Rapid platelet-function assay: an automated and quantitative cartridge-based method.

BACKGROUND: The platelet glycoprotein (GP) IIb/IIIa receptor is important in mediating platelet thrombus formation, and the GP IIb/IIIa antagonist abciximab (c7E3 Fab; ReoPro) is effective in preventing thrombotic ischemic cardiovascular complications of unstable angina and percutaneous coronary interventions. Small-molecule antagonists of GP IIb/IIIa based on the Arg-Gly-Asp (RGD) sequence show similar benefit, and some of these agents are orally active. However, there may be significant interindividual variation in response to such antagonists, especially with chronic oral therapy. It will be essential to balance the beneficial antithrombotic effect of these drugs with their potential for causing bleeding. In response to this need, we have developed a rapid platelet-function assay (RPFA), a point-of-care system that provides a quantitative measure of the competence of the GP IIb/IIIa receptor as reflected in the ability of platelets to agglutinate fibrinogen-coated beads. METHODS AND RESULTS: Polystyrene beads were coated with fibrinogen and placed in a cartridge along with a lyophilized peptide that activates the thrombin receptor. Anticoagulated whole blood was added to the cartridge, and then a microprocessor-controlled operation mixed the reagents and detected agglutination between platelets and coated beads. Quantitative digital results were displayed within 3 minutes. Because there is no dilution of the blood, the assay can be used to measure platelet activity in samples that have been treated with GP IIb/IIIa antagonists with high dissociation rates. RPFA results of whole-blood samples treated with different GP IIb/IIIa antagonists correlated well with both conventional turbidimetric platelet aggregation (r2=0.95) and the percentage of free GP IIb/IIIa molecules in the sample (r2=0.96). The mean difference in measurements between RPFA and aggregometry was -4% (+/-4% SD), and the mean difference in measurements between RPFA and free GP IIb/IIIa receptors was -2% (+/-6% SD). CONCLUSIONS: The RPFA provides rapid information on platelet function that mirrors turbidimetric platelet aggregation and reflects GP IIb/IIIa receptor blockade.

The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.

Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Quantitative prediction of transdermal iontophoretic delivery of arbutamine in humans with the in vitro isolated perfused porcine skin flap.

The use of the isolated perfused porcine skin flap (IPPSF), an alternative in vitro animal model, to predict the profile of the concentration of arbutamine in plasma samples from humans after transdermal iontophoretic administration of this novel catecholamine is described. The strategy involved administering the drug in the IPPSF (n = 8) and assaying concentrations of drug in the venous efflux versus time (IPPSF venous efflux profile). Intravenous infusion (n = 7) and transdermal studies (n = 32) were also conducted in humans. The IPPSF profile was then used as an input into an intravenous pharmacokinetic model obtained from the human experiments to predict the profile of concentration of drug in plasma versus time (plasma concentration-time profile) seen after iontophoretic administration. The IPPSF profiles were denormalized according to the parameters used in the human studies (i.e., multiplied by in vivo concentration, electrode area, current, and dosing time). For two different sets of iontophoretic dosing conditions, the concentration-time profiles that were predicted on the basis of the IPPSF study were compared with those seen after delivery to humans.

Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion.

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = -0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS)

A residency-based information system.

The medical literature has grown to an unmanageable size, and we need to develop information systems that provide better accessibility. An internal medicine residency program is an ideal setting for developing a system in which articles are screened for usefulness in clinical decision making and organized to be rapidly accessible and relatively inexpensive to use. In our residency training program, we use clinical problem-solving, journal club article review, and teaching conference preparation to generate appropriate articles from PAPERCHASE (MEDLINE) database searches. With the chief resident supervising the process, articles are then selected and organized in a computerized article file. The software program, Reference Manager (Research Information Systems, Inc., Encinitas, California), stores article citations and supports searches using standard, medical subject heading vocabulary key words. In an 18-month period, residents have collected more than 1800 references appropriate to clinical decision making and have established an article file that is now used daily as a medical information source.

Correlated measurement of platelet release and aggregation in whole blood.

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cost-benefit analysis of a plateletapheresis program.

We determined costs and benefits of a community donor plateletapheresis program (CDPP) designed to provide HLA-matched platelet transfusions for patients who were refractory to random-donor platelets (RDPs). Costs of establishing and maintaining the CDPP were $127,520 for the first year (1982). Benefits were expressed as cost savings attributed to the CDPP. After the program began, the use of RDP in the community was 17,458 units less than projected. Estimates of net cost savings during the first year ranged from $177,570 to $272,253 (1982 dollars; cost-to-benefit ratios were 1:1.39 to 1:2.14.) In a matched cohort study of marrow transplant patients, CDPP platelet transfusions were as effective as those from family donors while total platelet and red cell use was unchanged. In patients with acute leukemia treated with chemotherapy, significant reduction in both platelet and red cell use was seen after institution of CDPP support. We conclude that the CDPP is a cost-effective approach to platelet support.

