Clinical experience with transfusion of leukocyte-poor platelet concentrates prepared by filtration with prostacyclin

Repeated transfusions with platelets from randomly selected donors lead to HLA alloimmunization in about 50% of patients due to lymphocyte contamination of platelet concentrates. Attempts to remove the leukocytes from the platelet concentrates by additional centrifugation steps led to substantial loss of platelets. We report a new procedure for removal of almost all leukocytes with excellent platelet recoveries. Single donor concentrates are treated with 50 ng/mL prostacyclin to inactivate the platelets transiently. The concentrates are then passed through a cellulose-acetate filter to remove the leukocytes. In 30 concentrates this treatment reduced the contamination by leukocytes to less than 0.1 million per concentrate with a platelet recovery of 89% +/- 1% (mean +/- SEM). Thirty filtered platelet concentrates transfused to ten thrombocytopenic patients within one hour after filtration were well tolerated and led to corrected count increments of (22.0 +/- 1.1) X 10(6)/mL blood after one hour and normal survival thereafter. In four of five patients these concentrates reduced the bleeding time. We conclude that transient inactivation of platelets by prostacyclin enables optimal removal of leukocytes and may help to reduce alloimmunization during frequent transfusions with platelet concentrates.     

The effect of platelets in the activation of human blood coagulation factor IX by factor XIa

We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose- dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.     

Induction and enhancement by monocytes of antibody-induced modulation of a variety of human lymphoid cell surface antigens

We have previously reported that the addition of monocytes results in enhanced modulation of the T65 antigen when normal or leukemic lymphoid cells were cultured in vitro with the T101 monoclonal antibody. In the present investigation, we extend these findings to demonstrate that monocyte-enhanced modulation is a phenomenon that occurs with a variety of T and B lymphoid antigens identified by murine monoclonal antibodies. Two patterns of monocyte-enhanced modulation were observed: (1) augmentation by monocytes of existing antigen modulation by the T101 and anti-Leu-4 antibodies, and (2) induction by monocytes of previously unrecognized modulation with the anti-Leu-2 and anti-Leu-9 antibodies. Enhancement of modulation by monocytes was also detected with antibodies to surface IgM and HLA-DR antigens. Antigen modulation on lymphoid cell lines appeared to be more variable than on fresh cells, with or without monocytes. Monocyte-enhanced antigen modulation was not demonstrated with two monoclonal antibodies against solid tumors. Monocyte-enhanced modulation was shown to be dependent upon the Fc portion of the antibody, but independent of proteolytic or oxidative compounds released by monocytes. These findings indicate that the results obtained during in vitro studies of antigen modulation may vary with the source of cells and the extent to which monocytic cells are present. In addition, these findings suggest an enhanced role for Fc receptor-bearing cells of monocytic origin in antigen modulation following in vivo administration of monoclonal antibodies.     

Detection of sickle alpha- or beta0-thalassemia by studies of globin biosynthesis

Globin synthesis studies are useful in the analysis of thalassemia syndromes. We have applied globin synthesis and free alpha-chain pool studies of peripheral blood to characterize hematologic disorders where alpha- or beta-thalassemia was present in combination with HbS or HbC. In 60 non-thalassemic controls, the beta/alpha specific activity ratio was 1.01 +/- 0.06 (SD). In three patients with HbS-beta0-thalassemia, the (betas + gamma)/alpha ratios were 0.48-.067. In four patients with HbSS-alpha-thalassemia, the (BETAS/ALPHA RATIO was 1.26 +/- 0.18 (1.13- 1.53). The radioactive free alpha-chain pool in three patients with HbS- beta0-thalassemia was elevated (35.1%-53.0%), while three patients with HbSS-alpha-thalassemia had decreased free radioactive alpha-chain pools (3.2%-6.4%); both were significantly different from the mean (15.1% +/- 2.6%) of the 17 iron-sufficient controls. Simultaneous studies of the fraction of newly synthesized alpha chain contained in the free alpha- chain pool in peripheral blood and bone marrow demonstrated that this fraction was larger in peripheral blood than in marrow, and that the differences between thalassemia patients and controls previously found in bone marrow using these methods were also present in peripheral blood. The results indicate that even when family studies are not possible, patients with HbS in combination with alpha- or beta0- thalassemia can be differentiated from those with homozygous sickle cell disease by globin synthesis and free alpha-chain pool studies using peripheral blood.     

