Frank M. LaDuca

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found. Intra- and interest reproducibility was excellent (C.V.s less than 7%). By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.

The use of a HEMOCHRON® JR. HEMONOX™ point of care test in monitoring the anticoagulant effects of enoxaparin during interventional coronary procedures

AbstractBackground: Enoxaparin is increasingly used for the anticoagulation of patients undergoing percutaneous coronary intervention (PCI). Several reports have suggested the utility of using point of care tests in monitoring the anticoagulation levels of enoxaparin in patients undergoing PCI. The objective of this study was to evaluate a new point-of-care test (POCT) HEMONOX™ in monitoring the anticoagulant effect of enoxaparin in non citrated fresh whole blood samples from patients undergoing elective PCI procedure. Methods: Following IRB approval, blood samples were obtained from fifty-four patients who received two sequential intravenous doses of enoxaparin; 0.1 mg/kg followed 5 min later by 0.4 mg/kg for a total of 0.5 mg/kg. Blood was drawn at baseline and at 5, 10, 30 and 60 min post first bolus for evaluation in the clot-based POCT HEMONOX, ACT and aPTT and the chromogenic anti-Xa activity assay. Results: HEMONOX clotting time (CT) at baseline was 62.6 ± 6.2 secs, (n = 32) in healthy donors and statistically higher in PCI patients (71.6 ± 9.1 secs, p = 0.0001). The peak HEMONOX response that was always achieved at 10 min post bolus was >100 secs in all 54 patients, of these 83% yielded CT >150 secs (range: 150–466). There was no detectable anti-Xa activity level at baseline while peak HEMONOX CT corresponded to therapeutic levels (0.85 ± 0.14 U/ml; range: 0.61–1.34). Both HEMONOX CT and anti-Xa level significantly decreased at the time of sheath removal. HEMONOX CT at peak response suggested 3 patient subgroups with different levels of sensitivity to enoxaparin: low, intermediate and high responders. The correlation between anti-Xa activity level and HEMONOX CT was ≥0.85 in each patient subgroup when data from the 3 critical time points; baseline (absence of drug), peak response (10 min post bolus) and sheath removal (60 min post bolus) were analyzed. The correlation diminished to ≥0.83 when the analyses included data from all 5 time points [baseline, 5, 10, 30, and 60 min post bolus]. The HEMONOX test was the most sensitive POCT to measure the anticoagulant effects of enoxaparin. All patients completed PCI successfully. Conclusion: The HEMONOX test may be able to guide anticoagulation with enoxaparin during PCI.Abbreviated abstractThe HEMONOX assay is a one step whole blood coagulation test performed on the HEMOCHRON® Jr. Signature + POC system. The method was evaluated to monitor the anticoagulant level of enoxaparin in blood samples from patients undergoing PCI after receiving an intravenous dose of 0.5 mg/kg. The results suggest a clear distinction of HEMONOX CT between the baseline value of untreated patients and patients achieving therapeutic enoxaparin levels.