Plateletapheresis program. II. Computer selection of HLA compatible donors.

A rapid, cost-effective data entry and computerized matching system was developed for selection of HLA compatible cytapheresis donors for platelet-alloimmunized patients. The system was user-orientated with a computer-generated prompting system that facilitated program operation. Expansion of the number of compatible donors was achieved by treating splits and crossreactive HLA antigens as identical. By this mechanism, a 500-member donor pool easily supported the needs of a large patient population for single-donor cytapheresis platelets. An average of 3.4 compatible donors per patient was identified. For only two of the 48 (4%) thrombocytopenic recipients was transfusion support from the available donors inadequate to provide a posttransfusion platelet increment. At the end of 7 months, donation frequency for the 441 donors then in the pool averaged 2.0 +/- 2.4. This figure increased to 2.8 +/- 2.4 donations for the 176 panel members who actually underwent cytapheresis. Thus, the impact on donors participating in this program was not excessive.

Plateletapheresis program. I. Donor recruitment and commitment.

A dedicated cytapheresis donor program was developed to meet the need for granulocytes and single-donor platelets. A study was undertaken to determine the most effective way to recruit a panel of 500 HLA-typed committed volunteer cytapheresis donors. Factors analyzed included ease of donor recruitment, information necessary to assure donor commitment, rate and reasons for donor attrition, and donor reaction to participation in the program. Audiovisual educational materials were tested as a part of the recruitment process in terms of both motivating volunteer donors and guaranteeing their continued participation. Over a 16-month interval, 793 repeat donors of whole blood expressed an interest in joining the program. A simple brochure was enough to stimulate the majority of these donors (661 or 83%) to consider participation. Overall, 481 (61%) joined the program and were HLA typed. Of these, 311 (65%) required no additional information other than a brief phone exchange to answer questions. The remaining 170 (35%), at their request, received further structured education prior to enrollment. Actual participation in cytapheresis procedures was the major factor that maintained donor enthusiasm. Only 66 (16%) of the donors left the program during the first year; the majority were unable to participate because of circumstances beyond their control.

A field test of the sweat patch.

The sweat patch is a new, noninvasive method designed to estimate the ethanol consumption of drinking subjects. It consists of salt-impregnated absorbent pads protected by a plastic chamber with attached water-tight adhesive. The patch reportedly collects transepidermal fluid at a steady rate for up to 10 days. Recent laboratory research has indicated a linear relationship between the concentration of ethanol in transepidermal fluid and mean concentration of ethanol in blood. Levels of ethanol in the sweat patch allowed identification of persons drinking at least 0.5 g of ethanol/kg/day with 100% sensitivity and specificity. The study reported here was conducted to test the field effectiveness of this sweat patch in normal, active research subjects. First, several pretests were conducted to determine the optimal location of the patch on the body and its fluid uptake at various sites. A laboratory experiment using nonalcoholic subjects was conducted to replicate previous work, and methods of measuring ethanol concentration in the patch were refined. A field test of the patch was then carried out. Healthy active volunteers drank a single "moderate" dose of ethanol (0.5 g of ETOH/kg of body weight) and then remained abstinent for the next 3 days. A week later, a "heavy" dose (1.0/kg of body weight) was consumed. Only a trace of ethanol was detected in any of the patches worn in either experiment. The patch did not measure ethanol in the transepidermal fluid under field conditions. Thus, further design modifications and pilot testing are required before the full benefits of this unobtrusive measure of drinking can be realized.

Cellular abnormalities of folate deficiency.

To trace the development of folate-deficient abnormalities of morphology and DNA synthesis, Friend erythroleukaemia cells were grown in media containing 10(2), 10(3) and 10(4) ng of [3H]PteGlu1/ml and then transferred to folate-free media. Parameters examined were: intracellular folate levels; growth potential; morphology; dU suppression; and DNA content by flow microfluorimetry. The most sensitive indicators of folate-deficient cell growth were those related to DNA synthesis (dU and flow microfluorimetry). These became abnormal at intracellular folate levels of 0.2-0.5 ng/10(6) cells and markedly so below 0.1 ng/10(6) cells. Morphological criteria were less sensitive. Cells became megaloblastic at intracellular folate levels below 0.06 ng/10(6). The capacity of the cells to replicate in folate-free media was a function of the intracellular folate (ICF): duplications = 4.01 + ln(ICF)/0.67 (r = 0.993, P less than 0.001). These studies demonstrate that regardless of initial intracellular folate levels, cellular stigmata of folate deficiency appear when cellular folate falls below 3 X 10(5) molecules per cell (dU and flow microfluorimetry) and cells lose the capacity for further replication below 7-10 X 10(5) molecules. The intracellular folate level not only predicts early defects, but also determines the replicative capacity.

Folate utilization in Friend erythroleukemia cells.

