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Biological products are complex molecules and their testing presents unique technical challenges. Furthermore, there are certain expectations for the validation of methods applied for regulatory purposes. A few recent CBER policy initiatives which have an impact on the use of animal tests for biological products will be discussed, followed by an exposé of the MAPREC test for polio virus neurovirulence as an example of alternative test development and the validation process. Finally, some general comments about the validation process will be made.

Immunizations against most vaccine-preventable diseases will be needed indefinitely unless the disease is eradicated. Public acceptance of immunizations may be threatened as vaccine coverage increases and disease decreases, however, due to the increase in both causally and coincidentally related vaccine adverse events. The post-marketing surveillance for such events in the USA in response to the mandatory reporting requirements of the National Childhood Injury Act of 1986. While VAERS has many methodological limitations intrinsic to such systems, it can play an important role in helping to monitor vaccine safety and maintain public confidence in immunizations.

Factor VIII-related antigen (VIII R:Ag) has previously been identified on human platelets by fluorescein-labelled heterologous antibodies to factor VIII. However, it is not known whether these antibodies identify only VIII R:Ag or whether antigens associated with factor VIII coagulant activity (VIII:C Ag) are also present. The current studies utilize specific antibodies and their Fab' fragments to evaluate the presence of both VIII R:Ag and VIII:C Ag on human platelets and to study the functional role of the platelet-associated VIII R:Ag on ristocetin-induced platelet agglutination. Immunofluorescent studies demonstrate the presence of VIII R:Ag, but do not identify VIII:C Ag on platelets. The functional studies reveal that after incubation with either of the Fab' fragments, platelets retain their reactivity in the ristocetin cofactor assay. These results indicate that this population of VIII R:Ag is not essential for the ristocetin aggregation reaction and that no antigenic determinants for factor VIII coagulant activity (VIII:C) are detectable on human platelets by immunofluorescence.

We studied the bias and precision of serum cholesterol analysis by physician's office analyzers. Total imprecision (CV range, %) for analysis of serum pools was: Abbott Vision 1.5%-1.9%; Ames Seralyzer 3.9%-4.5%; BMD Reflotron 2.3%-3.8%; Chrometrics Cholesterol Test System 2.3%-2.8%; Kodak DT-60 1.6%-2.2%. The Ames Seralyzer exhibited an excessive between-run component of variation. We collected, from 109 volunteers, samples of venous serum, heparin-treated whole blood, heparin-treated plasma, and fingerstick whole blood, and analyzed each type (where possible) with each system; serum was also analyzed in duplicate (by a proposed Reference Method) at the Centers for Disease Control. For assays with serum, the BMD Reflotron and Kodak DT-60 exhibited negative bias. All systems gave lower results for plasma and whole blood than for serum. All systems except the Kodak DT-60 were less precise for analysis of patients' sera than for analysis of serum pools; between-specimen variables may influence the results of these systems.

Uteroglobin, a steroid-dependent, small molecular weight (15K) protein in the rabbit, inhibited thrombin-induced aggregation of both rabbit and human gel-filtered platelets (GFP). GFP aggregation by arachidonic acid was not affected by uteroglobin. There were no effects of uteroglobin on thrombin-induced clotting of plasma or purified fibrinogen, or inhibition of thrombin by antithrombin III. Additionally, preliminary results suggest that uteroglobin does not interfere with binding of thrombin to platelets. We suggest that inhibition of platelet aggregation by uteroglobin may function in preventing thrombosis and ensuring free flow of blood through the microvasculature of the uterus and the placenta and may induce some of the antimotility effects of progesterone on the uterus.

To investigate the specificity of terminal deoxynucleotidyl transferase (TdT) as a marker for lymphoblastic lymphoma in the non-Hodgkin's lymphomas, the authors assayed malignant cells from 142 patients with non-Hodgkin's malignant lymphoma, as well as cells from normal lymphoid tissue controls. Neoplastic cells from all 25 patients with lymphoblastic lymphoma were TdT positive. Of the remaining 117 patients with malignant lymphoma of other histologic subtypes, only one patient, with diffuse, large cell immunoblastic lymphoma, had TdT-positive cells. This patient's cells had positive results only by the enzyme assay and were TdT-negative by both immunofluorescence and immunoperoxidase assays. Of 56 patients whose cells were studied by both enzyme assay and an immunoassay method for TdT detection, a discordant result was obtained only on cells from the previously mentioned patient with large cell immunoblastic lymphoma. To determine the optimal immunoassay method, the authors studied malignant cells from 24 patients with non-Hodgkin's lymphoma for the presence of TdT by three different methods: immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin-complex immunoperoxidase. Fifteen of the 24 patients had lymphoblastic lymphoma. The avidin-biotin-complex method produced superior staining, with no false-negative results. False-negative results were seen with both the immunofluorescence and peroxidase-antiperoxidase methods. Other advantages of the avidin-biotin-complex method included a marked decrease in background and non-specific staining and the ability to use higher dilutions of antisera.

A patient who had a primary lung carcinoma in remission but had a solitary metastatic lesion to the cerebellum was referred to the neurosurgery service. He was noted to be thrombocytopenic, and further hemostatic evaluation revealed chronic disseminated intravascular coagulation. He was treated with heparin and platelet transfusions. Although the fibrinogen levels improved, the thrombocytopenia persisted. Four weeks after admission, repeat chest x-ray films and tomograms indicated progressing metastatic nodules. A review of the literature reveals a variety of hemostatic defects associated with tumor metastatic to the brain. This current case indicates the need for careful hemostatic evaluation in all patients with brain lesions.

Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of beta-thromboglobulin and platelet factor 4. The initial assays indicated that a beta-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the beta-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of alpha 1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases.

To determine the usefulness of terminal deoxynucleotidyl transferase enzyme activity as a biochemical marker in the study of lymphoma, we assayed 50 various malignant lymphomas as well as normal lymphoid tissue for this activity. Neoplastic cells from patients with Hodgkin's disease, Burkitt's tumor, Sézary's syndrome, chronic lymphocytic leukemia, leukemic reticuloendotheliosis and lymphocytic lymphomas (poorly differentiated and intermediate) were all without notable enzyme activity. Large-cell tumors of B-cell type were also negative. One of three large-cell tumors without B-cell or T-cell-surface specificity was positive (1.40 nmol per hour per milligram of DNA). Enzyme activity was consistently present only in lymphoblastic lymphomas (all six cases studied), with a mean activity of 7.80 nmol per hour per milligram of DNA. These six cases had heterogeneous surface immunomarkers. We conclude that terminal deoxynucleotidyl transferase is a biochemical marker that will be useful in the study of lymphoma.

The steady state passive efflux of salt from human red blood cells was measured in various low ionic strength media in which the osmotic pressure ranged from 200 to 600 milliosmolar. Sucrose was used as the nonpenetrating nonelectrolyte. If the flux is plotted against the log of the salt concentration, the data for each tonicity can be fitted by three straight-line segments separated by two sharp inflections, one at low external salt concentrations (0.1 to 0.3mM), confirming observations of LaCelle and Rothstein, and a second at higher salt concentrations (20 to 50 mM). As the osmolarity of the medium is increased, the inflection in every case seems to be uniquely determined by the membrane potential calculated from the Nernst equation with use of the chloride ratio. One inflection occurs at about 45 mV and the second at 170 mV in experiments at five different tonicities. Calculations from the Goldman equation suggest that the inflections represent potential-dependent changes to new permeability states. The osmotic pressure of the medium also influences the permeability. The coefficient is systematically reduced as the osmotic pressure is increased.