Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase.

BACKGROUND: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. METHODS: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. RESULTS: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS-), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS- patients receiving at least four doses at 320 IU m(-2) was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. CONCLUSION: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

Intra-operative parathyroid hormone monitoring in patients with parathyroid cancer.

BACKGROUND: Intra-operative parathyroid hormone (PTH) monitoring (IPM) is 97% accurate in predicting postoperative eucalcemia in sporadic primary hyperparathyroidism (SPHPT). However, its usefulness in parathyroid cancer has not been demonstrated. This study reports IPM accuracy during surgical resections for parathyroid cancer. METHODS: Eight of 556 consecutive patients with SPHPT underwent parathyroidectomy using IPM and had parathyroid cancer. Operative success was defined as eucalcemia > six months and operative failure/persistent cancer as hypercalcemia within six months of parathyroidectomy. The IPM criterion for operative success was defined as a >50% decrease of peripheral PTH levels from the highest either pre-incision or pre-excision values, 10 minutes after resection. RESULTS: In eight patients, 11 operations were performed. Ten operations (91%) resulted in >50% intra-operative PTH decrease. However, in only seven (70%) of these resections, eucalcemia was achieved for >6 months with five of these seven (71%) procedures being initial en bloc resections. The remaining 3/10 (30%) operations with >50% intra-operative PTH decrease resulted in operative failures. In the last operation, intraoperative parathormone monitoring (IPM) correctly predicted operative failure. IPM sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy in predicting outcome were 100, 40, 70, 100, and 75%, respectively. CONCLUSIONS: IPM with the criterion of >50% PTH drop from the highest level is less accurate in predicting operative success in parathyroid cancer when compared to SPHPT. A >50% intra-operative PTH level decrease in patients with parathyroid cancer, particularly in reoperative cases, is less predictive of complete resection. The initial recognition of this disease followed by proper resection remains essential in the treatment of parathyroid cancer.

Effects of an air pollution personal alert system on health service usage in a high-risk general population: a quasi-experimental study using linked data.

BACKGROUND: There is no evidence to date on whether an intervention alerting people to high levels of pollution is effective in reducing health service utilisation. We evaluated alert accuracy and the effect of a targeted personal air pollution alert system, airAware, on emergency hospital admissions, emergency department attendances, general practitioner contacts and prescribed medications. METHODS: Quasi-experimental study describing accuracy of alerts compared with pollution triggers; and comparing relative changes in healthcare utilisation in the intervention group to those who did not sign-up. Participants were people diagnosed with asthma, chronic obstructive pulmonary disease (COPD) or coronary heart disease, resident in an industrial area of south Wales and registered patients at 1 of 4 general practices. Longitudinal anonymised record linked data were modelled for participants and non-participants, adjusting for differences between groups. RESULTS: During the 2-year intervention period alerts were correctly issued on 208 of 248 occasions; sensitivity was 83.9% (95% CI 78.8% to 87.9%) and specificity 99.5% (95% CI 99.3% to 99.6%). The intervention was associated with a 4-fold increase in admissions for respiratory conditions (incidence rate ratio (IRR) 3.97; 95% CI 1.59 to 9.93) and a near doubling of emergency department attendance (IRR=1.89; 95% CI 1.34 to 2.68). CONCLUSIONS: The intervention was associated with increased emergency admissions for respiratory conditions. While findings may be context specific, evidence from this evaluation questions the benefits of implementing near real-time personal pollution alert systems for high-risk individuals.

Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and negative for inhibitors by Bethesda assay.

In patients with hemophilia A who have an inhibitor to factor (F)VIII measured by Bethesda assay, enzyme-linked immunosorbent assay (ELISA) can also be used to detect the inhibitor. In some studies non-inhibitory antibodies were also detected by ELISA in many patients who were negative by Bethesda assay. Our aim was to investigate whether there is a higher detection rate of FVIII antibodies by ELISA compared with Bethesda assay. We also compared outcomes using three different preparations of recombinant FVIII (rFVIII) to coat the microtiter plates for ELISA. Inhibitor detection by ELISA generally agreed with the Bethesda method. Only four of 26 patients with no clinical suspicion of an inhibitor and with no detectable inhibitor by Bethesda assay showed a non-inhibitory antibody by ELISA, and three of these were only weakly positive. Patients with severe hemophilia A and the intron 22 inversion (n = 21) did not show a higher incidence of non-inhibitory antibodies compared with those without that mutation. Finally, we found that the formulation of rFVIII has a small effect on ELISA performance, mainly in detection of low-level antibody. The results of the present study are in contrast to and fail to confirm previously published reports showing a higher incidence of non-inhibitory antibodies in hemophilia A.

