Harmonization of critical result management in laboratory medicine.

Unsafe medical care is a major source of disabling injuries and death throughout the world. The failure to notify, follow up, and action critical results, which signify life threatening situations, is of particular concern and may cause avoidable morbidity and mortality. International accreditation standards require pathology laboratories to have a system for the timely and reliable communication of critical results to clinical personnel responsible for patient care. In response, various practices and a number of different terminologies have been described in the literature. Increased attention to patient safety standards and multinational surveys, however, highlighted shortcomings and inefficiencies in existing communication systems. These failures and variations in practice call for clear guidance and harmonization of approaches in order to improve communications and to provide safer patient care. The objectives of this review are to create a harmonized terminology and to learn from international practices by systematically reviewing the best available evidence on existing approaches. Based on literature review findings we highlight key areas where harmonization is necessary and feasible and offer a conceptual framework and methods for designing better and more evidence-based systems for the timely notification of laboratory results that represent potential patient safety hazards.

Grading quality of evidence and strength of recommendations in clinical practice guidelines part 3 of 3. The GRADE approach to developing recommendations.

This is the third and last article in the series about the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to grading the quality of evidence and the strength of recommendations in clinical practice guidelines and its application in the field of allergy. We describe the factors that influence the strength of recommendations about the use of diagnostic, preventive and therapeutic interventions: the balance of desirable and undesirable consequences, the quality of a body of evidence related to a decision, patients' values and preferences, and considerations of resource use. We provide examples from two recently developed guidelines in the field of allergy that applied the GRADE approach. The main advantages of this approach are the focus on patient important outcomes, explicit consideration of patients' values and preferences, the systematic approach to collecting the evidence, the clear separation of the concepts of quality of evidence and strength of recommendations, and transparent reporting of the decision process. The focus on transparency facilitates understanding and implementation and should empower patients, clinicians and other health care professionals to make informed choices.

Quality assessment of clinical practice guidelines for adaptation in burn injury.

OBJECTIVES: A number of clinical practice guidelines (CPGs) are available for managing burn injury patients but clinical practice is highly variable. We report the first steps to trans-contextual adaptation of international burn CPGs to local settings. METHODS: Key clinical topics and questions to be covered in the final guideline were defined and prioritized. Systematic search between 1990 and 2008 retrieved 546 citations, of which 24 were CPGs on the general and intensive care of burn patients. Assessment of the clinical content of CPGs was carried out. Methodological quality of CPGs was evaluated using the AGREE instrument. RESULTS: Of the 24 CPGs evaluated, 10 (42%) were evidence-based. All major burn topics were covered by at least one CPG, but no single CPG addressed all areas important in terms of outcomes. According to the AGREE criteria, 2 CPGs (8%) were strongly recommended, 14 with provisos or alterations (58%) and the rest were not recommended for adaptation. CONCLUSIONS: Although existing CPGs for the management of burn may accurately reflect agreed clinical practice, most performed poorly when evaluated for methodological quality. Future CPG efforts addressing these methodological shortcomings would add substantially to the improved management of burned patients.

Grading quality of evidence and strength of recommendations in clinical practice guidelines: Part 2 of 3. The GRADE approach to grading quality of evidence about diagnostic tests and strategies.

The GRADE approach to grading the quality of evidence and strength of recommendations provides a comprehensive and transparent approach for developing clinical recommendations about using diagnostic tests or diagnostic strategies. Although grading the quality of evidence and strength of recommendations about using tests shares the logic of grading recommendations for treatment, it presents unique challenges. Guideline panels and clinicians should be alert to these special challenges when using the evidence about the accuracy of tests as the basis for clinical decisions. In the GRADE system, valid diagnostic accuracy studies can provide high quality evidence of test accuracy. However, such studies often provide only low quality evidence for the development of recommendations about diagnostic testing, as test accuracy is a surrogate for patient-important outcomes at best. Inferring from data on accuracy that using a test improves outcomes that are important to patients requires availability of an effective treatment, improved patients' wellbeing through prognostic information, or - by excluding an ominous diagnosis - reduction of anxiety and the opportunity for earlier search for an alternative diagnosis for which beneficial treatment can be available. Assessing the directness of evidence supporting the use of a diagnostic test requires judgments about the relationship between test results and patient-important consequences. Well-designed and conducted studies of allergy tests in parallel with efforts to evaluate allergy treatments critically will encourage improved guideline development for allergic diseases.

