Peter B. A. Kernoff

Laboratory control values for CD4 and CD8 T lymphocytes. Implications for HIV‐1 diagnosis

With the advent of standard flow cytometric methods using two‐colour fluorescence on samples of whole blood, it is possible to establish the ranges of CD3. CD 4 and CDS T lymphocyte subsets in the routine laboratory, and also to assist the definition of HIV‐1‐related deviations from these normal values. In 676 HIV‐1‐seronegative individuals the lymphocyte subset percentages and absolute counts were determined. The samples taken mostly in the morning. The groups included heterosexual controls, people with various clotting disorders but without lymphocyte abnormalities as well as seronegative homosexual men as the appropriate controls for the HIV‐1‐infected groups. The stability of CD4% and CD8% values was demonstrated throughout life, and in children CD4 values < 25% could be regarded as abnormal. The absolute counts of all T cell subsets decreased from birth until the age of 10 years. In adolescents and adults the absolute numbers (mean±s.d.) of lymphocytes, CD3, CD4 and CD8 cells were 1·90±0·55, 1·45±0·46, 0·83±0·29 and 0·56± 0·23 ± 109/l, respectively. In patients with haemophilia A and B the mean values did not differ significantly. In homosexual men higher CD8 levels were seen compared with heterosexual men and 27% had an inverted CD4/CD8 ratio but mostly without CD4 lymphopenia (CD4<0·4 ± 109/l). However, some healthy uninfected people were‘physiologically’ lymphopenic without having inverted CD4/CD8 ratios. When the variations‘within persons’ were studied longitudinally over a 5‐year period, the absolute CD4 counts tended to be fixed at different levels. As a marked contrast, over 60% of asymptomatic HIV‐1+ patients exhibited low CD4 counts <0·4 ± 109/l together with inverted CD4/CD8 ratios. Such combined changes among the heterosexual and HIV‐1‐seronegative homosexual groups were as rare as 1·4% and 3%, respectively. For this reason, when the lymphocyte tests show <0·4 ± 109/l CD4 count and a CD4/CD8 ratio of less than unity, the individuals need to be investigated further for chronicity of this disorder, the signs of viral infections such as HIV‐1 and other causes of immunodeficiency.

The natural history of human immunodeficiency virus infection in a haemophilic cohort

112 haemophilic patients infected with HIV were followed up with clinical and laboratory assessment between 1 December 1979 and 30 November 1988. Sixty‐six (59%) of the patients developed HIV‐related clinical symptoms and 22(20%) developed AIDS. Twenty (18%) of the patients developed p24 antigenaemia. Amongst the 59 patients whose date of seroconversion could be estimated the calculated 8‐year cumulative incidence of AIDS was 40% (symptoms 73%). For the whole cohort of 112 patients, the median slope of linear regression of the absolute T4 lymphocyte count was steeper for those with AIDS (‐0.113 × 109/1 per year) than for those without AIDS (‐0.054 × 109/1 per year) (P < 0.02). While 15 cases of AIDS developed during 58 patient‐years of follow up after falling below a T4 lymphocyte count of 0.2 × 109/1, only two cases occurred during 450 patient‐years before reaching this count. Thus the decline of the T4 lymphocyte count to 0.2 × 109/1 may be an appropriate additional end‐point for the assessment of new treatments for asymptomatic patients infected with HIV.

Homozygous protein C deficiency with delayed onset of symptoms at 7 to 10 months

We report an inbred family with two cases of homozygous protein C deficiency and review 11 other such cases. Both patients presented in the second half of their first year of life with recurrent rapidly disappearing ecchymotic skin lesions, disseminated intravascular coagulation, and venous thrombosis. Successful treatment has been achieved by frequent infusions of plasma or prothrombin complex then maintained with Warfarin. Homozygous recessive protein C deficiency usually presents in the neonatal period with purpura fulminans. Two cases have been described elsewhere which presented in the second decade of life with milder symptoms. The present cases appear to be intermediate in time of presentation and severity of symptoms. We also review the distinction that is now evident between recessive and dominant protein C deficiency.

Rapid radioimmunoassay for fibrinopeptide a in human plasma

Abstract A rapid method for the assay of fibrinopeptide A (FPA) in human plasma has been developed which allows results to be obtained within 2 1 2 hours of blood collection. Bentonite is used to remove fibrinogen from plasma. The assay is simple and sensitive. The normal range for plasma FPA (0.3 – 1.5 pmols/ml) is similar to that reported using the original method. There was a good correlation between results obtained by the rapid and original methods on samples obtained from patients with raised levels of circulating FPA and/or fibrinogen/fibrin degradation products, although the rapid assay tended to give slightly lower values. FPA levels in sequential samples drawn through 19-gauge ‘Butterfly’ needles become progressively lower in successive samples. A larger than usual discard is therefore needed to accurately assess basal levels. Comparisons between two antisera showed differences in affinity for FPA and cross-reaction with desaminotyrosyl FPA, but such differences are unlikely to cause problems when assaying clinical samples.

