Judith G. Pool

Assay of plasma antihaemophilic globulin (AHG).

THE introduction of the thromboplastin-generation test by Biggs and Douglas (1953) provided an entirely new and sorely needed basis for the quantitative assay of anhhaemophilic globulin (AHG). The procedure described by Biggs, Eveling and Richards (1955)~ modified by Pitney (1956) and by the original authors (Biggs and Macfarlane, 1957; Biggs, 1957)~ presents only two major impedimenis to adoption by the research laboratory engaged in long-term studies on AHG: the day-to-day reproducibdity is insufficiently good, and there is poor correlation between in-vitro and in-vivo measurements of AHG. The latter difficulty is, unfortunately, a serious, one, because the validity of an AHG assay can be demonstrated only in an AHGdeficient patient. Unless an anticoagulant is present, an assay should be able to measure the low level in the haemophiliac, measure the level in normal plasma to be used for transfusion and confirm the predicted rise in the patients level following the transfusion. This report describes further modifications of the reagents and procedure of Biggs, Eveling and Richards (1955)~ as well as statistical studies ofthe modified method which demonstrate its high level of precision and transfusion studies which demonstrate its validity.

Observations on Plasma Banking and Transfusion Procedures for Haemophihc Patients using a Quantitative Assay for Antihaemophilic Globulin (AHG)

MANY clinicians have been puzzled by the fdure of haemostasis in the haemophilic patient who has been transfused with apparently adequate amounts of freshly frozen plasma. Such failures become understandable, however, if a distinction is made between the amount of plasma administered and the amount of antihaemophilic globulin (AHG) administered. Information on the AHG levels in commonly used transfusion materials, on the AHG level required for haemostasis and on the survival of transfused AHG would permit rational planning of treatment. An essential requisite for such information is a reliable quantitative assay for AHG. In the precedmg paper (Pool and Robinson, 1959) a technique of assay is described which gives a coefficient of variation of less than 8 per cent (h=0.0366). Evidence is also presented from transfusion experiments that it is AHG and only AHG which is being measured. These same transfusion experiments cast doubt on the validity of the two commonly offered explanations of the inefficacy of transfusion. One of these postulates an abnormally rapid disappearance, within minutes, of transfused AHG, and the other proposes the presence of powerful anticoagulants in all severe haemopMc patients. The present report describes studies which indicate that it is the low AHG levels in the transfused plasma and the short (10-hour) half-time of the AHG in the recipient’s circulation that are responsible when therapy is ineffective. It also presents data on the causes of the low AHG levels in blood-bank frozen plasma and an analysis of the advisability of prophylactic transfusion therapy in haemophilia. It does not present any new information on the AHG levels required for haemostasis.

A Study of a Case of Congenital Hypoprothrombinaemia

BIGGS and Douglas (1953a) reviewed the reported cases of congenital hypoprothrombinaemia, and concluded that in none had the condition been adequately differentiated from deficiency of proconvertin (Factor VII), proaccelerin (Factor V) or other factors. They then described a case of acquired specific prothrombin deficiency. Since then, several reports of specific congenital deficiency have appeared, those of van Creveld (1954) and Quick, Pisciotta and Hussey (1955) being the most detaded. Since the discovery of the Stuart-Prower factor deficiency (Telfer, Denson and Wright, 1956; Hougie and Graham, 1956), all previously reported cases must be re-examined to exclude that diagnosis. The data of Biggs and Douglas, van Creveld, and of Quick and his colleagues are not complete enough for certainty, but it seems improbable that any of their patients was a case of deficiency of the Stuart-Prower factor. Nevertheless, there are some codct ing and puzzling observations in these communications, which will be considered in this report. In the present case of congenital hypoprothrombinaemia we have excluded deficiency of the Stuart-Prower factor and other anomalies previously described, and we have explored in detail the prothrombin consumption, prothrombinase* generation, and the turnover time of transfused prothrombin.