A large outbreak of scombroid fish poisoning associated with eating escolar fish (Lepidocybium flavobrunneum).

In August 2003, an outbreak of scombroid fish poisoning occurred at a retreat centre in California, USA. In a retrospective cohort study, 42 (75%) of the 56 dinner attendees who ate escolar fish (Lepidocybium flavobrunneum) met the case definition. Individuals who ate at least 2 oz of fish were 1.5 times more likely to develop symptoms than those who ate less (relative risk 1.5, 95% confidence interval 0.9-2.6), and to develop more symptoms (median 7 vs. 3 symptoms, P = 0.03). Patients who took medicine had a longer duration of symptoms than those who did not (median 4 vs. 1.5 h, P = 0.05), and experienced a greater number of symptoms (median 8 vs. 3 symptoms, P = 0.0002). Samples of fish contained markedly elevated histamine levels (from 2000 to 3800 ppm). This is one of the largest reported outbreaks of scombroid fish poisoning in the United States and was associated with a rare vehicle for scombroid fish poisoning, escolar.

The multidrug resistance P-glycoprotein. Oligomeric state and intramolecular interactions.

The human multidrug resistance P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of transporters, is frequently responsible for the failure of chemotherapy by virtue of its ability to export hydrophobic cytotoxic drugs from cells. Elucidating the inter- and intramolecular interactions of this protein is critical to understanding its cellular function and mechanism of action. Toward this end, we have used both biochemical and genetic techniques to probe potential oligomerization interactions of P-gp. Differentially epitope-tagged P-gp molecules did not co-immunoprecipitate when co-expressed in HEK293 cells or when co-translated in vitro, demonstrating that P-gp is monomeric in both the presence and absence of detergents. The two cytoplasmic domains of P-gp did not interact with each other in vivo when co-expressed as gene fusions in yeast. In contrast, the homologous domains of the transporter associated with antigen processing (TAP), which reside on separate polypeptides and must form a heterodimeric transporter (TAP1/TAP2), did interact in this system, suggesting a role for these domains in TAP dimerization. Implications for understanding the subunit organization of ABC transporters are discussed.

The interaction of the tyrosine kinase pp60src with membrane and cytoskeletal components.

To study the mechanism of oncogenic transformation we have investigated the association of pp60v-src with the cytoskeleton using a variety of mutants. Transformation is associated with the interaction of pp60v-src with the cytoskeleton, specifically in adhesion plaques. Biochemical analysis has shown a correlation between tyrosine-specific phosphorylation of the fibronectin receptor and the loss of surface-bound fibronectin, but no such correlation with phosphorylation of vinculin or talin. The role of the nontransforming protooncogene product pp60c-src was studied in platelets, which express large amounts of this protein. In resting platelets pp60c-src was soluble in detergent-containing buffers, however it became associated with the cytoskeleton, but not the membrane skeleton, after platelet activation. This association was inhibited by EDTA or RGDS peptides and was therefore dependent on occupancy of a platelet integrin, gpIIb/IIIa. The role of pp60c-src-associated tyrosine phosphorylation in platelet activation was also investigated. Inhibitors of tyrosine phosphorylation inhibited integrin-dependent platelet aggregation and second messenger production, indicating a close linkage between matrix receptor occupancy, pp60c-src association with the cytoskeleton and platelet function.

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.

Cytoskeletal assembly and vinculin-cytoskeleton interaction in different phases of the activation of bovine platelets.

