J. G. Huisman

ASSOCIATION BETWEEN BIOLOGICAL PROPERTIES OF HUMAN IMMUNODEFICIENCY VIRUS VARIANTS AND RISK FOR AIDS AND AIDS MORTALITY

49 individuals seropositive for human immunodeficiency virus (HIV) antibody were studied longitudinally for the relation between in-vitro properties of their sequential HIV isolates and clinical course before and after the development of AIDS. They were classified into three groups according to the syncytium-inducing capacity, replication rate, and host range of their HIV isolates. The most rapid progression to AIDS (median 15 months) and the lowest survival rate following AIDS diagnosis (median survival 12.5 months) were observed in individuals with high-replicating, syncytium-inducing HIV isolates, followed by individuals with high-replicating, non-syncytium-inducing isolates. In contrast, most individuals with low-replicating, non-syncytium-inducing HIV isolates remained symptom-free during the study period (median follow-up until AIDS diagnosis greater than 42 months), and the few individuals from this group in whom AIDS developed were still alive at the end of the study period (median survival greater than 34 months). In addition, AIDS patients from the three groups differed with respect to their symptoms. Zidovudine treatment in the symptom-free period seemed to delay the onset of AIDS in all risk groups, although stabilisation of CD4+ cell numbers was observed only in individuals with non-syncytium-inducing HIV variants.

Blood Donors with Indeterminate Anti‐p24gagReactivity in HIV‐1 Western Blot: Absence of Infectivity to Transfused Patients and in Virus Culture

Abstract. During a follow‐up period of 23–40 months, 7 regular blood donors had persistently, and 4 had intermittently indeterminate anti‐p24gagreactivity in human immunodeficiency virus (HIV)‐1 Western Blot. Serological testing and viral cultures revealed that these donors had no signs of infection for HIV‐1, HIV‐2, human T‐cell lymphotropic virus (HTLV)‐4, and HTLV‐1. Extensive interviewing and physical examination of these donors revealed neither risk factors, nor signs of HIV infection in the tested donors. Ten recipients, who were transfused with blood products from 6 of these 11 anti‐p24gag‐positive donors, were traced back. Six months after transfusion, no serological or clinical signs of HIV‐1, HIV‐2, or HTLV‐1 infection were observed in these patients. It is concluded that blood donors with persistent or intermittent anti‐p24gagreactivity in HIV‐1 Western Blot, without development of antibodies to other HIV‐encoded proteins in later blood samples, do not transmit the described retroviruses to transfused patients.

Detection of Early Anti-p24 HIV Responses in EIA- and Immunoblot-Negative Individuals

Abstract. A sensitive and specific radioimmunoprecipitation assay was developed for the detection and analysis of anti‐HIV antibody response in human sera with the use of 125I‐labelled purified HIV proteins with subsequent sodium‐dodecylsulfate gel electrophoresis (125I‐RIPA). The 125I‐RIPA was shown to be as specific but at least 1 log more sensitive with respect to the detection of gp41env and p24gag than the immunoblot analysis as tested in serum samples from several risk groups. Sequential sera were obtained from 9 individuals who seroconverted for HIV antibodies. In 4 individuals, antibody to p24gag was detected in earlier serum samples by the I25I‐RIPA than by EIA or immunoblot; in the other 5 individuals, the detection of p24gag concorded in enzyme‐linked immunosorbent assay (EIA), immunoblot and 125I‐RIPA. Moreover, in one of 78 randomly chosen EIA‐negative sera from individuals at high risk, antibodies to p24gag could be detected by the 125I‐RIPA. This early seroconversion was confirmed 3 months later by means of immunoblotting and EIA. The specificity of the 125I‐RIPA was further demonstrated by analyzing sequential EIA‐negative serum samples from 10 individuals at risk for AIDS, collected during 2 years at 3‐monthly intervals. All 80 serum samples were found to be negative in the 125I‐RIPA and the individuals revealed no signs of HIV infection. The I25I‐RIPA technique may be a valuable confirmatory assay in the serology of HIV infections. The sensitivity of this test provides a reliable measure of effective sensitivity when new‐generation screening tests are evaluated. In addition, this technique appears to be a specific and sensitive assay to detect antibodies to p24gag at very early stages of HIV infection.

Platelet‐Associated IgG in Thrombocytopenia: A Comparison of Two Techniques

Abstract. The results obtained in the analysis of 130 thrombocytopenic patients with a radioimmunoassay (RIA) for platelet‐associated IgG (PA‐IgG) and the platelet suspension immunofluorescence test (PIFT) were compared. The RIA was positive in 33 of 41 (82.9%) patients with idiopathic thrombocytopenia (ITP) and in 51 of 79 (64.4%) patients with secondary thrombocytopenia (STP). The PIFT was positive in 37 of the 41 (90.2%) ITP patients and in 57 of the 79 (72.2%) STP patients. Sensitivity and specificity for the diagnosis of ITP of both tests were comparable: 82.9 and 40.9% for the PA‐IgG(RIA) and 90.2 and 36.7% for the PIFT. A significant positive correlation was observed between the mean amount of PA‐IgG measured and the height of PIFT scores with anti‐IgG. Of 38 discrepancies between PA‐IgG(RIA) and PIFT with anti‐IgG, 15 were due to borderline results, 17 were associated with abnormal platelet‐size distribution and 20 were associated with occurrence of IgM antibodies. These results suggest influences of platelet fragments and/or aggregates on accurate measurement of PA‐IgG. Both fragments and aggregates escape from accurate platelet counting, while their contribution to the total IgG content remains. Therefore, a falsely elevated PA‐IgG (RIA) may be measured.

