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R B Counts

Publications and source records attributed to R B Counts.

25 records · Page 2Linked to original sources
PubMed1979

Hemostasis in massively transfused trauma patients.

Twenty-seven patients requiring massive transfusions were studied prospectively to determine whether administration of stored, modified whole blood induced a primary disorder of hemostasis evidenced by generalized microvascular oozing. Platelet counts fell in proportion to the number of units of blood transfused. In contrast, the levels of factors V and VIII correlated poorly with the units of blood transfused, 85% of the total variation in the levels being due to influences other than transfused blood. Levels of all other clotting factors were unrelated to the number of units of blood given. Eight patients developed abnormal bleeding. The cause appeared to be dilutional thrombocytopenia in five patients, and DIC in three. In six of the eight, bleeding was controlled with platelet concentrates alone. Two patients were given cryoprecipitate also. The most useful laboratory test for predicting abnormal bleeding was the platelet count. Fibrinogen levels should be followed as an aid in the diagnosis of DIC. The BT, PT, and PTT were not helpful in assessing the cause of bleeding, unless they were greater than 1.5 times the control value. We recommend that any patient receiving massive transfusions who develops diffuse microvascular bleeding be given platelet concentrates. Platelet counts as high as 100,000 may be required to control bleeding from surgical wounds. It is not necessary to supplement transfusions of stored, modified whole blood with fresh blood or fresh frozen plasma.

Adolescent↗
PubMed1978

Disulfide bonds and the quaternary structure of factor VIII/von Willebrand factor.

Human Factor VIII/von Willebrand factor, purified by calcium citrate-cellulose chromatography and 4% agarose gel filtration was subjected to sodium dodecyl sulfate gel electrophoresis on gels containing 2% acrylamide and 0.5% agarose. We find a series of multimers of which the apparent molecular weight of the higher members was congruent with5 million. The higher multimers were isolated by 2% agarose gel filtration. Treatment of the high molecular weight multimers with 2-mercaptoethanol at concentrations of 0.005-0.5% in the presence of 1% dodecyl sulfate resulted in a shift to lower molecular weight multimers. Between mercaptoethanol concentrations of 0.01 and 0.5%, the predominant species was the dimer of the basic subunit. Mercaptoethanol concentrations >0.5% were required to reduce the interchain disulfide bonds of the dimer. An artificial multimeric series was prepared by cross-linking von Willebrand factor subunits with dimethylsuberimidate. Comparison of the multimers produced by reduction with the multimers produced by cross-linking, demonstrated the absence of odd-numbered multimers from the reduced series. Thus, the protomeric unit appears to be the dimer. High molecular weight multimers had both ristocetin cofactor activity and Factor VIII procoagulant activity. Reduction of the protein in the absence of denaturing agents, caused a gradual shift to lower molecular weight species and a concomitant loss of von Willebrand factor activity. In contrast, Factor VIII activity was unchanged by reduction. These studies suggest that the moieties having von Willebrand factor activity and those having Factor VIII activities are covalently linked by disulfide bonds.

Blood Coagulation Factors↗
PubMed1976

Removal of heparin and protamine from plasma.

A simple chromatographic technique for rapid adsorption of heparin and protamine from plasma samples is described, allowing accurate interpretation of coagulation screening tests and specific clotting factor assays. With the use of columns of ECTEOLA-cellulose, up to 300 U. of heparin could be completely adsorbed from a 1 ml. plasma sample. When citrated nonheparinized plasma was passed over the ECTEOLA-cellulose columns, the thrombin, prothrombin, and partial thromboplastin times were unaffected. Levels of fibrinogen, prothrombin, and factors V, VII, VIII, IX, and XI average within 90 per cent of control, nonchromatographed samples. When heparinized plasma samples (0.1 and 1.0 U. per milliliter) were passed over columns, heparin was completely removed and the results of the screening tests and the specific factor assays were the same as for the chromatographed nonheparinized samples. In addition, heparinized samples with decreased factor VIII activity maintained their pretreatment factor VII activities after heparin removal. Blood samples containing heparin were obtained from two patients during open-heart surgery. Following heparin adsorption on ECTEOLA-cellulose columns, factor VIII activity levels remained above 60 per cent during cardiopulmonary by-pass. The presence of protamine sulfate in plasma samples prolonged the prothrombin and partial thromboplastin times while slightly shortening the thrombin time. The protamine effect persisted after ECTEOLA-cellulose, but could be removed by a similar column of carboxymethyl-cellulose. The latter resin had no effect on screening tests or on assays of factors VIII or IX activity. The combination of the two resins was then used to remove the separate inhibitory effects from heparinized plasma samples to which protamine had been added.

Adsorption↗
PubMed1975

Solid-phase immunoradiometric assay of factor-VIII protein.

A solid phase immunoradiometric assay for factor-VIII protein has been developed using 125I-labelled rabbit antibody made against highly purified factor VIII. Antibody specific for high molecular weight factor-VIII protein was isolated using an immunoadsorbent consisting of highly purified factor VIII bound to diazotized m-aminobenzyl (oxymethyl)-cellulose. The purified anti-factor VIII antibody was labelled with 125I while bound to the immunoadsorbent and then eluted at pH 2.9. Dilutions of plasma samples for assay were incubated for 48 h in anti-factor VIII antibody-coated tubes. The tubes were washed to remove unbound proteins and the 125I-labelled, purified antibody was added. After 48 h incubation the tubes were washed to remove unbound antibody and counted. The concentration of immunoreactive factor-VIII protein in pooled normal human plasma was determined to be 8 microgram/ml. The minimum amount of factor VIII measured by the assay is less than 0.16 ng. Factor-VIII protein was present in normal concentration in haemophilia A plasma, and in reduced concentration in von Willebrand's disease plasma. The method has the advantages of improved sensitivity, specificity, efficiency and economy as compared with previous factor-VIII immunoassays.

Animals↗
PubMedDate not supplied

Preparation of cryoprecipitated factor VIII concentrates.

Factors affecting the yield of factor VIII in cryoprecipitate have been investigated in the context of a blood component program. Both in vitro and in vivo measurements were used to assess the effects of critical variables on the yield of factor VIII activity. Variables such as anticoagulant, plastic bag, mixing during collection, and platelet contamination had no significant effect on yield of factor VIII activity in cryoprecipitate. Among the most critical factors affecting yield were storage time of whole blood and procedures for freezing, thawing, and reconstitution. The following procedures were found to assure a 60 per cent recovery of factor VIII in cryoprecipitate: 1)processing of whole blood within six hours of collection; 2)use of a technique to freeze plasma within 30 minutes either in a -70 C ethanol bath or -85 C freezer; 3)rapid thawing (1 1/2 hour or less) in a 4 C circulating water bath; 4)centrifugation at 4,500 X g for 10 minutes at 4 C followed by draining of the supernatant in a 4 C cold room; 5) storage of the precipitate at -20 C until ready for use; 6) thawing in a 37 C water bath for at least 15 minutes followed by addition of 20 ml of 0.15 M saline for a 20 minute period at room temperature, and gentle mixing before pooling units for transfusion. The recovery of factor VIII in cryoprecipitate appears to be limited to about 65 per cent by its solubility in plasma at 4 C. Therefore, further effort to increase the amount available for treatment should involve improving the supply of plasma for its preparation and decreasing the cost of processing.

Blood Cell Count↗