The Effect of Several Variables on Cryoprecipitated Factor VIII (AHG) Concentrates

Because of current interest in the new CPD anticoagulant and in acidified platelet‐rich plasma for the production of platelet concentrates, the effect of these changes on cryoprecipitation of Factor VIII was investigated. It was found that use of CPD anticoagulant results in Factor VIII concentrates at least as good as those prepared from corresponding ACD plasma. It was also found that cryoprecipitation of Factor VIII is strikingly affected by pH, with the yield being very low at pH 6.0 and rising steadily to a plateau at about pH 6.8, with no further change up to pH 8.0. Acidified plasma can be restored to normal for production of cryoprecipitates by neutralization of the added acid with NaOH. A survey of five possible diluents for the cryoprecipitates showed that normal saline and retained supernatant plasma both function as well as citrated saline; good stability at room temperature was demonstrated for the AHG in all diluents examined.

Treatment of Hemophilia with Factor VIII Concentrates

THE treatment of hemorrhagic episodes in hemophilic patients requires administration of the coagulation protein that is deficient — namely, antihemophilic globulin (factor VIII), derived from the p...

Ethionine-induced depression of plasma antihemophilic globulin in the rat.

Summary and Conclusions Parenteral administration of ethionine markedly depressed the AHF content of the blood in rats. This effect probably represents interference with synthesis of the protein rather than increased destruction. Although other coagulation factors were affected, the ethionine action did not involve all serum proteins, as shown by paper electrophoresis. Extensive tissue damage in ethionine-treated animals was confined to the pancreas, liver and bone marrow. Neither total pancreatectomy in dogs, nor carbon tetrachloride-induced liver damage in rats had any effect on plasma AHF levels. Studies are in progress to determine a possible role of the reticulo-endothelial system.

The effect of delayed refrigeration on red blood cells, platelet concentrates and cryoprecipitable AHF.

The effect of delaying blood processing for six hours while maintaining it at ambient temperature was investigated. Blood drawn from volunteers on two occasions was processed immediately (I) or after a six‐hour delay (D). The effects of the delay on the efficacy and safety of red blood cells, platelet concentrates and cryoprecipitable AHF were studied. There was a more rapid decrease in 2,3‐DPG in the delayed group during 21 days of refrigerated storage. ATP levels declined at similar rates. 24‐hour survival of 51CR‐labeled autologous cells was slightly better (p = .05) in the (I) group but excellent for both. Total platelets, per cent recovery and pH at 72 hours were identical for both groups. All cultures were sterile. There was no difference in total AHF recovered or per cent recovery between the two groups. To increase the availability of blood components, a six‐hour processing delay seems warranted.

Assay of the Immune Inhibitor in Classic Haemophilia: Application of Virus-Antibody Reaction Kinetics

The kinetics of virus neutralization by antibody are shown to apply to factor‐VIII neutralization by the immune antibody of haemophilic patients. The analysis established for virus neutralization has been used as a model for designing a general system for inhibitor assay based on mixtures in which antigen is present in excess, rather than with antibody in excess, as has been the case with systems previously proposed. The new system permits conversion of inhibitor potency results from other systems based on the use of different mixtures of factor VIII and inhibitor as long as factor VIII is in excess and as long as constant physical conditions of time and temperature of incubation are applied. It also takes account of the fact that some antibodies reveal a‘residual resistant fraction’ of antigen with which they cannot react. The procedure for converting assay data into‘Oxford units’ is described.

Ineffectiveness of intramuscularly injected Factor 8 concentrate in two hemophilic patients.

PROPHYLACTIC therapy of hemophilia is handicapped by the short half-life of factor VIII in the circulation (eight to fifteen hours). However, even 1 per cent of the normal level probably confers so...

Women in Medicine

The situation is improving; at Stanford, a conscious effort has significantly increased female representation on the faculty and raised to 25% the proportion of women in the 1972–73 entering class. But negative attitudes still persist, often among women themselves. In the final analysis women must define the special needs that must be met if they are to pursue medicine as a career while still leading full personal lives.