75%, moderately impaired oxygen transport function after transfusion, exert a hemostatic effect, and increase the plasma volume. Liquid preserved platelets can be stored at room temperature with agitation for only 2 days to have acceptable in vivo survival 2 hours following transfusion, normal lifespan, and a hemostatic effect to reduce the bleeding time in thrombocytopenic patients. RBCs frozen with 40% W/V glycerol at -80 °C for at least 10 years, thawed, and deglycerolized in the Haemonetics ACP215 can be stored in Nutricel at 4 °C for 2 weeks with a 24-hour posttransfusion survival of 75%, moderately impaired oxygen transport function after transfusion, exert a hemostatic effect and increase the plasma volume. Leukoreduced single donor platelets treated with 6% DMSO, the supernatant DMSO removed prior to freezing at -80 °C for 2 years, thawed, and diluted with 0.9% NaCl or AB plasma have a bimodal population of platelets: one population has reduced in vivo survival, but increased hemostatic effect and the other has normal in vivo survival. AB plasma can be stored at -80 °C for at least 14 years, thawed, and stored at 4 °C for 24 hours with acceptable in vitro function of clotting proteins. The data reported by the NBRL, Boston, Mass. over the past 50 years and the 15-year experience by the Netherlands military now recommend that FDA, ARC, HHS and DOD should support the use of universal donor frozen group O Rh positive and group O Rh negative RBC, frozen group O platelets and frozen AB plasma from male donors. The frozen blood products will eliminate the severe adverse events of mortality and morbidity associated with the current FDA approved red blood cell products, platelet products, and plasma.

Role of nitric oxide in the prevention of severe adverse events associated with blood products.

The reduction in vitro of nitric oxide binding to the globin portion of hemoglobin (SNOHb) in fresh and liquid preserved red blood cells has been reported to be responsible for the severe adverse events (SAEs) associated with red blood cell transfusion. No in vivo data were reported that the reduction in SNOHb in red blood cells following transfusion was irreversible. In addition, no clinical data were reported that the reduction in SNOHb in red blood cells produced severe adverse events (SAEs) in recipients.

Exploring the feasibility of selective depletion of T lymphocyte subsets by whole blood immunoadsorption cytapheresis.

Normal turnover of T lymphocytes is slow relative to other blood cells. Consequently, the physical removal of circulating leucocytes by thoracic duct drainage, repeated leukapheresis or blood filtration results in T cell depletion and immunosuppression. However, clinical use of such procedures is impractical compared with immunosuppressive drugs or radiation. None the less, immunosuppression by physical depletion of T cells, avoiding the systemic toxicities of drugs and radiation, might have clinical advantages if immunophenotypically distinct T cell subsets could be depleted selectively. Recent advances in targeted plasma protein apheresis using adsorbent macrobead columns prompted us to determine whether analogous techniques might permit CD4+ T lymphocytes to be removed selectively from whole blood. To explore this possibility, we linked murine anti-human-CD4 and isotype-identical control monoclonal antibodies (mAbs) to agarose, polyacrylamide and polystyrene macrobeads (150-350 microm) and then evaluated the selectivity, specificity and efficiency of macrobead columns to remove CD4+ T cells from anti-coagulated whole blood at varying mAb densities and flow rates. We also examined saturation kinetics and Fc-oriention versus random coupling of mAbs to macrobeads. Sepharose 6MB macrobead (250-350 microm) columns proved to be most effective, selectively removing up to 98% of CD4+ T cells from whole blood. Moreover, depletion efficiency and selectivity were retained when these columns were reused after elution of adherent CD4+ cells. These studies indicate that selective depletion of T lymphocyte subsets by whole blood immunoadsorption apheresis using mAb-linked macrobead columns may be feasible on a clinical scale. It is possible that such apheresis techniques could achieve targeted forms of immunosuppression not possible with drugs or radiation.