The human cardiac mast cell: localization, isolation, phenotype, and functional characterization

We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.     

Improvement of platelet storage conditions by using new polyolefin containers

BACKGROUND : Storage of pooled platelet concentrates (PCs) with yields above 3.0 × 10¹¹ platelets per unit in a 1-L PL-732 polyolefin container for 5 days often results in a drop in pH to below 6.0. Recently, new oxygen-permeable platelet containers (1-L PL-2410, 1-L and 1.5-L Compoflex) have been developed. The maximal platelet storage capacities of the new containers and the PL-732 were compared. STUDY DESIGN AND METHODS : Large platelet pools (n = 27) with platelet concentrations between 1.2 and 1.4 × 10¹¹ per L were made from 3 to 5 PCs prepared from buffy coats. The pools were divided in equal volumes among the PL-732 and the three new platelet containers. Platelet counts in the PCs ranged from 1.0 to 5.0 × 10¹¹ per unit. All PCs were stored on a flatbed shaker at 22 ± 2°C and evaluated on Days 1, 3, 5, and 7 by measuring platelet count, pH, pO₂, pCO₂, HCO₃-, glucose, lactate, platelet swirling, and soluble p-selectin. RESULTS : Day 7 storage of PCs (n = 6) with yields between 3.0 and 4.0 × 10¹¹ platelets in PL-732 showed mean ± SD pH values of 5.93 ± 0.05 and lactate values of 32.3 ± 7.9 mmol per L; in 4 of these 6 PCs, pH was below 6.0. In contrast, storage of these PCs in 1-L PL-2410 and 1.5-L Compoflex containers and of 2 of these 6 PCs in 1-L Compoflex containers showed pH values above 6.8. Lactate values were 15.5 ± 1.3, 15.3 ± 1.8, and 19.5 ± 4.7 mmol per L, respectively (p < 0.001 vs. PL-732). The platelet storage capacity of the new containers with platelet yields between 4.0 and 5.0 × 10¹¹ per unit (n = 6) was evaluated. Day 7 storage of these PCs in the 1.5-L Compoflex showed an average pH value of 6.74 ± 0.20; in 2 of 6 PCs, pH was below 6.8. The average pH value in the PL-2410 was 6.38 ± 0.31, and in all PCs, pH was below 6.8. Average lactate values were 17.8 ± 5.7 and 25.8 ± 5.6 mmol per L (p < 0.05), respectively. Soluble p-selectin values on Day 7 of storage increased approximately twofold in all PCs. CONCLUSION : The new oxygen-permeable containers showed platelet quality comparable to that with the PL-732 and for longer storage periods and at higher platelet counts.     

Effects of stimulation frequency,amplitude, and impulse width on muscle fatigue

Introduction: We investigated the effect of stimulation intensity (in percent of maximal tolerated stimulation current, mTSC), frequency, and impulse width on muscle fatigue. Methods: Using a randomized crossover design, 6 parameter combinations (80% mTSC, 80 Hz , 400 μs; 60% mTSC, 80 Hz , 400 μs; 80% mTSC, 20 Hz , 400 μs; 60% mTSC, 20 Hz , 400 μs; 80% mTSC, 80 Hz , 150 μs; 60% mTSC, 80 Hz , 150 μs) were tested in both legs of 13 athletic men (age 26 ± 2.3). The slope of the linear regression line over all tetani (FIS) and the number of tetani whose force was above 50% of the initial tetanus (FIN) were used to quantify fatigue. Results: FIS and FIN were significantly lower in high‐frequency protocols. No effects on FIS and FIN were found for intensity and impulse width. Conclusions: Stimulation frequency, but not impulse width or intensity, affected fatigue kinetics. Muscle Nerve 53 : 608–616, 2016     

丹参酮-预胶化淀粉共研磨混合物的研究

目的：提高难溶性药物丹参酮（Tanshinone TAN)的溶出度。方法：将TAN与预胶化淀粉（Pregeletiniged Starch PGS)共研磨制备成共研磨混合物,测定了TAN原料药和共研磨混合物的溶出度,通过扫描电镜分析了丹参酮的存在状态。结果：共研磨混合物的溶出度较TAN原料药有显著提高,共研磨3h的共研磨混合物中,TAN以无定形态或超微颗粒附着于载休表面。结论：将TAN与PGS共研磨能有效地改善TAN的溶出度,且方法简便。