Howard M. Reisner

Probabilistic classification of hemophilia A carriers by discriminant analysis

Abstract The linear discriminant procedure is described for obtaining, on the basis of F.VIII-related antigen determinations and F.VIII coagulant activity, appropriate formulas for calculating the probability that a woman is a carrier for hemophilia A. In applying the method to two separate bodies of data, a significant effect of age was observed on the F.VIII-related activities, and allowing for this increased the accuracy of discrimination. Tests for validity demonstrated that in each of the two sets of data a linear discriminant was valid provided: (i) allowance was made for the effect of age where appropriate, (ii) logarithmic transformations were taken of both the F.VIII-related antigen determinations and the coagulant activity, and (iii) allowance was made for the fact that the variance of the discriminant is significantly larger in the carriers than in the normals.

Measurement of human factor IXa activity in an isolated factor X activation system

To determine the functional properties of factor IX isolated from the plasma of CRM+ hemophilia B patients, an assay system using proteins isolated from human plasma had to be developed which would be amenable to kinetic studies under a variety of experimental conditions. The present study describes the activation of factor X by factor IXa, isolated from normal human plasma, in an assay system which allows manipulation of calcium, phospholipid and factor VIIIa concentrations. Initial rate measurements with factor VIIIa present in the system were made by incubating factor VIII with factor Xa immediately before factor IXa assay. With this approach, the initial rate of factor X activation was constant, with little evidence for a lag period. Within the framework of the assay system described in the present study, it should be possible to examine not only genetic variants of factor IX, but also variants of factor VIII, as well as providing a means of routine factor IXa and factor VIII(a) assays.

Use of a simple visual assay of Willebrand factor for diagnosis and carrier identification.

Summary. A visual assay of factor VIII‐related Willebrand factor (VIIIR:WF) is described which utilizes formaldehyde‐fixed platelets, end points being read in microflocculation tiles. Four dilutions of a sample can be assessed simultaneously, and the correlation with aggregometric assays is high (r=0.91). Measurement error is 8.0% for a single assay in triplicate and less than 5% if an assay is repeated three times. The method has been used for 2 years by the coagulation genetics group at Chapel Hill for diagnosing subjects with von Willebrands disease and assigning genotypes to members of families transmitting this disorder. Its utility in classifying known carriers of haemophilia A has also been examined, both in conjunction with assays of VIII:C and in a three‐way test with assays of VIII:C and VIIIR:Ag. As predicted by the Lyon hypothesis, the rate of false negative diagnosis was higher than false positive diagnosis, but the overall rate of misclassification on single plasma samples was 7/51=13.7%. The error rate was the same whether discrimination was based upon assays of VIII:C vs. VIIIR:Ag, VIII:C vs. VIIIR:WF, or VIII:C vs. VIIIR:Ag vs. VIIIR:WF, the same individuals being misclassified by each method. The observed rate of misclassification was well within the rates reported by others and very similar to our previous experience. We have concluded that this method of assaying VIIIR:WF is highly useful for diagnosing vWd, detecting inhibitors to VIIIR:WF, and examining large numbers of column fractions. It is a useful supplement, although it cannot yet substitute for, assays of VIIIR:Ag in detecting carriers of haemophilia A.

Lymphoblastoid cell-produced immunoglobulins: Preparative purification from culture medium by hydroxylapatite chromatography

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.

Genetic counselling in haemophilia by discriminant analysis 1975-1980

Between January 1975 and January 1980 we counselled 214 possible or obligatory carriers of haemophilia A in an attempt to provide the counsel they needed and to determine the utility of a probabilistic method of assigning the heterozygous genotype (carrier detection). We found the method of assignment to be quick and easy to use, and the single output (the final probability favouring carriership P(C)) to be understood by most counsellees. The final probabilities obtained were either very high or very low in 80% of the women, which allowed us to give clear counsel in four instances in five. The final probabilities could also be used to relate Mendelian expectation to observation within each of three subsets of women (18 mothers of sporadic haemophiliacs, 78 sisters, and 62 more distant relatives), while the aberrant likelihood ratios of 6/36 (17%) of the obligatory carriers provided an estimate of the false negative diagnostic rate owing to lyonisation. There was no significant age effect on VIII:C or VIIIR:Ag levels of obligatory carriers, and the VIII:C levels of the obligatory carriers who had received the gene from their fathers did not differ from those of the obligatory carriers who had received the gene from their mothers. The ratios of high:low probabilities among the sisters and distant relatives of haemophiliacs conformed to genetic expectation, while the ratio among mothers of sporadic haemophiliacs suggested that their expectations of carriership were greater than 80%. Twenty of the 214 counsellees (9%) were pregnant on the first visit, and 13 of those with low P(C)s (0·0-0·33) went to term and delivered 11 non-haemophilic children. Four with P(C)s between 0·50 and 1·00 requested amniocenteses, and one male was aborted. Three who were obligatory carriers also requested amniocentesis which led to the abortion of a second male. Seven women who were assessed before pregnancy and found to have high P(C)s returned after becoming pregnant. All requested amniocentesis (one twice) and fetoscopy was requested for six of the seven males discovered, one male having been aborted before fetoscopy became available. Of the six males who were fetoscoped, three normal males reached term, one normal male was born prematurely, and one haemophilic male and one normal male did not survive fetoscopy.