Comment: Prothrombin Time Monitoring Devices

TO THE EDITO R : Baclofen is indicated as a muscle relaxant and an antispasmodic for the alleviation of spasticity in patients with multiple sclerosis or spinal cord disease. The manufacturer1 reports that doses of baclofen approximately 13 times the recommended human dose could increase the incidence of omphaloceles (ventral hernias), substantial reductions in food intake and weight gain, and incomplete sternebral ossification in rat fetuses. At seven times the recommended human dose, baclofen was associated with an increased incidence in unossified phalangeal nuclei of forelimbs and hindlimbs in rabbit fetuses; reduction in mean fetal weight occurred in offspring of mice receiving 17 or 34 times the human daily dosage of baclofen.2 There are no adequate controlled studies of baclofen use in pregnant women, and the drug should be used during pregnancy only when the potential benefits justify the possible risks to the fetus (Category C).1 , 2 We present the results of treatment with intrathecal baclofen in a pregnant woman. Case Report. In June 1992, a 34y e a r-old woman was in a traffic accident and experienced a complete fifth cervical vertebra (C5)–level medular injury, resulting in severe spasticity according to the Ashworth scale for rigidity evaluation and the Penn scale for spasticity. Because the spasticity was resistant to treatment with oral drugs (baclofen 30 mg q8h, diazepam 5 mg q8h), the patient met the criteria for treatment with intrathecal baclofen. A Medtronic pump (Syncromed, Minneapolis, MN) with intrathecal baclofen was implanted in April 1993. Oral baclofen and diazepam were discontinued after intrathecal baclofen was initiated. In September 1 9 9 6 , the patient became pregnant and was given intrathecal baclofen 140 μg/d throughout the pregnancy; her spasticity remained under control. Delivery was by Caesarean section performed at 36 weeks’ gestation. The newb o rn was a boy with APGAR scores of 9 at one minute and 10 at five minutes; his weight (2155 g), length (47 cm), and head circumference (33 cm) were within the n o rmal range for gestational age. He did not display any major or minor extern a l m a l f o rmations, and ultrasonographic examinations of head, heart, and abdominal o rgans were normal. Karyotyping did not disclose chromosomal anomalies. Xrays of bones at 20 months of age did not show osseous malformations. Neurologic examination and psychomotor development up to the age of 24 months were within the normal range. Three months after the previous delivery, the patient became pregnant again. During this pregnancy, it was not necessary to modify the dosage of intrathecal baclofen. The pregnancy was uncomplicated, and delivery was by Caesarean section at 34 weeks’ gestation. The infant was a boy with APGAR scores of 8 at one minute and 10 at five minutes; weight (2240 g), length (46 cm), and head circumference (32 cm) were within the normal range for gestational age. Physical examination as well as laboratory studies (identical to those of the previous infant) were n o rmal. At 12 months of age, he showed normal psychomotor development. The patient is currently receiving intrathecal baclofen 154 μg/d. Discussion. In the first pregnancy, the risk/benefit ratio of stopping the intrathecal baclofen infusion was ethically valued, and maintaining the treatment was considered the best option since spasticity was controlled and discontinuing the treatment might be dangerous for the patient and even more dangerous for the fetus. The patient was informed of the risks and benefits of treatment with intrathecal baclofen and her autonomy was respected. The administration of intrathecal baclofen seemed more eff e ctive and safer than oral treatment in this patient because she did not respond adequately to oral treatment; baclofen crosses the blood–brain barrier in only small amounts following oral administration,2 and intrathecal baclofen produces effective cerebrospinal fluid concentrations while plasma concentrations are 100 times less than those occurring with oral adm i n i s t r a t i o n .3 In addition, one case4 of treatment with intrathecal baclofen 1000 μg/d was described in a woman who gave birth to a healthy girl.4 Given the positive results of our patient’s first pregnancy, the treatment was continued during the second pregnancy. The treatment with intrathecal baclofen during pregnancy was successful in controlling severe s p a st i c i t y, and there was no display of teratogenicity in these two children.

Assessing heparin neutralization following cardiac surgery: sensitivity of thrombin time-based assays versus protamine titration methods

Adequate assessment of heparin neutralization following cardiac surgery is critical in reducing the patient’s exposure to protamine. Both excessive protamine and residual heparin have been associated with postoperative bleeding and poor patient recovery. The activated clotting time (ACT) is the preferred intraoperative heparin monitor, while both protamine titration (i.e. a protamine-containing ACT) and thrombin time methods have been used to detect circulating residual heparin after protamine administration. Following initial protamine dosing using the protamine response test (PRT), postoperative monitoring was employed in the operating room prior to transport of the patient to intensive care. Two point-of-care assays, the thrombin time (TT) and the protamine dose assay (PDA), were evaluated to determine their relative heparin sensitivity and their usefulness to quantitate protamine dose. The PDA, which is based on the ACT, was shown in laboratory and clinical studies to detect residual heparin above 0.25 units/ml and to quantify additional minidoses of protamine (as low as 25 mg) required to obtain complete heparin neutralization. Differential evaluation of the TT and heparin neutralized thrombin time (HNTT) was shown in laboratory studies to be more sensitive to small amounts of residual heparin than the ACT. Clinical evaluations confirmed that additional protamine is required in approximately 12% of cardiac surgical cases managed using the PRT system. Both the PDA and TT/HNTT provided useful postoperative assessment of the adequacy of heparin neutralization. The TT/HNTT had slightly improved heparin sensitivity even in the presence of significant fibrinogen loss. These point-of-care assays provide the opportunity to optimize heparin and protamine management in the cardiac surgery patient.

