An attempt to standardize the APTT for heparin monitoring, using the P.T. ISI/INR system of calibration. Results of a 13 centre study.

Five APTT reagents, a heparin clotting assay and a chromogenic assay have been tested at 13 centres using lyophilised plasma containing different levels of added heparin (in vitro samples), and fresh normal plasma samples and samples from patients receiving heparin therapy (ex vivo samples). Marked differences in sensitivity between the reagents to in vitro and ex vivo samples were found. When calibration of the reagents was attempted using orthogonal regression analysis based on the P.T. ISI/INR system, the results were disappointing. Whichever reagent was accepted as standard, the between laboratory differences in ISI's were very large, and the within laboratory and between laboratory CV's were unacceptably high. Regression lines through normal and patient clotting times were commonly non parallel or skewed. From the results of only 5 out of more than 30 commercial reagents, it is concluded that the application of such a system would give no confidence in any individual result and could be misleading.

Severely heated therapeutic factor VIII concentrate of high specific activity.

A new method for the manufacture of a heated factor VIII concentrate of high specific activity (2-6 IU factor VIII:C/mg protein) has been developed. Addition of heparin to cryoprecipitate extract at acid pH precipitated fibrinogen and fibronectin. Factor VIII was then recovered from the supernatant by precipitation with glycine and sodium chloride. After re-solution and desalting on Sephadex G-25, the concentrate was sterile-filtered and lyophilised. The dried product was stable to heating in the final container at 80 degrees C for 72 h. Data from 25 consecutive batches of concentrate prepared from 1,200-1,500 kg plasma pools are presented. The mean final yield of heated product was 190 IU factor VIII:C/kg plasma. The concentrate has been found to be safe and effective in clinical use.

Clinical experience with a pasteurised human plasma concentrate in factor XIII deficiency.

A three-day-old infant presented with umbilical haemorrhage. Factor XIII deficiency was diagnosed. When one month old she commenced prophylactic injections of pasteurised factor XIII concentrate of human plasma origin. During two and a half years treatment there were no haemorrhagic episodes and factor XIII concentrate was well tolerated. Satisfactory post infusion factor XIII levels were achieved. Three transient elevations of aspartate transaminase occurred, the cause of which has not been established. There was no evidence of transmission of hepatitis or H.I.V. infection. A brother, born one year later, is also affected and commenced prophylactic therapy with the same factor XIII concentrate. Experience in these two infants suggests the product is efficacious.

Studies on the effect of heat treatment on the thrombogenicity of factor IX concentrates in dogs.

This study examines the effects of heat treatment for 72 h at 80 degrees C on the potential thrombogenicity of lyophilized human coagulation factor IX concentrates. Since heating generated minor amounts of thrombin, concentrate was prepared with antithrombin III addition prior to heat treatment. Changes in coagulation parameters were followed prior to and after infusion of 100 iu/kg of heated and unheated concentrates to dogs. All batches produced a transient fall in platelet count during infusion and a delayed rise in plasma fibrinopeptide A, accompanied by a minor prolongation of the activated partial thromboplastin time. Such changes were less marked for heated batches. Control infusion of a 'failed' factor IX concentrate showed an additional fall in fibrinogen, rise in fibrin degradation products and a more rapid rise in fibrinopeptide A, while thrombin infusion caused an even more dramatic intravascular coagulation. These studies indicated no increase in the potential thrombogenicity of freeze dried factor IX concentrates as a result of heat treatment.

Pasteurisation induced changes in an antithrombin III concentrate.

The changes that take place when a therapeutic antithrombin III (AT III) concentrate is heated in the presence of citrate ion have been assessed. There is some loss of heparin cofactor antithrombin activity and of heparin binding ability. Protein aggregates are also formed during heating. These aggregates are not AT III but impurities in the concentrate.

A pasteurised concentrate of human plasma factor XIII for therapeutic use.

A therapeutic concentrate of factor XIII containing both A and B sub-units has been prepared from 300 kg pools of human plasma. The process starts from a cold-ethanol fraction from cryoprecipitate supernatant and therefore does not interfere with the recovery of other clinically valuable plasma proteins. Factor XIII is purified approximately 600-fold from plasma by precipitation with sodium citrate and by the removal of fibrinogen by brief heating. The product has been pasteurised in sorbitol solution to inactivate blood-borne viruses, ultrafiltered to remove sorbitol, adsorbed with bentonite and freeze-dried in a formulation meeting requirements for intravenous injection.

Heat-treated NHS factor VIII concentrate in the United Kingdom--a preliminary study.

Three patients who have been given intermediate purity NHS heat-treated factor VIII concentrate have been followed prospectively for 7-10 months. None had previously received more than six donor units of blood products containing factor VIII. There were no clinical side effects from concentrate administration, haemostasis was satisfactory and no patient developed clinical or laboratory evidence of hepatitis or HTLV III/LAV infection. Heat treatment resulted in the loss of slightly more than 20% of factor VIII activity but in vivo recovery of factor VIII and half disappearance times were within the expected range.

A pasteurized antithrombin III concentrate for clinical use.

A method for large-scale production of a pasteurized antithrombin III (AT III) concentrate for therapeutic use has been adapted from published methods. It includes the following steps: (1) batchwise adsorption onto heparin-Sepharose from plasma depleted of cryoprecipitate and prothrombin complex; (2) chromatographic elution at high salt concentration; (3) pasteurization for 10 h at 60 degrees C in the presence of added citrate ion; (4) desalting on Sephadex G-25, and (5) sterile filtration and freeze-drying. Seven batches prepared in this manner gave a mean yield of 269 U AT III/kg plasma. The product passed all the usual animal safety and pyrogenicity tests and has been used successfully in several courses of treatment of congenital deficiencies.

Methods of assessing factor VIII content of stored fresh frozen plasma intended for preparation of factor VIII concentrates.

Minimally destructive methods were sought to assess the factor VIII content of fresh frozen plasma intended for large-scale fractionation and stored in five-liter polyethylene packs after pooling approximately 23 plasma donations. Although factor VII distribution in the frozen pack was not perfectly homogenous, a central "core" through the frozen pack gave a representative sampel of the entire contents of the pack. Coring was compared with other methods of pack sampling before large-scale cryoprecipitation. The survival of factor VIII was studied in three grades of stored plasma which had been separated and frozen within 4 hours, 8 hours, and 18 hours of blood donation. Plasma frozen after overnight storage of blood is a satisfacory starting material for the preparation of factor VIII and factor IX concentrates.