Rationale and evolution of therapy with porcine factor VIII:C

Highly purified porcine factor VIII:C (FVIII:C) concentrate prepared by polyelectrolyte fractionation has been available for therapeutic use since 1980. Over the last decade substantial international experience has confirmed the value of porcine FVIII:C in management of hemophilia with inhibitors, and recent studies have underlined its particular effectiveness in treating patients with the acquired form of the disease. The rationale for use of porcine FVIII:C is based on a twofold premise. First, most inhibitors interact less strongly with porcine FVIII:C than they do with the human factor; cross-reactivity is especially low, and often negligible, among patients with acquired disease. Second, when measurable levels of circulating FVIII:C can be achieved, the likelihood of clinical hemostasis is maximized. In a variable proportion of patients with the congenital disease, anamnestic rises in titers of the inhibitor against human FVIII:C may follow treatment with the porcine factor, and this phenomenon may constrain therapy. These events seem to occur rarely in persons with acquired inhibitors, however, thus broadening therapeutic application of porcine FVIII:C to these patients. Although anamnesis often is perceived as a limitation, significant untoward transfusion reactions are highly unusual after porcine FVIII:C therapy. Although early experience with this form of treatment centered on management of major bleeding episodes and hemostatic crises, use of porcine FVIII:C has more recently been extended to more routine bleeding problems, immune tolerance induction regimens, prophylaxis, and home therapy. These and other advances are discussed.

Serological response and detection of viraemia in acute hepatitis C virus infection

The serological response during acute hepatitis C virus (HCV) infection was examined by enzyme-linked immunosorbent assay (ELISA) in sequential serum samples from 13 haemophiliacs following their first exposure to factor VIII concentrates contaminated with HCV. The commercially available C100-3 peptide and a new 22 kDa recombinant protein (p22) encoded by the nucleocapsid region of the viral genome were used for antibody detection, whilst a nested polymerase chain reaction (PCR) method was used for the detection of viraemia. In addition, eight sporadic cases of acute HCV infection were studied. The results in haemophiliacs demonstrated that seroconversion to the C100-3 antigen occurred in only one-third of the patients within 12 weeks of disease onset, but all of the patients had a diagnostic serological response to p22 during this phase of the disease. The new test was positive in all the sporadic cases at a time when the commercially available test was negative. Although PCR offers a sensitive method for the detection of recent HCV infection, the complex methodology makes it unsuitable for diagnostic laboratories. The new ELISA test with p22 may therefore have a useful diagnostic role in acute disease.

AIDS and children: Some of the issues in haemophilia care and how to address them

Haemophilia is a life-long, life-threatening inherited condition for which there is treatment but no cure. It affects not only the patient but all members of the family. This paper focuses on how AIDS/HIV, which shares these characteristics with haemophilia, has affected children with HIV, their siblings, those who have remained antibody negative and those with HIV infected fathers. The impact of haemophilia and AIDS on the family is considered and the ripple effect of AIDS on health care staff is addressed.

Hepatitis C antibody assay in a longitudinal study of haemophiliacs.

Summary. Stored sera from 28 patients with inherited coagulation disorders who had developed non‐A non‐B hepatitis (NANBH) following a first exposure to clotting factor concentrates and 15 similar, but unmatched, patients who had received blood products but had normal transaminases on sequential testing were tested using the Ortho enzyme‐linked immunosorbent assay (ELISA) anti‐HCV assay. Twenty‐seven of the 28 patients with NANBH were anti‐HCV positive after exposure. In 10 of those in whom dates of first exposure and seroconversion were well‐defined, the median time interval to NANBH was 4 weeks (range 1–7) and to anti‐HCV seroconversion was 11 weeks (range 7·5–14·5). None of the 15 patients without NANBH developed anti‐HCV. This first generation Ortho ELISA anti‐HCV assay showed 96% sensitivity and 100% specificity and has potential use as an adjunct in the surveillance of new clotting factor products.

The application of polyethylene glycol to radioimmunoassays used in haemostasis

Use of polyethylene glycol 6000 in the second stages of double antibody radioimmunoassays for fibrinopeptide A, B-thromboglobulin and platelet factor 4 facilitates the more rapid separation of free from bound antigen, and allows precipitating antiserum to be used in greater dilution. At a final PEG 6000 concentration of 5%, separation of free from bound antigen was complete within 1 hour, and antisera could be used in dilutions 3-9 times greater than those recommended by the manufacturers. PEG 6000 had a negligible effect on the affinity of first stage antibodies for their respective antigens. Radioimmunoassays using PEG 6000 were sensitive to protein concentration, failure to adjust standards to similar protein concentrations as those of test samples causing artefactually low results.

A flow cytometric analysis of fibronectin binding to platelets from patients with peripheral vascular disease

Flow cytometry using fluorescently-conjugated antibodies has been used as a sensitive technique to study the expression of glycoproteins on the surface of resting and activated platelets (8-10). This technique permits analysis of single cells and thus can be used to detect heterogeneous expression of cell surface antigens or ligands (11). This paper reports the results of a flow cytometric analysis of platelet bound fibronectin to resting and thrombin-stimulated platelets from a group of 13 patients with peripheral vascular disease.