Prostaglandins in the preparation of blood components.

Prostaglandin E(1) significantly improved the separation of blood components in blood bags. The recovery in vivo and the life-span values of the platelets were not altered. The hemostatic effectiveness of platelets treated with prostaglandin was shown to be normal in man.

Thrombotic thrombocytopenic purpura in southeastern New England.

Eight patients with thrombotic thrombocytopenic purpura (TTP) originating within a 25-mile radius had their conditions diagnosed in a three-year period at a community teaching hospital in southeastern New England. In the preceding ten years, only one case of TTP had occurred in the same hospital. A niece-uncle relationship was present in two patients, and lymphocyte typing showed that they both shared an HLA haplotype. In the remaining patients, no social, familial, or environmental connection was established. Three patients died, all of whom were female. Six patients received exchange plasmapheresis with excellent responses in five. Autopsies in the three fatal cases showed widespread organ involvement with TTP but did not disclose evidence of any common underlying disease. This unusual occurrence should alert physicians to the possibility of localized outbreaks of TTP and the necessity of considering this diagnosis in all patients with unexplained thrombocytopenia.

Adoptive cellular immunotherapy for the treatment of malignant gliomas.

UNLABELLED: The median survival for adults with recurrent primary malignant gliomas is 56 weeks following surgery, radiation, and chemotherapy. Generally, reoperation can extend the median survival an additional 26-32 weeks. We have developed an aggressive treatment program that utilizes low doses of interleukin-2 (IL-2) combined with ex vivo activated killer cells (LAK) infused via an indwelling catheter placed into the surgical resection cavity. Autologous leukocytes were collected during a standard 3-4 h, outpatient leukapheresis procedure, then activated ex vivo for 4-5 days with high doses of IL-2. The treatment protocol consisted of two 2-week cycles of therapy over a 6-week period. Patients with stable disease or objective response on follow-up MRI scans were retreated at 3-month intervals. Acute and cumulative IL-2-related toxicities were observed, but limited, and included fever, headache and transient neurologic irritation. Corticosteroid levels and usage were strictly controlled during immunotherapy, although higher doses were used intermittently to mitigate toxicity. Biologic changes included lymphocytic infiltration, regional eosinophilia, tumor necrosis, and the localized production of IL-2, IFN-gamma and IL-12, demonstrated by in situ hybridization and immunohistochemistry. SUMMARY: IL-2 plus autogeneic LAK cells can be safely administered intracavitary to treat high grade primary brain tumors with limited toxicity within the central nervous system. Six out of 28 patients had long-term survival of greater than 2 years post-reoperation plus immunotherapy with 2 patients alive over 8 years. The presence of a marked regional eosinophilia appeared to correlate with increased survival and may be predictive of a biologic and therapeutic response. Regional adoptive immune therapy was well tolerated and should be considered an option for patients with high-grade tumors refractive to standard therapeutic approaches.

Idiopathic hemochromatosis presenting as amenorrhea and arthritis.

Idiopathic hemochromatosis in young adults has been increasingly recognized over the last three decades. Younger patients with hemochromatosis frequently have presenting problems other than diabetes, cirrhosis, and hyperpigmentation. A young woman with idiopathic hemochromatosis is described. Arthritis and secondary amenorrhea developed at age 20, and liver biopsy showed hemochromatosis at age 29. Further work-up revealed that the amenorrhea was due to underproduction of pituitary gonadotropins. The patient was treated with phlebotomy. Estrogen and progesterone replacement was begun because of severe osteoporosis. Serum iron studies may be useful in young patients with unexplained amenorrhea and/or arthropathy.

Immune thrombocytopenia and Gaucher's disease.

A 35-year-old Ashkenazi woman with Gaucher's disease was evaluated for persistent thrombocytopenia. The diagnosis of Gaucher's disease was made by bone marrow aspiration and confirmed by the determination of glucocerebrosidase levels in leukocytes and cultured skin fibroblasts. Studies of platelet-associated IgG and in vivo platelet survival demonstrated immune-mediated destruction of platelets consistent with immune thrombocytopenic purpura. A trial of prednisone had no effect on the platelet count. Total splenectomy resulted in a complete and prolonged remission. The clinical implications of Gaucher's disease and concurrent immune thrombocytopenic purpura are discussed.

Hemodynamic oxygen transport and 2,3-diphosphoglycerate changes after transfusion of patients in acute respiratory failure.

