Assessment of a closed wash system developed for processing living donor femoral heads.

NHS Blood and Transplant Tissue and Eye Services (TES) and Scottish National Blood Transfusion Services Tissues and Cells Directorate (TCD) currently bank whole, frozen femoral head bone from living donors who are undergoing primary hip replacement surgery. When required, the bone is issued to a surgeon still frozen on dry ice (- 79 °C). Consequently, the femoral head bone is not processed, is not sterilised and at the time of issue, it contains donor blood, bone marrow and associated cells. We have previously shown that, cut, shaped and washed bone from deceased donors can be processed to remove up to 99.9% of blood, bone marrow and associated cells (Eagle et al. 2015). However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone; therefore in this report, a method has been developed in which whole femoral heads can be washed to remove donor blood and bone marrow components. Processing results in excess of 99% bone marrow component removal-soluble protein, haemoglobin and DNA; the procedure is performed inside a closed system, thereby eliminating the need for terminal sterilisation by irradiation. In addition, uniaxial testing demonstrated no difference in compressive strength between washed and unwashed bone. We suggest that this washed bone may be capable of improving incorporation after grafting without disturbing biomechanical properties of the graft.

Intra-operative washing of morcellised bone allograft with pulse lavage: how effective is it in reducing blood and marrow content?

The use of unprocessed bone carries a risk of transmission of blood borne diseases. Although models of infectivity are unproven, a theoretical risk of transmission of variant Creutzfeld-Jakob Disease, a human prion disease, exists as probable blood borne transmission has been reported in three cases. The aim of our study was to determine the effectiveness of standard operating theatre pulse lavage in removing protein, fat and double stranded Deoxyribonucleic acid (dsDNA) from morcellised bone allograft. Twelve donated femoral heads were divided into halves and milled into bone chips. One half of the bone chips were washed with pulse lavage, whereas, the other half acted as control. In order to determine the amount of protein, fat and dsDNA present in the washed and unwashed samples, a validated multistep washing protocol was used. Using the validated technique, simple intra-operative washing of morcellised unprocessed bone allograft removed a significant amount of the protein (70.5%, range: 39.5-85%), fat (95.2%, range: 87.8-98.8%) and DNA (68.4%, range: 31.4-93.1%) content. Intra-operative washing of morcellised bone allograft with pulse lavage may thereby reduce the theoretical risk of prion and other blood borne disease transmission. Combined with the known improved mechanical characteristics of washed allograft, we would recommend pulse lavage as a routine part of bone allograft preparation.

Studies on the stability of VIII:C during the manufacture of a factor VIII concentrate for clinical use.

The stability of VIII:C was investigated by monitoring samples taken at different points from a routine process for the manufacture of factor VIII concentrate and by examining the stabilising influence of a number of product formulations. Loss of VIII:C over process-finishing procedures (formulation, 0.22 micron filtration, dispensing) was associated with a citrate-induced inactivation which could be prevented by controlling the ionised calcium concentration of the solution. These results were obtained using a one-stage clotting assay but were not observed using a two-stage assay. No evidence for activation was found in vitro (e.g. by FPA generation and VIII:C stability) and the yield increase suggested by the one-stage assay was supported by results from a controlled clinical evaluation.

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.

Control of large-scale plasma thawing for recovery of cryoprecipitate factor VIII.

Cryoprecipitation is commonly used as the primary step in the preparation of clinical factor VIII concentrates; yet recovery is usually very low. Much of this loss is due to poor temperature control and a process of continuous plasma thawing has been designed to overcome this. A substantial improvement has resulted, with an increase in both yield and purity of factor VIII:C of over 50% in comparison to a conventional batch thaw process.

Changes in factor VIII complex activities during the production of a clinical intermediate purity factor VIII concentrate.

Samples taken at various stages of preparation of intermediate purity factor VIII concentrate were assayed for factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIIIC:Ag) and factor VIII related antigen (VIIIR:Ag). The antigen results were used to assess mechanical loss during fractionation as these markers are relatively stable. In contrast VIII:C is sensitive to both mechanical and inactivation losses. The major loss of factor VIII occurred during the cryoprecipitation and extraction step and was due to both mechanical loss and inactivation. Losses before and after this step were largely due to inactivation of the factor VIII. Assay of VIIIR:Ag in concentrates presented problems and a modified technique is suggested.

Calibration of the limulus test for blood products.

The Protein Fractionation Centre has been using the Limulus Amoebocyte Lysate test for detecting the presence of pyrogens for some five years. All tests on final plasma products have been carried out in parallel with the EP rabbit pyrogen test. Good correlation between both tests has been obtained with products such as distilled water and crystalloid solutions. However, it is more difficult to correlate results when dealing with plasma proteins. Each product appears to react differently. Some normally pass the limulus test undiluted while others, such as immune globulins and albuminoid products, have to be diluted to various concentrations before they pass. A preliminary statistical analysis has been carried out on the results of pyrogen testing on Stable Plasma Protein Solution, firstly to ascertain that a significant correlation does exist between the LAL test and the rabbit test, and secondly to try to obtain "cut off" values for the limulus test which compare well with the official EP temperature rises in the rabbit test.