Measurement of aspirin concentrations in portal and systemic blood in pigs: effect on platelet aggregation, thromboxane and prostacyclin production.

Low doses of enteric-coated aspirin were administered orally to pigs. Plasma aspirin concentrations measured in blood obtained simultaneously from permanent catheters in a systemic artery and portal vein for 6 hours after dosage showed a large variation in the plasma aspirin concentration: time profile between pigs. After 50 mg single dose the ratio of the arterial: portal area under the plasma concentration versus time curve (AUC) was 0.63 +/- 0.08 (mean +/- SE, n = 6). In three pigs which received all three dosage regimens, the arterial: portal AUC ratios were 0.48 +/- 0.05 after 50 mg single dose, 0.52 +/- 0.02 after 100 mg single dose and 0.47 +/- 0.02 after 100 mg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65 mM) was completely abolished after chronic aspirin administration of 100 mg daily. Thromboxane production (pg/10(6) platelets) induced by this stimulus decreased from 536 +/- 117 before aspirin to 57 +/- 14 after aspirin (mean +/- SE, n = 4; p = 0.03). Aortic prostacyclin synthesis, measured as 6-keto PGF1 alpha (ng/disc after 10 min incubation), was 1.66 +/- 0.28 (mean +/- SE, n = 4) in untreated pigs and 0.95 +/- 0.25 (n = 5) in treated pigs (p = 0.07). Results from this study support the idea that a difference between aspirin concentrations in the portal and systemic circulations can be achieved. Whether this can be translated into a clinically useful differential effect on the vessel wall compared to the platelet remains to be determined.

Potentiation of ADP-induced aggregation in human platelet-rich plasma by 5-hydroxytryptamine and adrenaline.

1. We have used dose-response curves to quantitate the potentiation of adenosine 5'-diphosphate (ADP)-induced aggregation and thromboxane (TXA2) generation by 5-hydroxytryptamine (5-HT) and adrenaline in human citrated platelet-rich plasma. We have also quantitated the inhibition of these responses by aspirin, ketanserin and yohimbine, singly and in pairs. 2. Ketanserin (5 microM) inhibited TXA2 production and the second wave of platelet aggregation induced by a range of concentrations of ADP alone. This indicates that endogenous 5-HT, released from the platelet dense granules, contributes significantly to responses induced by ADP. 3. When 5-HT (10 microM) was added before ADP, a lower concentration of ADP was required to cause 50% aggregation and TXA2 generation. The ratio of ADP concentrations (CR) to cause 50% aggregation in the presence and absence of 5-HT was 2.1 when only added 5-HT was considered, and 5.0 when endogenous 5-HT was also taken into account. 4. Potentiation of ADP-induced aggregation by 5-HT also occurred in the presence of aspirin, resulting in a CR of 2.3. As expected, ketanserin inhibited potentiation by 5-HT in the presence and absence of aspirin. Although aspirin caused substantial inhibition of aggregation induced by ADP and 5-HT (CR 3.4), further inhibition occurred when ketanserin was also present (CR 6.5). 5. A subthreshold concentration of adrenaline (0.25 microM) caused substantial potentiation of ADP-induced aggregation in the absence (CR 4.0) and presence (CR 2.0) of aspirin. As expected, yohimbine (9 microM) inhibited this potentiation.Maximum TXA2 generation induced by ADP increased from 32.5 to 59.4 pg per 106 platelets when adrenaline was present. Aggregation induced by ADP and adrenaline was markedly inhibited by aspirin (CR 5.1) but was further inhibited when yohimbine (9 microM) was also present (CR 10.0).6. Results from this in vitro study show ketanserin and yohimbine have the potential to be used in combination with aspirin as antithrombotic agents in vivo.