Effects of centrifugal and roller pumps on survival of autologous red cells in cardiopulmonary bypass surgery.

BACKGROUND: Either a roller pump or a centrifugal pump can be used in the extracorporeal circuit during surgery with cardiopulmonary bypass. In this study, we assessed the effect of these two pumps on the 24-h post-transfusion survival values of autologous red blood cells (RBC). STUDY DESIGN AND METHODS: Fourteen male patients subjected to extracorporeal bypass procedures were studied. In seven patients, the autologous red cells were collected following the cardiopulmonary bypass procedure using the roller pump, and in seven patients, autologous red cells were collected following the cardiopulmonary procedure using the centrifugal pump. The 24-h post-transfusion survival values of the autologous RBC were measured using the 51 disodium chromate/99m technetium double isotope procedure. The effects of the extracorporeal bypass procedures using the roller pump and the centrifugal pump were also assessed by the measurements of hematocrit, platelet count, plasma hemoglobin, and serum lactate dehydrogenase levels. RESULTS: The 51 disodium chromate 24-h post-transfusion survival values of the autologous RBC were similar whether the roller pump or the centrifugal pump was used in the extracorporeal circulation, as were the hematocrit, platelet count, plasma hemoglobin and serum lactate dehydrogenase levels. CONCLUSION: The 24-h post-transfusion survival values of autologous RBC, measured by the 51 disodium chromate/99m technetium double isotope procedure, were not significantly different, whether the roller pump or the centrifugal pump was used in the extracorporeal circuit using membrane oxygenators during cardiopulmonary surgical procedures.

Expired liquid preserved platelet releasates retain proliferative activity.

BACKGROUND: Donated platelets for clinical use currently have a shelf life of 5 days as the result of possible bacterial contamination and loss of hemostatic function. Platelet releasates contain multiple growth factors that have been shown to accelerate wound healing. We sought to demonstrate that although expired platelets can no longer sustain hemostasis, they serve a longer term role as a reservoir of growth factors that could be harnessed in wound healing applications. MATERIALS AND METHODS: Liquid preserved human platelets were activated from 1 to 21 days after collection using zeolite and were then analyzed for their ability to stimulate human fibroblast proliferation, which is an in vitro serogate of growth factor activity and wound healing potential. Total protein content, the concentration of platelet-derived growth factor (PDGF) and transforming growth factor-beta were also measured. RESULTS: Activated liquid preserved platelet releasates significantly stimulated fibroblast proliferation. Twenty-one-day-old platelets were as stimulatory as 2-day-old platelets. Total protein concentration, PDGF, and transforming growth factor-beta concentrations remained constant throughout the 21-day course. Western blot analysis using an antibody against human PDGF revealed minimal protein degradation over time. CONCLUSIONS: These data demonstrate that although the role of platelets as hemostatic agents degrades rapidly with time, platelets' ability to serve as a reservoir for growth factors remains intact for at least 3 weeks. These growth factors could be collected, stored, and used as a topical agent to promote healing of chronic and recalcitrant wounds.

Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effect of agonists, pH, and plasma and saline resuspension.

BACKGROUND: Some of the tests used to assess the quality of fresh and preserved platelets (PLTs) include PLT number, PLT morphology, pH of the PLT medium, PLT response to hypotonic stress, and PLT aggregation to agonists. This study was performed to assess the function of fresh and preserved PLTs by their response to aggregation and their production of thromboxane A2 after in vitro stimulation with agonists. STUDY DESIGN AND METHODS: PLTs isolated by apheresis procedures were stored at 22 degrees C for as long as 5 days and then frozen with 6 percent dimethyl sulfoxide, stored at -80 degrees C, thawed, washed, and resuspended in medium. The effects of agonists and the pH and composition of the medium on PLT aggregation and PLT production of thromboxane A2 after stimulation were measured. RESULTS: The agonists and the pH and composition of the medium affected both the aggregation response and the production of thromboxane A2 by the fresh and preserved PLTs. PLT aggregation response to arachidonic acid (AA) and adenosine diphosphate (ADP) was significantly lower in the cryopreserved PLTs than in the fresh and preserved PLTs. After stimulation with AA and ADP, the cryopreserved PLTs produced more thromboxane than did the fresh and liquid-preserved PLTs. CONCLUSIONS: The agonists and the pH and composition of the medium affected the response to aggregate and produce thromboxane in vitro in both the fresh and the liquid-preserved PLTs. PLT thromboxane A2 production may be a better in vitro test than PLT aggregation to assess PLT function in vivo.