The goals of management of patients with respiratory failure include improving arterial oxygenation with PEEP and red cell transfusion to maintain oxygen carrying capacity, both of which contribute to improving tissue oxygen delivery. However, standard CPD-stored blood is rapidly depleted of 2,3 diphosphoglycerate (2,3 DPG) and ATP, with resultant inadequacy of the red cell oxygen transport function. In 15 patients requiring mechanical ventilation with PEEP whose initial Hct less than or equal to 35%, we studied the effect of transfusion of 7 ml/kg of CPD-stored packed red blood cells on hemodynamic and oxygen delivery variables, pulmonary venous admixture (QA/QT), and erythrocytic P50, 2,3 DPG and ATP concentrations. Hemodynamics were not significantly altered by transfusion. 2,3 DPG decreased significantly from 14.5 +/- 1.1 to 13.1 +/- 1.5 mcmol/g Hb (mean +/- SD, p less than 0.05). There was no significant change in P50 or ATP. QA/QT rose significantly, from 20.1 +/- 7.8 to 28.9 +/- 12.3% (mean +/- SD, p less than 0.02). In our patients, an increase in arterial oxygen content obtained by transfusion was not followed by any associated decrease in cardiac work, as implied by solution of equations for oxygen delivery and oxygen consumption. The rise in QA/QT is undesirable in patients requiring PEEP, since it complicates management of their mechanical ventilatory support.

Enhanced red cell oxygen transport in a pregnant woman with hemoglobin SD disease.

An anemic pregnant black primigravida with hemoglobin SD disease was studied throughout pregnancy and parturition. Hemoglobin SD disease was established by agar gel electrophoresis at pH 6.2. The patient carried to term a healthy femlae infant, an unusual occurrence in a patient homozygous for hemoglobin SD disease. The increase in the red cell concentration of 2,3 disphosphoglycerate (2,3 DPG) of 18--21 micrometer/g Hb (normal 12 +/- 2 micrometer/g Hb) was associated with a decrease in red cell oxygen affinity. The patient's in vitro P50 value ranged between 35 and 45 mm Hg (normal 28 +/- 1 mmHg). The function of red cells with increased 2,3 DPG levels and decreased affinity for oxygen in a pregnant woman with hemoglobin SD disease is discussed.

A microimmunofluorescent assay to detect human granulocyte antigens and antibodies.

Granulocyte antibodies are important in the pathogenesis of neonatal granulocytopenia, in some varieties of idiopathic granulocytopenia, and in transfusion reactions, particularly since the development of leukocyte transfusion therapy for granulocytopenic patients. Previous assays for the detection of granulocyte-specific antigens and antibodies were cumbersome, complex, and unreliable. Thus we have now described a new microimmunofluorescent method to assay human granulocyte antigens and their antibodies. This sensitive and reliable technique allows for the preservation of human granulocyte antigenicity for at least 24 hours. The granulocyte-specific antigens of a Caucasian population, residing in metropolitan New York, were studied by this microimmunofluorescent method. We report the distribution of granulocyte-specific antigens in this population, together with the corresponding gene frequency. This microimmunofluorescent test should improve our understanding of antigenic systems restricted to single cell populations.

Neonatal alloimmune thrombocytopenic purpura.

We reviewed 58 literature reports of neonatal alloimmune thrombocytopenic purpura (NAITP). The mortality rate was 9%. The total incidence of suspected intracranial hemorrhage was 28%. We reviewed 17 sibship cases for the relation of birth order to treatment and outcome. Among firstborn affected infants (n = 17) the mortality rate and incidence of central nervous system sequelae were 24 and 47%, respectively, compared to rates of 5 and 15%, respectively, in their younger affected siblings (n = 20). The improved outcome in the latter group appeared to be related to more frequent cesarean section delivery and more frequent and earlier use of corticosteroids and maternal platelet transfusions in the neonate. Sensitive assays of maternal platelet alloantibody are now available, but they lack specificity for NAITP affecting the current gestation. There are two reports in which sensitive assays revealed rising titers of maternal platelet alloantibody during advancing gestation. We propose further study to determine if this is specific for the antepartum diagnosis of NAITP.

Red cell 2,3-diphosphoglycerate and oxygen affinity of hemoglobin in patients with thyroid disorders.

We measured red blood cell 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP), and the P50 value in vitro of the oxyhemoglobin dissociation curve, which is the oxygen tension at half saturation of hemoglobin, in order to quantitate red blood cell oxygen transport function in individuals who were diagnosed as hypothyroid, euthyroid, or hyperthyroid based on measurements of thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and their clinical status. Hypothyroid (mean T4 2.8 microgram/dl, T3 49 ng/dl, TSH 37 microU/ml) and hyperthyroid (mean T4 14 microgram/dl, T3 271 ng/dl, TSH less than 0.7 microU/ml) patients had normal red cell 2,3-DPG and ATP levels and normal P50 values in vitro. The known changes in oxygen consumption produced by alterations in thyroid hormone levels in patients with hypothyroidism or hyperthyroidism did not affect red blood cell oxygen transport function.