Endothelial Cell Hybrids and the Suppression of Factor VIII Related Antigcn Exprcssion

Hybrid somatic cell clones have been generated by fusing human vascular endothelial cells in primary culture to cells of four rodent lines. Factor VIII related antigen (VIIIR:Ag) was clearly demonstrable in the cultured endothelial cells, even when they had been co‐cultured with rodent cells. Hut in none of 14 hybrid clones was VII1R; Ag detectable. Isozyme analyses for human chromosome markers show that all the assayed human chromosomes were represented among the hybrids, and that various subsets of human chromosomes have been deleted from individual hybrid clones. It may be concluded, therefore, either that VIIIR:Ag production depends on a particular combination of human chromosomes not represented in any of the hybrids, or that the rodent cells contribute some agent which intracellularly blocks VIIIR:Ag expression.

The genetics of blood coagulation

Hemostasis is the term applied to the process that regulates the loss of blood from the circulatory system following injury. It involves three interrelated physiological mechanisms: constriction of blood vessels, aggregation of blood platelets to damaged subendothelial surfaces, and the formation of fibrin clots. Together these produce the vascular plug that prevents further bleeding. Abnormal function of one or more of the separate mechanisms may result in excessive bleeding or hemorrhage.

The Separation of Willebrand Factor from Factor VIII‐Related Antigen

Summary. The three activities associated with factor VIII—coagulant (VIII:C), antigenic (VIIIR:Ag), and platelet agglutinating or Willebrand factor (VIIIR:WF)— have been separated by sequential antibody affinity chromatography, utilizing a rabbit antibody to factor VIII and a spontaneous human antibody to VIII:C. Normal plasma differentially lost its factor VIII‐related antigen following passage over the rabbit antibody column. Subsequent passage of the VIIIR:Ag‐depleted plasma over the human antibody column resulted in the loss of VIII:C activity, with retention of the Willebrand factor activity, antigen being partially recovered from the heterologous antibody column. These experiments demonstrate that it is possible to separate two of the factor VIII activities, VIIIR:Ag and VIIIR:WF, which are usually regarded as properties of a single molecule.

A radioimmunoassay for the Gm(b0) allotype of human immunoglobulins

A radioimmunoassay for the human allotype Gm(b0) which provides a sensitive and quantitative measurement of the level of this IgG3 genetic marker has been developed. The assay system can detect 15 nanograms of Gm(b0) IgG3 protein and is not inhibited by immunoglobulins of other allotypes and isotypes. Using this assay, good correlation was found between IgG3 and Gm(b0) levels in homozygous Gm(f, b0) sera and gene dosage effects could be confirmed. The correlation between Gm(b0) levels and IgG3 in Negroid Gm(a, b0) sera was not as good. This reduced correlation has been attributed to antigen differences in the IgG3 Gm markers characteristic of some Negroid Gm(a, b0) sera.

Immunoassays for pentamidine and related compounds: Development of a facile inhibitory ELISA suitable for clinical use

Aromatic dicationic drugs have a broad spectrum of activity against protozoal and fungal pathogens including Pneumocystis carinii, Leishmania mexicana amazonensis, Cryptosporidium parvum and Cryptococcus neoformans. Pentamidine serves as the exemplar for an extensive collection of newly synthesized related compounds, which have reduced toxicity and a wider range of target organisms. Assays of pentamidine and related compounds have depended on HPLC‐tandem mass spectrometry (HPLC‐TMS) for the quantitation and identification of drug and metabolites. Immunoassays for pentamidine would have many advantages over the HPLC methods including relative simplicity of assay format and required equipment, convenience in sample preparation and reduction in time and cost of assays.