ProTime self-management yielding improvement of fluency and quality of life

Patient self-management (PSM), as the standard of care for vitamin K-antagonist therapy management in Germany requires a detailed, point-of-care (POC) device-specific training program to ensure quality patient care. In a multi-center trial using the ProTime System (Training program plus POC device), 105 patients were enrolled to evaluate efficacy of training, knowledge retention, patient satisfaction and quality of life (QoL). Patients returned to the centers 1, 3 and 6 months after training to complete questionnaires and demonstrate INR test proficiency. Training assessment employed self-evaluation and comparison of POC results between PSM and professional operators. Patient satisfaction and QoL were assessed using a modification of the questionnaire described by Sawicki and the SF12v2 QoL Survey, respectively. Patients demonstrated statistically significant improvements in knowledge post training (p < 0.001) and retained the acquired information (p = NS vs. post-training; N = 45) after 6 months. Trained patients yielded equivalent INR results to professional operators (r = 0.92) with little or no bias across all clinic visits. Compliance with weekly testing improved from 1 to 3 months (p = 0.03), remaining at the required weekly frequency through 6 months. Average patient satisfaction improved significantly during the first month and remained constant thereafter. There was a statistically significant improvement in the Physical Component Summary of SF12 between baseline and 3/6 month assessments in all centers. In conclusion, PSM requires a comprehensive system including appropriate disease and POC device training. Such a system fosters compliance, improved knowledge about underlying disease, patient satisfaction and QoL.

Histidyl-tRNA synthetase, the myositis Jo-1 antigen, is cytoplasmic and unassociated with the cytoskeletal framework☆

The myositis-specific anti-Jo-1 autoantibody, which is directed against histidyl-tRNA-synthetase, is found in 30% of polymyositis patients. The Jo-1 antigen has been reported to be a nuclear antigen by some authors. On the contrary we show that less than 2% of the total histidyl-tRNA and lysyl-tRNA synthetase activities are associated with purified rat liver nuclei or the hepatocyte intermediate filament-nuclear fraction. In the presence of polyethylene glycol, in which the high Mr multi-enzyme complex containing lysyl-tRNA synthetase is insoluble, 65% of the lysyl-tRNA synthetase and only 15% of histidyl-tRNA synthetase activities remained associated with the cytoskeletal framework. The Jo-1 antigen exhibited a diffuse granular cytoplasmic distribution in cultured rat hepatocytes as determined by indirect immunofluorescent microscopy. Hence, the Jo-1 antigen is cytoplasmic and unassociated with the cytoskeletal framework or high Mr synthetase complex in situ.

Bedside measurement of factor VIII:C activity in individuals with hemophilia A

Factor VIII replacement therapy for patients with hemophilia A is conventionally monitored using a plasma‐based factor VIII:C assay (a modified activated partial thromboplastin time [APTT] test). The plasma factor VIII assay requires the preparation of plasma from citrated whole blood and measurement of the clotting times of mixtures of patient plasma, factor VIII‐deficient substrate, and APTT reagent. Results are not routinely available in less than 1.5 hr, reducing the clinical value of the laboratory data regarding the ability to immediately adjust patient therapy. Results from the whole blood factor VIII assay, performed on a portable coagulation analyzer and using test tubes prefilled with the necessary APTT and factor VIII‐deficient reagents, are available within 5–7 min. This immediate determination of the factor VIII:C level from citrated whole blood provides the opportunity to greatly reduce turnaround time and improve the efficacy of factor VIII replacement therapy. Based on clotting time, factor VIII:C activity is read from a standard curve. A clinical evaluation of this whole blood test was performed in two hemophilia centers. A high degree of correlation was seen (r = 0.813, n = 220) between the whole blood values obtained and conventional laboratory results. This level of correlation was superior to that obtained when comparing two different plasma‐based systems (r = 0.753, n = 23). Factor VIII:C activity levels measured using the whole blood assay system were similar, irrespective of the test operator (laboratory technologist, nurse clinician, or patient). This study indicates that the whole blood factor VIII assay provides results comparable to those of conventional plasma‐based assays, but in a more rapid and efficient manner. It provides an opportunity to reduce unnecessary patient consumption of replacement preparations, hence reducing the cost of hemophilia A maintenance and prophylaxis regimens, and to reduce overall patient exposure to human blood products.

Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures.

We have reported a rapid method for the quantitation of proteins secreted in culture media (F.M. LaDuca, C.V. Dang, and W.R. Bell (1986) Anal. Biochem. 158, 262-267). Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture. The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin. Since heparin is known to affect certain cellular functions, the fates of [35S]methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments. Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin. At 8 h during the chase, there was a 40-50% reduction in fibrinogen-antigen in spent culture medium lacking heparin. The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis. In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen. The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation. Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.