In order to study the generation, factors controlling endogenous folate pools, and their functional importance, Friend erythroleukemia cells were grown in media containing 100; 1,000; and 10,000 ng/ml of tritiated pteroylglutamic acid (3H)PteGlu1 and then studied in unlabeled media with varying amounts of PteGlu1. The intracellular folate pool was directly proportional to the PteGlu1 in which the cells were incubated. At equilibrium, greater than 95% of the labeled intracellular folate pool chromatographed as polyglutamyl folate, regardless of the exogenous folate concentration. The functional importance of the intracellular folate pool was studied by varying the endogenous pool and the exogenous (media) supply. The ability of the cells to replicate in the absence of exogenous folate was directly proportional to the intracellular polyglutamyl folate pool. The maximal rate of replication, however, required exogenous PteGlu1 in addition. The cell doubling time was the most important determinant of intracellular folate turnover; changes in the intracellular pool size and the extracellular folate concentration had no effect on the turnover time. In a rapidly proliferating tissue, the onset of functional folate deficiency will be determined by dilution of intracellular polyglutamates among progeny until a critical level is reached.

Bone marrow necrosis in acute leukemia.

2 patients with premortem marrow necrosis in acute leukemia are discussed. A review of the course of each patient plus those in the literature suggests that premortem marrow necrosis may not be a poor prognostic sign in acute lymphoblastic leukemia but generally precedes a prolonged and fatal pancytopenia in acute myelogenous leukemia. The technetium-99m rhenium sulfur colloid marrow scan was found to be of value in assessing the extent and degree of necrosis of the marrow as well as in documenting and predicting marrow recovery following chemotherapy.

Use of a physiologic pharmacokinetic model of glucose homeostasis for assessment of performance requirements for improved insulin therapies.

Methods are presented for assessing insulin therapies using a physiologic pharmacokinetic model of glucose homeostasis in man. The model is composed of simultaneous differential equations that represent physiologic compartments and spaces in which glucose and insulin are distributed and undergo metabolic reactions. The model is used to simulate clinical experiments in which blood glucose concentration is controlled by artificial device therapies. Predictions of the theoretical model for responses of normal and diabetic individuals to standard intravenous and oral glucose tolerance tests are compared to clinical data. Reasonable agreement is obtained between predictions of the computer simulations and clinical data for normal individuals. The responses of a diabetic person to oral glucose tolerance tests are simulated by removal of the pancreas from the glucose homeostasis model and introduction of insulin into the model by a prescribed therapy. Model simulations reaffirm expectations concerning the poor blood glucose control attainable by intramuscular insulin injection. Simulations of blood glucose regulation by an artificial pancreas using closed-loop feedback control for controlling insulin delivery rate reveal hyperinsulinemia that results in a net shift in the deposition of a glucose load from liver to peripheral tissues. Simulations of this system in which the time delay for glucose measurement is varied from 1.5 to 30 min show that increases in sensor delay result in progressive loss in glucose regulation, exacerbation of hyperinsulinemia, and increased insulin requirements.

Enterohepatic circulation of methotrexate in rats in vivo.

The pharmacokinetics of the methotrexate enterohepatic cycle were studied in rats in vivo. For plasma levels of methotrexate between 10(-5) and 10(-8) M, biliary levels were directly proportional and concentrated 27-fold. When labeled methotrexate was administered in doses sufficient to achieve plasma levels of 10(-6) M, approximately 50% of methotrexate appeared in the bile in normal animals and up to 80% appeared in anephric animals. In spite of the high percentage of administered methotrexate which appeared in the bile, complete interruption of the enterohepatic cycle in otherwise normal animals did not affect the plasma decay curve of a bolus of methotrexate. The increased biliary excretion which occurred in animals with renal impairment was utilized with possible therapeutic implications. Bile drainage in these animals rapidly decreased plasma methotrexate levels compared to nondrained controls. This suggests that interruption of the methotrexate enterohepatic cycle may provide an alternative for the management of methotrexate toxicity associated with renal insufficiency.

The role of the enterohepatic cycle in folate supply to tumour in rats.

The importance of the folate enterohepatic cycle in governing the supply of folate to implants of a rapidly-growing tumour were studied in a new animal model. Following enteric administration of tritiated pteroylglutamic acid, [3H]PteGlu1, tumour uptake of labelled folate was limited to CH3[3H]H4PteGlu1 produced by the gut mucosal cells during absorption or subsequently recirculated through the enterohepatic cycle. 50% of the labelled folate reaching the tumour nodules in the first 6 h after enteric administration first circulated through the enterohepatic cycle. In addition, labelled folate taken up by tumour was immediately incorporated into a polyglutamyl folate pool. There was no evidence for a release of labelled folate from tumour for recirculation to the liver. Therefore the liver and folate enterohepatic cycle appear to play a major role in regulating the supply of folate to rapidly proliferating tissues such as tumour by acutely storing folate from the diet and then secreting it into bile for reabsorption and transport to tissue.