Survival of baboon biotin-X-N-hydroxysuccinimide and (111)In-oxine-labelled autologous fresh and lyophilized reconstituted platelets.

BACKGROUND AND OBJECTIVES: In accordance with Food and Drug Administration (FDA) regulations, platelets can be stored in the liquid state at 22 degrees C for only 5 days. Platelets frozen with 6% dimethylsulphoxide (DMSO) can be stored at -80 degrees C for 2 years, and platelets frozen with 5% DMSO can be stored at -150 degrees C for 3 years. Studies are being conducted to determine the effects of lyophilization of platelets. In the present study, we assessed the survival of autologous lyophilized-reconstituted platelets in the baboon. MATERIALS AND METHODS: We studied fresh baboon platelets and baboon platelets that had been treated with paraformaldehyde, frozen, lyophilized, thawed and reconstituted. Aliquots of these platelets were labelled with (111)In-oxine or biotin-X-N-hydroxysuccinimide (biotin-X-NHS) before autotransfusion, and measurements were made of the in vivo recovery and lifespan. We also evaluated the response of fresh and lyophilized platelets to in vitro agonists by measuring the level of platelet surface markers and heterotypic aggregates in the peripheral blood following the autotransfusions. RESULTS: The (111)In-oxine- or biotin-X-NHS-labelled lyophilized, reconstituted platelets exhibited survival times of less than 15 min. These platelets did not respond to stimulation with agonists to decrease platelet GPIb and increase platelet P-selectin and platelet GPIIb-IIIa levels 1 min post-transfusion and they accumulated more procoagulant factor V than did the fresh platelets. CONCLUSIONS: Lyophilized reconstituted baboon platelets labelled with (111)In-oxine or biotin-X-NHS before autotransfusion exhibited an in vivo circulation time of less than 15 min. Further study of the lyophilized, reconstituted platelets is required to evaluate their haemostatic function.

In vitro testing of fresh and lyophilized reconstituted human and baboon platelets.

BACKGROUND: Studies have been performed on human fresh, liquid-preserved, and cryopreserved platelets (PLTs) to assess PLT-adhesive surface receptors, PLT membrane procoagulant activity, PLT aggregation, and thromboxane production. Lyophilization has been developed as a method to preserve PLTs. This study was performed to evaluate these measurements on human and baboon fresh and lyophilized reconstituted PLTs. STUDY DESIGN AND METHODS: In both human and baboon fresh and lyophilized PLTs, aggregation response and PLT production of thromboxane A2 were measured after stimulation, and PLT surface markers P-selectin, glycoprotein (GP) Ib, GPIIb-IIIa, and factor (F) V were measured before and after stimulation. RESULTS: Fresh PLTs responded to the dual agonists arachidonic acid and adenosine diphosphate (ADP) to aggregate and produce thromboxane A2, and in both the PLT surface markers P-selectin and GPIIb-IIIa increased and GPIb decreased after stimulation. Neither human nor baboon lyophilized reconstituted PLTs aggregated to dual agonists, and neither produced thromboxane A2, increased PLT surface markers P-selectin or GPIIb-IIIa, or decreased PLT GPIb after stimulation. Nevertheless, after recalcification the lyophilized reconstituted PLTs accumulated FV to a significantly greater degree than fresh PLTs. CONCLUSIONS: Lyophilized reconstituted PLTs exhibited modification of the PLT membrane that interfered with aggregation and thromboxane production, prevented increases in PLT P-selectin and GPIIb-IIIa and decreases in GPIb after stimulation, and increased FV accumulation after recalcification. The in vitro data suggest that lyophilized PLTs may have reduced in vivo survival. In vivo studies are needed to determine the survival and function of lyophilized PLTs.

