False reactivity in GTI Pak Plus ELISA kits due to the presence of anti-mouse antibody in patients' samples.

The development of commercially available ELISA kits (GTI, Inc., Waukesha, WI) that use antigens adhered to microtiter plate wells by the use of mouse monoclonal antibodies made it possible for hospital transfusion service laboratories to test for platelet- and/or HLA-specific antibodies without reliance on reference laboratories. However, human anti-mouse antibodies (HAMAs) may cause false reactions in ELISAs. We designed a study to determine the impact of HAMAs on these ELISAs. Samples from 210 patients were evaluated from January 1995 to April 2002; 79 (38%) were found to be positive for HLA- and/or platelet-specific antibodies. Thirty (38%) of these positive samples,as well as ten negative samples that served as controls, underwent HAMA neutralization/inhibition procedures before being retested by ELISA. One (10%) of the control samples was reactive after treatment. When the samples that were positive in routine testing were treated to neutralize/inhibit HAMAs, reactivity was unchanged in 20 (67%); reactivity was eliminated in eight (27%) of the samples tested. All of the specimens that showed a reduction or elimination of their reactivity after neutralization/inhibition had an initial optical density (OD) ratio < 3.0 whereas those that remained unchanged in reactivity had an OD ratio > 7.0 (p < 0.05). Reactivity present only in the treated samples was observed in three (10%) of the positive samples tested;one was additionally reactive with HLA antigen only and two with glycoprotein Ia/IIa. The presence of HAMAs should be considered when antibodies against more than one platelet-specific glycoprotein are detected and if the optical density ratio is < 3.0.

Development of anti-Bw6 reactivity in patients receiving r-GCSF: a preliminary report.

Recombinant granulocyte colony-stimulating factor (r-GCSF) is used in autologous bone marrow/peripheral blood progenitor cell transplantation (ABMT/PBPC) to shorten the period of neutropenia. As these patients require platelet transfusions, their sera may be monitored for the presence of platelet/human leukocyte antigen (HLA) antibodies. Sera of some patients on r-GCSF (16 microg/kg/day) became difficult to evaluate in vitro for the presence of HLA and platelet antibodies because of apparently anomalous reactions in solid phase red cell adherence (SPRCA) assays. SPRCA tests were positive only when platelets were adhered to the polystyrene plates in the presence of glucose; when other simple sugars were used, the patients' sera failed to react. HLA Bw6-positive platelets were more likely than HLA Bw6-negative platelets to be reactive (p <.001). The transfusion of HLA Bw6-positive platelets to patients displaying this in vitro reactivity (positive patients) resulted in a 50 percent lower corrected count increment (CCI) than those given to patients without it (negative patients; p = <.001). When all transfusions were considered, the CCI for those of the positive patients was decreased 73 percent when compared to the control patients (p =.0005). The presence of the antibody also was associated with a twofold increase in the number of platelet transfusions given (p =.0005). ABMT/PBPC patients receiving r-GCSF may develop unexpected reactions on SPRCA antibody screens and poor responses to transfusion of Bw-6-positive platelets.

Frequency of thrombocytopenia associated with gentamicin therapy.

Many drugs, including antibiotics such as gentamicin, have been associated with the development of a drug-induced thrombocytopenia. Serologic methods for detection of drug-dependent platelet antibodies (DDPAs) are not routinely performed, and the incidence of such antibody-mediated thrombocytopenia is not known. As we routinely perform solid-phase red cell adherence assays for the detection of DDPAs, a study was designed to determine the incidence of gentamicin- associated platelet antibodies (GAPAs) in our institution. Adult patients who received gentamicin from 1/1/98 to 7/31/98 were evaluated for inclusion in the study. Testing for the presence of GAPAs was performed if the patient had a decrease in platelet count while receiving gentamicin or if the platelet count increased or decreased within 3 days of the last gentamicin therapy. Patients receiving gentamicin without development of thrombocytopenia were tested as controls. During the study period, 926 patients received gentamicin, with 324 (35%) being evaluated for the presence of GAPAs; GAPAs were identified in 25 of 659 patients (4%) eligible for the study. All control samples were found to lack GAPAs. If only patients exhibiting changes in platelet counts are considered, the incidence increases to 7.7 percent, with females apparently being almost twice as likely to develop GAPAs than are males. Gentamicin-associated thrombocytopenia is not an infrequent occurrence in hospitalized patients.

Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays.

We developed a simple modification of the solid-phase red cell adherence (SPRCA) assay system that can be used to identify drug-dependent platelet antibodies (DDPAs) reactive by either the hapten or immune complex reaction mechanisms. Between January 1994 and August 1996 we tested sera from 173 patients [123 (71%) with unexplained thrombocytopenia and 50 (29%) because of poor responses to platelet transfusions not explicable by alloimmunization or the clinical situation] for DDPAs possibly associated with the receipt of 61 different drugs. We correlated positive results with patients' clinical courses. DDPAs were identified in samples from 138 (80%) of the patients tested. Antibodies reactive only by the hapten mechanism were identified in 51 (37%) of those sera exhibiting positive reactions. The clinical courses of 108 (78%) patients were evaluable. Discontinuation of the implicated drug(s) resulted in prompt (<5 d) resolution of the thrombocytopenia or improvement in response to transfusion in all of these patients. In four cases thrombocytopenia returned upon re-exposure to the implicated drug. This adaptation of SPRCA provides a simple means of investigating the possibility of DDPAs and documents a higher frequency of these antibodies than has previously been suspected.

Detection of drug-dependent platelet antibodies by use of solid-phase red cell adherence techniques.

Many drugs have been reported to cause drug-dependent thrombocytopenia, either by the immune complex or by hapten mechanisms. Testing for the presence of these platelet antibodies has not been considered feasible for transfusion services because their presence was thought to be rare, and their detection involved complex and costly methods. We have developed a new technique for detection of these antibodies that can be performed without the need for specialized and expensive instrumentation. A solid-phase red cell adherence assay was used to detect drug-dependent platelet antibodies active by either the immune complex or the hapten mechanism. Three cases were evaluated for the presence of drug-dependent platelet antibodies. Two patients presented with thrombocytopenia that could not be attributed to other causes. The third case was evaluated for the presence of drug-dependent antibodies after poor responses to platelet transfusions. In these three cases, discontinuation of the implicated drugs, i.e., porcine heparin, quinine sulfate, amoxicillin, Bactrim, and albuterol, was followed by a correction of thrombocytopenia or improved platelet transfusion response within 72 hours. This test methodology and protocol has proven very useful in avoiding transfusions with little likelihood of benefit, and in identifying drugs interfering with platelet recovery or survival. Further investigations with this technique may expand our knowledge of the capability of this technique and of the observed frequency of drug-related immunologic platelet destruction.

Effect of storage time on clinical efficacy of single-donor platelet units.

Decrements in platelet function and recovery are known to accumulate during the 5-day storage period, and the clinical response to platelets transfused after several days' storage has been suggested by some researchers to be less than that seen with platelets stored for shorter periods. The clinical response to single-donor platelet (SDP) units (as measured by corrected count increments [CCIs] and intertransfusion intervals) was investigated in autologous bone marrow transplant patients. Twenty-seven consecutive autologous bone marrow transplant patients with a variety of hematologic and solid organ malignancies were evaluated for posttransfusion CCIs after 419 SDP transfusions of units stored for 1 to 5 days. Patients were not excluded from the study because of clinical condition, such as fever or sepsis. The mean 15-minute posttransfusion CCI for SDP units stored for only 1 day (11,006 +/- 5,157) was no different than that for units stored for 5 days (10,225 +/- 4,481; p > 0.05); 24-hour posttransfusion CCIs were also not different if the SDP unit had been stored for 1 day or 5 days (6229 +/- 4489 vs. 4786 +/- 2759; p > 0.05) or for any intermediate period. Nor were intertransfusion intervals affected by storage time. While platelets may exhibit a progressive lesion during the 5-day storage period, these changes do not result in a decreased clinical response.

Selection of platelets for refractory patients by HLA matching and prospective crossmatching.