Quantification of Platelet‐Bound Alloantibodies by Radioimmunoassay: A Study on Some Variables

Abstract. Several variables that may affect accurate measurement of platelet‐associated IgG (PA‐IgG) were studied using a radioimmunoassay of the consumption type. The amount of PA‐IgG of washed, unfixed normal donor platelets was 1.0 ±0.9 fg IgG/platelet (mean ±2 SD). Upon storage of washed platelets in a buffer containing EDTA, the amount decreased significantly to 0.2 ± 0.2 fg IgG/platelet. Simultaneously, an increase in modal platelet volume was observed. Similar results were obtained when platelets were fixed with paraformaldehyde (PFA). We postulate that this decrease in PA‐IgG is caused by the release of plasma IgG entrapped by the surface‐connected canicular system of the platelet, when the platelets swell during storage in EDTA or fixation with PFA. This presence of varying amounts of entrapped plasma IgG may cause the wide discrepancies in PA‐IgG found in normal donor platelets as well as platelets from ITP patients by other investigators. A good quantification of platelet‐bound alloantibodies was possible with our assay when platelets were routinely fixed to diminish the amount of nonspecific PA‐IgG. This was demonstrated with different anti‐Zwa (=anti‐PlA1), anti‐Baka and anti‐HLA sera. We also observed that fragments of platelets as well as fragments of cells of other types can cause aspecifically increased Pa‐IgG values and can thus interfere with the proper measurement of platelet‐bound antibodies in all kinds of immunoassays in general.

Diagnosis and Prevalence of HIV‐2 Antibodies in Different Population Subsets in The Netherlands

Abstract. A serum panel obtained from male homosexuals (n = 278); i.v. drug abusers (n = 99), patients attending a VD clinic (n = 390), blood donors who visited Central or West Africa (n = 573), blood donors who had sexual contact with natives from Central or West Africa (n = 38), blood donors from Surinam, South America (n = 481), individuals positive for anti‐HIV‐1 (n = 94), individuals with indeterminate HIV‐1 western blot (WB) reactions (n = 73), African patients with AIDS or AIDS‐related symptoms (n = 30), and random Dutch blood donors (n = 555), was tested with HIV‐2 ELISA (ELAVIA‐2). Of these 2,611 samples, 32 (1.2%) were repeatedly reactive. Antibodies to gp140/105env were found in 4/32 by HIV‐2 WB, and in 1/4 by radioimmunoprecipitation (RIPA). These 4 HIV‐2 WB‐positive samples were also reactive with gp160/120env in HIV‐1 WB, suggesting cross‐reactivity. In a spot test with synthetic peptides of the transmembrane glycoprotein of both HIV types, 3/4 were only HIV‐1 positive and 1/4 was strongly HIV‐2 positive and weakly HIV‐1 positive. In inhibition assays with soluble HIV‐1 or HIV‐2 synthetic peptides in HIV‐1 and HIV‐2 peptide ELISA, cross‐reactivity was excluded, which indicates an HIV variant or HIV‐1/HIV‐2 double infection. It is concluded that for the moment HIV‐2 infection is at low prevalence in risk groups in The Netherlands, and that in addition to WB and RIPA, synthetic peptide assays are useful for differentiation between HIV‐1 and HIV‐2 antibodies.

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia

A patient is described with a disturbance of platelet function comparable to that in Glanzmanns thrombasthenia. Platelet aggregation and binding of fibrinogen to the patients platelets were defective and thrombin‐induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex‐dependent anti‐GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22°C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two‐dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.

Monitoring of Platelet Contamination in Filtered Red Blood Cell Concentrates

Abstract. Monitoring of contaminating platelets, granulocytes, and lymphocytes in leukocyte‐poor red blood cell concentrates is usually done by counting in an electronic particle counter. Sensitivity and specificity of this technique are compromised by the contamination of the preparations with other cell types and particles thereof. In this report we studied platelet contamination in filtered red blood cell concentrates by use of a radioimmunoassay for detection of the platelet glycoprotein complex Ilb‐IIIa. Our results indicate that platelets and/or fragments thereof, not detectable for particle counters, are present in blood cell concentrates. This finding might have important implications for the preparation of pure red blood cell concentrates to avoid unwanted immunization after transfusion.