Vinculin is an Mr 130 kDa protein that has been implicated in membrane-cytoskeleton interaction in various cell types. It has been demonstrated that vinculin is not a cytoskeletal component in resting platelets, but part of it becomes associated with the cytoskeleton during thrombin-induced activation. In this study, using a quantitative immunoblotting technique, the relation of vinculin to the cytoskeleton in different phases of activation of bovine platelets was explored, and the process of incorporation of vinculin into the cytoskeleton was related to that of cytoskeletal assembly. The assembly of cytoskeleton proceeded at a significantly faster rate than the association of vinculin with it, which shows that the latter process is not due to passive trapping of vinculin into the Triton-insoluble residue, but certain biochemical changes had to occur before such an interaction became possible. When the formation of pseudopodia was prevented by cytochalasin B, but neither aggregation nor the release reaction induced by thrombin were inhibited, the recovery of vinculin in the Triton-insoluble residue even increased. In both time- and thrombin-concentration-dependent studies, poor correlation was found between vinculin-cytoskeleton association and the extent of aggregation. Activation with phorbol-myristate-acetate, which is a strong stimulus for aggregation but produces only a slight release in the granular content, resulted in the association of only a negligible amount of vinculin with the cytoskeletal fraction. The incorporation of vinculin into the cytoskeletal fraction of thrombin activated platelets started with the release reaction but still proceeded, and the greatest part of the reaction occurred after secretion had gone to completion.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of integrin mobility and cytoskeletal association in normal and RSV-transformed chick embryo fibroblasts.

The mobility of the integrin receptor in trypsinised chick embryo fibroblasts (CEF) was investigated using the CSAT monoclonal antibody. The binding of CSAT to trypsinised CEF followed by incubation at 37 degrees C resulted in patching and then capping of the receptor. This capping was dependent on cellular metabolism, since agents such as sodium azide or 2-deoxyglucose inhibited the process. Whereas about 95% of unclustered integrin was soluble in Nonidet P40-containing buffers, after capping more than 25% of surface integrin became detergent-insoluble, indicating a physical association with cytoskeletal elements. Thus the crosslinking of integrin via its beta subunit is sufficient, to induce cytoskeletal association. Unusually, the microfilament-disrupting drugs cytochalasins B and D potentiated CSAT-induced capping in terms of both cell number and the conformation of caps on individual cells. Double immunofluorescent staining demonstrated that in cytochalasin-treated cells both F-actin and talin co-localised with surface CSAT-integrin clusters. The co-distribution of these cytoskeletal components with surface integrin was retained in cytoskeletal preparations, although there was no quantitative increase of either talin or vinculin in the cytoskeletons. The cocapping of talin with integrin clusters on CEF could also be observed in the absence of cytochalasins. No differences were found in capping efficiency, talin and actin co-localisation or cytoskeletal association of surface-modulated integrin in Rous sarcoma virus (RSV)-transformed cells compared with untransformed counterparts, although differences in the response to cytochalasins were observed. These results provide novel evidence for a physiologically relevant association of integrin with cytoskeletal components and its regulation by surface configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine-specific phosphorylation of gpIIIa in platelet membranes.

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali-resistant phosphoproteins and the majority of pp60c-src were in the surface membrane fraction. An alkali-resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti-phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non-reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c-src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody-purified pp60c-src. Our results indicate that tyrosine-specific phosphorylation of gpIIIa by pp60c-src may play a role in the regulation of platelet function.

Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes.

We have investigated the localization and phosphorylation of the fibronectin receptor in chick embryo fibroblasts transformed either by wild-type Prague C strain Rous sarcoma virus, which induces a rounded, less adhesive phenotype with a loss of surface fibronectin and gross cytoskeletal changes, or transformed by rASV2234.3, a variant which induces a flat, adhesive morphology with the retention of surface fibronectin and more normal cytoskeleton. Immunofluorescence showed co-distribution of pp60v-src with integrin and fibronectin fibrils in rASV2234.3-transformed cells. Total levels and surface expression of integrin were unchanged in both transformed cell types compared with untransformed cells. However, whereas integrin band 3 (beta 1 subunit) in Prague C-transformed cells was hyperphosphorylated on tyrosine, this was reduced virtually to normal in rASV2234.3-transformed cells. A similar differential phosphorylation of integrin band 3 could be found in membranes phosphorylated in vitro. rASV2234.3 pp60v-src was less efficient in phosphorylating a synthetic peptide containing the putative integrin tyrosine phosphorylation site, indicating that this variant pp60v-src has an altered substrate specificity compared to Prague C pp60v-src. The correlation between tyrosine-specific phosphorylation of integrin and loss of surface fibronectin suggests that this could play an important role in the induction of the transformed phenotype.