ADP, adrenaline and serotonin stimulate inositol 1,4,5-trisphosphate production in human platelets.

Although adenosine diphosphate (ADP) is a well-known stimulus of platelet aggregation, it is not the generally accepted view that ADP stimulates phosphatidylinositolbisphosphate (PtdIns(4,5)P2) hydrolysis. Using a very sensitive competitive receptor binding assay for inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), we have detected Ins(1,4,5)P3 production at early ( < 10 s) time points after stimulation of human platelets by the weak agonists ADP, adrenaline and serotonin (5-hydroxytryptamine, 5-HT). When adrenaline or 5-HT was combined with ADP in the presence of aspirin, there was a significant potentiation of ADP-induced platelet aggregation, but there was no potentiation of Ins(1,4,5)P3 generation. Also, the increases in intracellular calcium (Ca2+) concentrations stimulated by ADP were not potentiated by adrenaline in the presence of aspirin. Therefore, the synergism between the purinergic and adrenergic pathways of platelet activation occurs downstream from PtdIns(4,5)P2 hydrolysis and intracellular Ca2+ mobilization, although prior to platelet aggregation.

The antiplatelet effect of daily low dose enteric-coated aspirin in man: a time course of onset and recovery.

We have studied the onset and recovery of inhibition of platelet function by low dose aspirin. Enteric-coated aspirin 50mg daily was administered to five human volunteers for five weeks and then 100mg daily was given for a further five weeks. We studied platelet aggregation and thromboxane formation in response to a range of stimuli: ADP, adrenaline, arachidonate and collagen, and also measured thromboxane formation after coagulation of whole blood (serum thromboxane). The onset of inhibition of platelet aggregation was progressive over several days for each of the four platelet stimuli, and was synchronous with the inhibition of thromboxane formation. Maximum inhibition occurred by day three for the weak stimuli ADP and adrenaline, by day five for the stronger stimuli arachidonate and collagen, but did not occur until day eight for serum thromboxane. Further inhibitory effects on both aggregation and thromboxane generation were observed after 100mg daily. Two weeks after the cessation of aspirin the responses to collagen and arachidonate and serum thromboxane had returned to normal. Platelet aggregation in response to the weaker stimuli, ADP and adrenaline, still showed detectable inhibition two weeks after cessation of aspirin, but had returned to normal by four weeks. These experiments provided no evidence for an effect of aspirin on platelets separate to its effect on cyclooxygenase. The onset and recovery of inhibition of platelet function by low dose aspirin was dependent on the strength of the stimulus studied.

Destruction of ristocetin cofactor by coagulation at 4 degrees C.

Ristocetin cofactor (VIIIR:RCo) and factor VIII-related antigen (VIIIR:Ag) were measured in anticoagulated and non-anticoagulated blood incubated at 4 degrees C, room temperature (RT) or 37 degrees C for 24 hours. A marked decrease in VIIIR:RCo, to almost undetectable levels, and a smaller decrease in VIIIR:Ag occurred when whole blood clotted at 4 degrees C. These changes were slight or absent when blood clotted at RT or 37 degrees C. VIIIR:RCo lost at 4 degrees C was not recoverable by further incubation at 37 degrees C but the less-marked loss of VIIIR:Ag was partially recovered. In blood which had clotted at 4 degrees C there was a change in the electrophoretic profile of VIIIR:Ag on crossed immunoelectrophoresis: there was more anodal migration of the VIIIR:Ag peak, consistent with a decrease in the mean molecular size. Further experiments showed that the decrease in VIIIR:RCo during coagulation at 4 degrees C preceded the decrease in fibrinogen levels. In cell-free plasma VIIIR:RCo also decreased markedly when coagulation occurred at 4 degrees C. The results show that loss of VIIIR:RCo occurs when blood is allowed to clot at 4 degrees C: this is not due to cryoprecipitation and does not require the presence of blood cells. The data suggest that it is probably caused by plasma proteases activated early in the coagulation pathway.

Haematological characteristics of the common marmoset (Callithrix jacchus jacchus).