Restoration of red blood cell volume following 2-unit red blood cell apheresis.

BACKGROUND AND OBJECTIVES: Blood donors who weigh at least 130 lbs (59 kg) and have a haematocrit of at least 40 V per cent can donate 2 units of blood, from which a 360-ml volume of red blood cells (RBC) can be isolated. This study was carried out in seven healthy male blood donors to assess the restoration of the RBC volume 1 month following a 2-unit RBC apheresis procedure. MATERIALS AND METHODS: RBC volumes were measured prior to donation and 4 weeks after the 2-unit RBC apheresis procedure without oral iron supplementation. RESULTS: Four weeks after the removal of 2 units of RBC from the male donors not supplemented with oral iron, the RBC volume was restored to 92% of the precollection value. The 360-ml volume of RBC collected represented 12-19% of the donor's original RBC volume. CONCLUSIONS: Male donors can safely donate 2 units of RBC and will restore a mean of 92% of their RBC volume within 1 month without iron supplementation.

Circulation and distribution of autotransfused fresh, liquid-preserved and cryopreserved baboon platelets.

BACKGROUND AND OBJECTIVES: Studies were carried out in five healthy male baboons to determine the 111indium oxine (111In-oxine) survival of autologous fresh, liquid-preserved and cryopreserved platelets. Simultaneous organ-distribution studies were performed to determine the percentage uptake of platelets by the spleen and/or liver. MATERIALS AND METHODS: Each of five baboons was transfused, on three different occasions, with autologous fresh platelets stored at 22 degrees C for 18 h, liquid-preserved platelets stored at 22 degrees C for 5 days and washed previously frozen platelets, labelled with 111In-oxine. RESULTS: In vivo recovery at 2 h was 81% for the fresh platelets, 54% for the previously frozen platelets and 44% for the 5-day-old liquid-preserved platelets. The weighted mean life span was 5.4 days for fresh platelets, 4.2 days for previously frozen platelets and 2 days for liquid preserved platelets. Increased radioactivity was detected over the liver 2 h after transfusion for both the previously frozen and liquid-preserved platelets. CONCLUSIONS: Cryopreserved platelets and liquid-preserved platelets stored at 22 degrees C for 5 days had reduced survival 2 h post-transfusion and reduced life span values compared to fresh platelets. In addition, the finding of increased radioactivity over the liver in the baboons that received cryopreserved and liquid-preserved platelets suggested that the liver was the site for removal of the non-viable platelets.

Induction of platelet formation from megakaryocytoid cells by nitric oxide.

Although the growth factors that regulate megakaryocytopoiesis are well known, the molecular determinants of platelet formation from mature megakaryocytes remain poorly understood. Morphological changes in megakaryocytes associated with platelet formation and removal of senescent megakaryocytes are suggestive of an apoptotic process. Previously, we have established that nitric oxide (NO) can induce apoptosis in megakaryocytoid cell lines. To determine whether there is an association between NO-induced apoptosis and platelet production, we exposed Meg-01 cells to S-nitrosoglutathione (GSNO) with or without thrombopoeitin (TPO) pretreatment and used flow cytometry and electron microscopy to assess platelet-sized particle formation. Meg-01 cells treated with TPO alone produced few platelet-sized particles (<3% of total counts), whereas treatment with GSNO alone produced a significant percentage of platelet-sized particles (22 +/- 4% of total counts); when combined with TPO pretreatment, however, GSNO led to a marked increase in platelet-sized particle production (48 +/- 3% of total counts). Electron microscopy confirmed that Meg-01 cells treated with TPO and GSNO yielded platelet-sized particles with morphological features specific for platelet forms. The platelet-sized particle population appears to be functional, because addition of calcium, fibrinogen, and thrombin receptor-activating peptide led to aggregation. These results demonstrate that NO facilitates platelet production, thereby establishing the essential role of NO in megakaryocyte development and thrombopoiesis.

Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS+ for the preparation of platelet gel.

BACKGROUND AND OBJECTIVES: We compared three methods of isolating platelet-rich plasma (PRP) using the Haemonetics Cell Saver 5 and one method of isolating PRP by plateletpheresis using the Haemonetics MCS+. PRP contains both platelets and fibrinogen, which are used in the preparation of haemostatic agents. MATERIALS AND METHODS: When the Haemonetics Cell Saver 5 was used, 500 ml of blood from each of 30 normal volunteer donors was collected into 70 ml of citrate-phosphate-dextrose (CPD) anticoagulant. In a further 14 normal volunteers, the Haemonetics MCS+ was used to isolate PRP by plateletpheresis using an acid citrate dextrose (ACD) to blood ratio of 1 : 9. In a separate study, CPD-anticoagulated whole blood from another 30 volunteers was used for measurement of fibrinogen levels in the plasma and cryoprecipitate. RESULTS: A larger volume of PRP can be collected using the Haemonetics Cell Saver 5 than by using the Haemonetics MCS+. The platelet concentration and the total number of platelets were higher in the PRP isolated using the Haemonetics MCS+ than in the PRP isolated by the three methods used with the Haemonetics Cell Saver 5, with differences in platelet concentration and PRP volume among the four methods. The mean fibrinogen level in the plasma was 253 mg % +/- 47 (SD) and in the cryoprecipitate was 1085 mg % +/- 304 (SD). CONCLUSIONS: The most appropriate method of PRP isolation for preparation of platelet gel is dependent upon the specific surgical procedure to be undertaken and the patient's needs.

Survival and function of baboon RBCs released from clotted blood and washed before autologous transfusion.

BACKGROUND: One alternative to an allogeneic transfusion is the salvaging of the patient's own shed blood. In this study, baboon blood was allowed to clot and the RBCs that were released from the clotted blood lysed with and without urokinase were washed before autologous transfusion. STUDY DESIGN AND METHODS: Forty-four studies were done in 13 baboons (Papio cynocephalus or Papio anubis) over a 3-year period. In 24 studies, a 50-mL volume of blood was collected without an anticoagulant and stored at 22 degrees C for as long as 72 hours before washing and autologous transfusion. In 20 other studies, a 50-mL volume of blood was collected without an anticoagulant and allowed to clot for 30 to 60 minutes. Urokinase, ranging from 2,500 to 10,000 units per mL, was added, and the blood was stored at 22 degrees C for 24 hours before washing and autologous transfusion. RESULTS: RBCs that were stored at 22 degrees C without urokinase for 24 hours exhibited an in vitro recovery value of 45 percent, a (51)Cr 24-hour posttransfusion survival of 86 percent, and an index of therapeutic effectiveness of 39 percent. The (51)Cr T(50) value was normal at 14 days, and RBC oxygen-transport function was slightly reduced. RBCs that were stored at 22 degrees C for 24 hours with 10,000 units per mL of urokinase exhibited an in vitro recovery value of 89 percent, a (51)Cr 24-hour posttransfusion survival value of 86 percent, and an index of therapeutic effectiveness of 76 percent. The (51)Cr T(50) value was normal at 14 days, and the RBC oxygen-transport function was only slightly reduced. CONCLUSION: Autologous baboon RBCs isolated from clotted blood treated or not treated with urokinase and washed before transfusion have excellent survival and normal or only slightly reduced oxygen-transport function.