Serum concentrations of erythropoietin measured by radioimmunoassay in hematologic disorders and chronic renal failure.

Bioassays for human erythropoietin are cumbersome, time-consuming, and insensitive. The purification of human erythropoietin (EP) had provided small quantities of highly bioactive EP (approximately 70,000 U/mg) required for the development of an EP radioimmunoassay (RIA). The RIA for EP described in this investigation, can detect 5 mU/ml of EP in the assay tube; the serum concentration of EP in normal individuals ranged from less than 18 to 81 mU/ml with a mean value of 29 mU/ml. In contrast, nine patients with severe aplastic anemia had markedly elevated serum EP concentrations with a mean value of 3,487 mU/ml, range 984--6,434 mU/ml. In this RIA, patients with Polycythemia vera had consistently undetectable EP concentrations, less than 18 mU/ml. Eleven patients with chronic renal failure had a higher mean serum EP concentration (40.5 mU/ml) than normal individuals, but the range (less than 18-115 mU/ml) overlapped that of normals. By immunologic and gel chromatographic criteria, EP measured in serum was similar to standard urinary EP. The EP immunoassay that we have developed has sufficient sensitivity and specificity not only to quantitate the elevated serum EP levels found in aplastic anemia but also to discriminate decreased from normal serum concentrations of EP in most circumstances. This simple, reliable RIA has provided the necessary framework upon which to increase our understanding of the importance of EP in hematopoiesis.

Neonatal alloimmune thrombocytopenic purpura: a case report.

We report a case of a sibling pair with neonatal alloimmune thrombocytopenic purpura. Serial antepartum platelet alloantibody quantitation by an enzyme-linked immunoabsorbent assay revealed rising antibody titers during advancing gestation. We discuss the implications of this finding in the antepartum diagnosis of neonatal alloimmune thrombocytopenic purpura, a rare, but frequently fatal disorder.

Altered platelet function and circulation induced by amphotericin B in leukemic patients after platelet transfusion.

Four adults, recently diagnosed, acute leukemic patients received 40 separate platelet transfusion treatments before, during, and after intravenous administration of amphotericin B. Fourteen of these platelet transfusions were administered while the patients also received amphotericin B; 26 platelet transfusions were given before or after amphotericin B therapy. The mean platelet increment for platelet transfusions administered while the patients were untreated with amphotericin B was 58 +/- 6 per cent. While the same patients were being treated with amphotericin B, the mean platelet increment was significantly decreased to 29 +/- 8 per cent (t = 3.1; p less than 0.005). In addition, when the patients were not being given amphotericin B, a highly significant negative relationship (r = -0.77; p less than 0.001) between the values for bleeding time and venous platelet count was present. In contrast, during amphotericin B treatment there was no correlation between the patients' bleeding times and venous platelet counts. We propose that amphotericin B therapy has a direct toxic effect on the function and circulation of transfused platelets. These toxic effects are quickly corrected by discontinuing this antifungal antibiotic.

Platelet transfusion therapy and circulating immune complexes.

30 platelet transfusions were administered to 9 thrombocytopenic patients with acute leukemias. Using the Raji cell radioimmunoassay, the serum concentration for circulating immune complexes (CIC) were determined immediately before and 10--12 h after each transfusion therapy. Elevated serum concentrations for CIC were present in 14 of the pretransfusion samples. After platelet transfusion therapy, a significant (p < 0.02) decrease in the values of CIC occurred. In these 14 transfusion studies the mean percent platelet increment was considerably lower (0.10 > p > 0.05) than the mean for the 16 transfusion studies in which the pretransfusion values for CIC were normal. These results show that CIC probably play an important role in removing transfused platelets from the circulation. Conversely, platelets are able to remove CIC from the circulation.

Plasma levels of beta-thromboglobulin and platelet factor 4 in relation to the venous platelet concentration.

The plasma levels of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) were determined in patients with various hematologic malignancies, and the results were related to simultaneously determined venous platelet counts. All studied patients were in a steady state. The plasma beta-TG concentrations were determined on 69 occasions and the values ranged from 0 to 82 ng/ml. In 33 instances, the venous platelet count was <25 x 10 (9/1) and in two thirds of these samples beta-TG was undedectable. The highest values for plasma beta-TG were found in patients with the highest venous platelet counts. A highly significant correlation (r=0.77, p <0.001) between the values for plasma beta-TG and venous platelet count was present. The plasma concentrations for PF-4 ranged from 0 to 50 ng/ml. Similarly, there was a highly significant relationship (r=0.78, p<0.001) between the values for PF-4 and venous platelet concentration. We conclude, if the plasma levels of beta-TG and PF-4 are used as markers of platelet activation in vivo, it is necessary to simultaneously consider the platelet concentration in the collected blood.