Blood cell indices and parameters of haemostasis were studied in the common marmoset. The majority of the results were similar to those found in man. Differences from man were that the prothrombin time was shorter in the marmoset, higher concentrations of aggregating stimuli were required to cause platelet aggregation, and marmoset platelets did not aggregate under the influence of adrenalin. There was sexual dimorphism evident in the data for fibrinogen concentration and for platelet count, both of which were higher in females than in males. Marmoset platelets were very similar in ultrastructure to those of man.

A study of the utilization of research in practice and the influence of education.

This paper reports part of a multi-phase study which aimed to investigate the extent to which nurses utilize research and to identify factors associated with research utilization. The findings presented examine the influence of education upon research utilization. Firstly, a survey of registered nurses working in general medical and surgical wards in Scotland was conducted. 680/936 (72.6%) nurses returned self-report questionnaires to measure the level of utilization of 14 research based practices and assess the presence of potential influencing factors. This was then followed up through interviews with a sub-sample of nurses. An association was found between a higher educational level and research utilization. The nurses reported that in courses as opposed to study days, they were expected to engage in study and read and complete course work whereas attendance at study days could be an entirely passive experience and was often more of a morale booster. Nurses who read at least one journal regularly, had had more study leave, or had attended research courses also had a higher level of research utilization.

Anticoagulation and the GP patient.

The use of anticoagulant drugs requires a knowledge of the essential elements of their pharmacology and mechanisms of action. This article illustrates the marked differences for the drugs warfarin, heparin and aspirin.

Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A.

In some mild haemophilia A patients (discrepant phenotype), coagulation FVIII levels by one-stage assay (FVIII-1st) are more than double those by classical two-stage coagulation assay (FVIII-2st), and may fall within the normal range. Our aim was to assess automated two-stage chromogenic FVIII assays (FVIII-chr) for diagnosis of mild discrepant haemophilia A. Three chromogenic FVIII kits (Biophen, Coamatic and Dade-Behring) were evaluated, using recommended and extended incubation times. Samples were tested from patients with discrepant haemophilia (n = 7) and equivalent mild haemophilia (agreement between FVIII-1st and FVIII-2st, n = 4). For equivalent haemophilia, FVIII-chr were consistent with FVIII-1st and FVIII-2st for all kits at all incubation times. For discrepant haemophilia, using recommended incubation times, mean FVIII-chr using Biophen reagents was 22 IU/dl (range 13-31), with Coamatic 26 (17-34) and with Dade-Behring 41 (33-47), compared with 36 (27-44) for FVIII-1st and 8 (6-9) for FVIII-2st. FVIII-chr decreased as incubation time was increased with Biophen and Coamatic, but decreased less with Dade-Behring. FVIII-chr using the Dade-Behring kit gave similar results to FVIII-1st and is not suitable for diagnosis of mild discrepant haemophilia A. FVIII-chr by Biophen and Coamatic kits is suitable for diagnosis of these patients, especially with an extended incubation time.

Methodology and outcomes of platelet aggregation testing in Australia, New Zealand and the Asia-Pacific region: results of a survey from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program.

Platelet aggregometry is widely used to investigate platelet function but its performance is poorly standardized between laboratories. The aim of this work was to document the platelet aggregation methods used in specialist laboratories enrolled in the Haematology Quality Assurance Program of the Royal College of Pathologists of Australasia. A questionnaire requesting many details of methodology was distributed and from the responses, we determined a consensus view. Consensus was defined here as >70% agreement among respondents in answer to a question and this was seen for a number of aspects of the preanalytical, analytical and interpretive phases. However, for many questions there was a wide variation in responses. Sixteen laboratories provided a breakdown of the types of abnormal results typically seen in a 12-month period. In these laboratories a total of 1400 patients were tested and 390 (27%) had abnormal platelet function. Although it was common to diagnose the cause as aspirin or an aspirin-like defect or a release/storage pool disorder, the range of experience was wide and other rare defects were reported. We conclude that whilst there are a number of points of agreement between laboratories in platelet function testing, standardization could be improved.

The extent of nursing research utilization in general medical and surgical wards.