Survival, function, and hemolysis of shed red blood cells processed as nonwashed blood and washed red blood cells.

BACKGROUND: Shed nonwashed blood and shed washed red blood cells (RBC) are being used as alternatives to allogeneic liquid-preserved RBC for patients during thoracic and cardiovascular surgical procedures. METHODS: Mongrel dogs were bled a volume of blood into the abdominal cavity and the shed blood was reinfused as nonwashed blood or washed RBC. The 51Cr RBC volumes were measured before, immediately after, and 24 hours after the exchange transfusion to assess the recovery of the shed RBC and the 24-hour posttransfusion survival. Compatible dogs were given allogeneic transfusions of 51Cr-labeled nonwashed blood and washed RBC, and 24-hour posttransfusion survival and half-life were measured. RESULTS: Immediately after the 100% exchange transfusion, the recovery value was 62% for the nonwashed shed blood and 82% for the washed RBC. Both the nonwashed blood and the washed RBC had 24-hour posttransfusion survival values of 90% and normal oxygen transport function after the exchange transfusion. Compatible allogeneic 51Cr-labeled nonwashed blood and washed RBC had normal 24-hour posttranfusion survival and 51Cr half-life values. CONCLUSIONS: The survival, function, and hemolysis of shed nonwashed blood and shed washed RBC were similar to fresh blood in the dog that underwent a 100% exchange transfusion.

Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction.

BACKGROUND: Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. METHODS AND RESULTS: Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (n=10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (n=9) had more circulating monocyte-platelet aggregates (34.2+/-10.3% [mean+/-SEM]) than patients with no AMI (n=84, 19.3+/-1.4%, P<0.05) and normal control subjects (n=10, 11.5+/-0.8%, P<0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI. CONCLUSIONS: As demonstrated by 3 independent means (in vivo tracking of activated platelets in baboons, human coronary intervention, and human AMI), circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.

Anemia-induced increase in the bleeding time: implications for treatment of nonsurgical blood loss.

BACKGROUND: Preoperative bleeding time (BT) does not correlate with postoperative bleeding in patients subjected to surgical procedures. A significant positive correlation has been reported between the BT 2 hours after cardiopulmonary bypass surgery and the nonsurgical blood loss during the first 4 hours after bypass surgery. This study was done to investigate the effect of Hct and platelet count on the BT measurement in normal, healthy men and women. STUDY DESIGN AND METHODS: To assess the relative effect of RBCs and platelets on the BT, 22 healthy male and 7 healthy female volunteers were subjected to the removal of 2 units of RBCs (360 mL), followed by the return of the platelet-rich plasma (PRP) from both units and the infusion of 1000 mL of 0.9-percent NaCl. Four of the men and all seven women received their RBCs 1 hour after their removal. Shed blood levels of thromboxane B(2) (TXB(2)), 6-keto prostaglandin F(1 alpha), and peripheral venous Hct were measured. BTs were measured in 15 men and 13 women before and after a plateletpheresis procedure to collect 3.6 x 10(11) platelets per unit. RESULTS: The 2-unit RBC apheresis procedure produced a 60-percent increase in the BT associated with a 15-percent reduction in the peripheral venous Hct and a 9-percent reduction in the platelet count. The plateletpheresis procedure produced a 32-percent decrease in the platelet count, no change in peripheral venous Hct, and no change in the BT. After the removal of 2 units of RBCs, the shed blood TXB(2) level decreased significantly. Reinfusion of 2 units of RBCs restored the BT and restored the TXB(2) level to the baseline levels. CONCLUSION: The acute reduction in Hct produced a reversible platelet dysfunction manifested by an increase in BT and a decrease in the shed blood TXB(2) level at the template BT site. Return of the RBCs restored both the BT and the shed blood TXB(2) level to normal. The platelet dysfunction observed with the reduction in Hct was due in part to a reduction in shed blood TXB(2) and other, unknown mechanisms.