A multi-site clinical study compared platelets chosen for refractory patients by prospective platelet crossmatching using stored donor platelets and HLA-based selection. Seventy-three patients who were refractory to random-donor platelets received two plateletpheresis components, one chosen by HLA-based criteria and the other by crossmatching. Patients were carefully evaluated to exclude nonimmune factors that could adversely affect transfusion results. Each of the five study sites used a crossmatch procedure with which it had experience. Results from this study indicate the following: 1) The overall rate of successful transfusion was similar when an HLA-based method of donor selection that includes all grades of matching and mismatching was compared to a crossmatch-based method of donor selection. 2) HLA-based selection that restricts recipients to grade A and BU matches was superior to a selection method based upon crossmatching alone. Donor selection based on HLA matching (grades A or BU) was also superior to selection based on any degree of HLA mismatching (grades BX, C, or D). 3) Selection of donors based on HLA-cross-reactive groups (defined by in vitro serologic crossreactivity) was no more successful than that based on grade C and D mismatches and was no more successful than selection by crossmatching alone. 4) Lymphocytotoxic and platelet antibodies were not detected in many of the enrolled patients, even though patients demonstrating nonimmune factors were eliminated from the study. It can be concluded that HLA-compatible (grades A and BU) platelets provide optimal support for refractory patients, but that crossmatch-selected platelets are acceptable as an alternative component.

Depressed interferon synthesis in skin fibroblasts from lung cancer patients.

Skin fibroblast cell cultures, derived from male adult lung cancer patients, an adult control population, and a newborn population were examined for their susceptibility to transformation with Kirsten murine sarcoma virus and their ability to respond to an interferon inducer (poly I.poly C). An association between sensitivity to viral transformation and induction of interferon was observed. Cultures derived from lung cancer patients demonstrated an increased sensitivity to virus transformation and a decreased ability to respond to interferon induction as compared with age-matched controls and newborns.

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

Characteristics and aldehyde dehydrogenase activity of four rat hepatoma cell lines produced by diethylnitrosamine-phenobarbital treatment.

Recent studies in our laboratory have shown that five established rat hepatoma cell lines provide a wide spectrum of tumor-associated aldehyde dehydrogenase (ALDH) activity representative of the range of activities of this enzyme seen in primary rat hepatocellular carcinomas. Four newly established rat hepatoma cell lines, RLT-2M, RLT-3C, RLT-9F, and RLT-5G, were derived from a primary hepatocellular carcinoma. The primary tumor was induced by a single injection of diethylnitrosamine (15 microM/g body weight) to a 1-d-old female S-D rat followed at weaning by chronic phenobarbital treatment. RLT-2M was established from outgrowths of minced tumor pieces. RLT-3C, RLT-9F, and RLT-5G were cloned from RLT-2M by the serial endpoint dilution. All four lines have been maintained in culture for over 100 passages. The ALDH phenotype in both the primary tumor and the four new cell lines was determined by total activity assay, gel electrophoresis, and histochemistry. By total activity assay, the primary tumor did not possess significant tumor-ALDH activity. In contrast, the four new cell lines expressed tumor-ALDH activity. However, they differed in their basal ALDH activities and in ALDH inducibility by 3-methylcholanthrene, benzo(a)pyrene, and phenobarbital. Additionally, significant decreases in tumor-ALDH activity occurred when cells from each line were passaged in vivo. The four lines have been characterized by light and electron microscopic morphology, tumorigenicity, chromosome number, doubling time, and colony formation efficiency in soft agar.

Depressed interferon synthesis in skin fibroblasts from lung cancer patients.

Skin fibroblast cell cultures, derived from male adult lung cancer patients, an adult control population, and a newborn population were examined for their susceptibility to transformation with Kirsten murine sarcoma virus and their ability to respond to an interferon inducer (poly I X poly C). An association between sensitivity to viral transformation and induction of interferon was observed. Cultures derived from lung cancer patients demonstrated an increased sensitivity to virus transformation and a decreased ability to respond to interferon induction as compared with age-matched controls and newborns.