There has been extensive speculation about the lack of research utilization in nursing but little attempt to quantify this phenomenon outside of North America. The current demands for evidence-based practice necessitate research utilization as one element of the process. As part of a larger project, this study aimed to describe the extent of research utilization by registered nurses in general medical and surgical wards in the Scottish Health Service. A postal survey was conducted for nurses to self-report their level of utilization of 14 research-based practices. The 14 practices represented examples of direct, indirect and methodological utilization of research. A research utilization score was constructed for each of the 14 practices and a total mean score constructed for all 14 practices. A random two-stage stratified sampling resulted in a total sample of 936 nurses from 25 hospitals. A 73% response rate was achieved. The total mean research utilization score for all nurses across all 14 nursing practices suggests that on average, nurses had heard, believed in and were beginning to use the practices. The sampling technique over-represents nurses in large hospitals and charge nurses, hence a weighting calculation on all scores was completed. There was little difference in weighted and unweighted scores. Scores on individual practices ranged from 60% (405/680) of nurses never having heard of a practice to 85% (574/680) always using a practice. This approach provides a valid and reliable method of assessing the extent of nursing research utilization. In several of the practices, nurses are making significant attempts at research-based practice. The level of research utilization compares favourably with research completed in North America and provides a baseline for United Kingdom and other country studies.

Platelet function in platelet concentrates and in whole blood.

Platelet function was studied in CPD whole blood stored at 4 degrees C for one and three days and in platelet concentrates stored at room temperature for the same periods of time. Comparisons were made of platelet shape, nucleotide content, beta-thromboglobulin (beta TG) liberated during storage, and platelet aggregation in response to ADP, collagen, sodium arachidonate and ristocetin. It was found that in whole blood the shape of the platelets was less discoid than in platelet concentrates. However, platelet aggregation in response to ADP, collagen, and sodium arachidonate was preserved better in whole blood than in platelet concentrates. Platelet nucleotides were the same in whole blood as in platelet concentrates, but the plasma levels of beta TG were less in whole blood. The results show that as judged by aggregation, beta TG release and nucleotide content, platelets from whole blood were at least as functional as those from platelet concentrates. However, platelets from whole blood had lost their discoid shape, which suggests that they would have a short survival in the circulation.

Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymorphisms (RFLP).

BACKGROUND: Haemophilia A is a sex-linked bleeding disorder carried by unaffected females. Currently, the two main methods used for the determination of carrier status in women from families with haemophilia A are bioassays and DNA-based assays using restriction fragment length polymorphisms (RFLP). AIM: The aim of this paper was to assess the current usefulness of these two methods. METHODS: Bioassays measured factor VIII coagulation activity by a two-stage coagulation assay and von Willebrand antigen by immunoelectrophoresis. RFLP were determined with two intragenic probes (p114 and p486) and two linked probes (St14 and DX13). Data were analysed using a Bayesian analysis to allow for all possible recombination events. We also incorporated an estimate for the risk of mosaicism into calculations in isolated haemophilia families. Both bioassays and RFLP were used to determine carrier status in 63 women, 31 from known haemophilia families and 32 from families of isolated cases. The techniques were assessed for their ability to classify the patients as normal (p < 0.2) or carrier (p > 0.7). Where applicable, intron 22 inversion was also tested. RESULTS: In the known families, six women could not be classified after bioassay, but all could be classified by RFLP. Of the 32 women from families of isolated cases, eight were unclassified by bioassay and 12 were not definitely classified using RFLP. However, RFLP was useful in determining that a recent mutation had occurred in six of the eight families in which DNA from the grandparents was available. CONCLUSION: For diagnosis of carriers of haemophilia, RFLP is the preferred method in familial haemophilia, but is less useful in isolated haemophilia.

Direct mutation analysis as the preferred method for carrier diagnosis in families with isolated cases of haemophilia B.

Approximately one-third of haemophilia B cases are described as isolated due to their occurrence in families with no prior history of the disorder. In this report, two families with isolated haemophilia B were studied by the standard method of restriction fragment length polymorphism (RFLP) analysis coupled with factor IX activity and antigen levels with the aim of achieving carrier diagnoses. The limitations of using this approach in the determination of carrier status were highlighted by diagnostic problems arising in both families. The problems included difficulty in interpreting bioassay results, homozygosity for the RFLP marker in a key family member and the possibility of germline mosaicism. Unequivocal carrier diagnosis in the two families was ultimately achieved by direct mutation analysis.

Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant.

The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s. To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.