Therapeutic transfusions of previously frozen washed human platelets.

The platelets used in this study were collected by serial centrifugation, and within four hours of collection were frozen with 5% dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute by storage in a mechanical refrigerator at -80 C. The frozen platelets were stored for four to ten weeks before thawing and washing. After washing, the units were kept at room temperature for six to eight hours before transfusion. The units were pooled, and an average of eight units was given to each of four patients, with a range of three to 14 units per transfusion. In vitro recovery after washing was about 65 per cent and in vivo recovery of the 51chromium labeled (51Cr) platelets was about 35 per cent. The infusion of these previously frozen washed platelets corrected prolonged bleeding times in patients. The homologous platelets were transfused along with other blood products to treat patients with hematologic disorders. The circulation and function of the donor platelets were influenced by compatibility of the platelets, the quality of platelet preservation and the patient's disease state.

Retention of HLA antigens on previously frozen human platelets.

Recent technical achievements allow satisfactory cryopreservation of human platelets that, after thawing and washing, maintain functional viability for therapeutic use. We have now demonstrated that such previously frozen platelets retain full HLA antigenic activity in spite of the combination of freezing, thawing, and washing procedures and the presence of the surface-active cryopreservative DMSO. Platelets stored at 4 C without DMSO and previously frozen platelets from the same donors were tested in parallel for quantitative expression of HLA antigens. In all cases, the previously frozen platelets were quantitatively equal or superior to the platelets stored at 4 C with regard to their capacity to specifically reduce the HLA antibody activities of selected typing sera against a panel of antigen-positive lymphocytes.

Physiologic effects of transfusing red blood cells with high or low affinity for oxygen to passively hyperventilated, anemic baboons: systemic and cerebral oxygen extraction.

Anemic, passively hyperventilated baboons were given preserved red blood cells either with increased or with slightly reduced affinity for oxygen to restore the red cell volume. In the high affinity group there was a 50% increase in cerebral blood flow immediately after the transfusion, but there was no significant change in the low affinity group. The cardiac output decreased slightly in the low affinity group, and increased slightly but insignificantly in the high affinity group. Two hours after transfusion the cerebral blood flow had returned to normal in the high affinity group. In both groups there was a decrease in arterial blood pH and an increase in Po2 in blood from the pulmonary artery and the jugular vein after transfusion. A 40% restoration of the 2,3 DPG level occurred within 4 hours of the transfusion of red cells with high affinity for oxygen, and this rapid increase was associated with increases in blood pH and inorganic phosphorus levels. Preserved red cells with high affinity for oxygen and low 2, 3 DPG levels significantly increased the cerebral circulation during the 2-hour posttransfusion period. These findings lend support to the recommendation that preserved red cells with normal or elevated 2,3 DPG levels be administered to patients in hemorrhagic or septic shock, and to patients subjected to extracorporeal circulation during cardiac surgery in order to lessen the demand for increased blood flow and to ensure adequate tissue oxygenation during the postoperative period.

Physiologic effects of hyperventilation and phlebotomy in baboons: systemic and cerebral oxygen extraction.

Eighteen anesthetized baboons were studied to determine the effects of passive hyperventilation and phlebotomy on oxygen transport. After 1 hour of hyperventilation a significant increase in the red cell affinity for oxygen occurred in vivo. This was not associated with any significant changes in cardiac output, oxygen consumption, or in lactic acid production. There was a 40% decrease in cerebral blood flow, a 10 mm Hg decrease in the pulmonary artery Po2 level, and a 17 mm Hg decrease in the jugular venous Po2 level. After 1 hour of hyperventilation, the plasma inorganic phosphorus level decreased significantly, the red cell ATP level decreased slightly, and the red cell 2. 3 DPG level increased significantly, indicating that inorganic phosphorus had been removed from the blood during hyperventilation. Passive hyperventilation was maintained, and the baboons were bled 32% of their red cell volume. The blood volume was partially restored with nonbuffered isotonic saline. One hour after the phlebotomy and volume restoration (2 hours of hyperventilation) there were no changes in oxygen consumption, cardiac output, cerebral blood flow, or blood lactate levels, but the pulmonary artery Po2 level was decreased by 15 mm Hg, and the jugular venous Po2 level was decreased by 20 mm Hg. Systemic oxygen consumption was not affected by the significant decrease in pulmonary artery Po2.