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February 2008

Editor:
Michael Bissell, MD, PhD

Protein S proficiency testing
Using oral fluid samples to detect measles virus strains
Statins and vein graft intimal hyperplasia
Interference of blood cell lysis on coagulation testing
Structure of the tumor suppressor p53
Complement factor I and Staphylococcus aureus phagocytosis
Automated preparation of platelet concentrates

Protein S proficiency testing Protein S proficiency testing

Both functional (activity) assays and antigenic (quantitative immunologic) assays test for protein S deficiency. Free protein S antigen assays measure only unbound (free) protein S, which is the active form of protein S. Total antigen assays measure bound (inactive) and unbound (free, active) protein S. Protein S deficiencies are quantitative (type I) or qualitative (type II). Type I deficiencies are characterized by decreased functional and antigenic protein S levels. Type II deficiencies are characterized by decreased functional protein S levels but normal antigen levels. A third type of protein S deficiency, known as type III or type IIa, is characterized by decreased functional and free antigen levels with normal total antigen levels. Therefore, type II deficiencies are not detected by free protein S antigen assays, and type II and type III deficiencies are not detected by total protein S antigen assays. The authors conducted a study that suggests that some total protein S assay results could be normal even in the presence of a type I deficiency. The North American Specialized Coagulation Laboratory Association (NASCOLA) provides proficiency testing, or external quality assessment, to North American coagulation laboratories using the European Concerted Action on Thrombosis (ECAT) International Thrombophilia External Quality Assessment Scheme proficiency testing program. To assess current laboratory practices in North America and evaluate the performance of different reagent-instrument combinations for protein S testing, the authors analyzed the protein S results from all NASCOLA proficiency testing for 2002 and the first half of 2003. Furthermore, the authors compared the performance of NASCOLA laboratories with that of ECAT laboratories. They also performed a written survey of NASCOLA laboratories to further assess laboratory practices for protein S testing. The authors found that the free protein S antigen assays and the Diagnostica Stago Staclot protein S assay were extremely accurate in detecting a heterozygous type I protein S deficiency. Another functional protein S assay and most total protein S assays were less reliable, depending to some extent on the instrument used. All assays used by NASCOLA laboratories identified normal protein S specimens. NASCOLA laboratories performed at least as well as European Concerted Action on Thrombosis laboratories on the proficiency tests. The results suggest that the diagnosis of heterozygous protein S deficiency may be problematic with some assays. Many total protein S antigen assays do not add to the diagnosis and can be unreliable for protein S deficiency subtyping. Better standardization of functional and antigenic assays is needed.

Van Cott EM, Ledford-Kraemer M, Meijer P, et al. An analysis of North American Specialized Coagulation Laboratory Association proficiency testing. Am J Clin Pathol. 2005: 123: 778– 785.

Reprints: Dr. Elizabeth Van Cott, Division of Clinical Laboratories, Dept. of Pathology, Massachusetts General Hospital, Jackson 235, 55 Fruit St., Boston, MA 02114

Using oral fluid samples to detect measles virus strains Using oral fluid samples to detect measles virus strains

Vaccination programs have controlled measles infection in many parts of the world, and the transmission of measles virus has been interrupted in some countries, including the United States and Australia. Despite these achievements, measles continues to be a major childhood disease, causing nearly 800,000 fatalities annually. This has prompted the World Health Organization to undertake a phased approach to eliminating measles virus in regions of the world where the infrastructure for measles molecular surveillance is lacking. Laboratory diagnosis of measles infection is achieved primarily through the demonstration of measles virus-specific antibodies. In the measles virus genome, genetic variability exists in the nucleoprotein (N) and hemagglutinin (H) genes, with the greatest degree occurring in the carboxy-terminal end of the expressed N gene. This variability can be used in molecular typing of wild-type measles virus strains. When coupled with epidemiologic techniques, molecular characterization has proven to be a powerful adjunct to controlling measles by providing the means to identify the infected source and subsequent transmission pathways of the virus. To obtain a better understanding of the global picture of measles virus genotypes, it is necessary to expand measles surveillance to remote areas of the world that lack the infrastructure for appropriate collection, processing, storage, and shipment of specimens for virologic testing. Measles virus initially infects and replicates in the respiratory epithelium. The virus extends to local lymph nodes, where it is amplified, resulting in viremia. A few days before the onset of rash, virus can be cultured from the mucous membranes of the nasopharynx, conjunctivae, and mouth of an infected individual, suggesting that the respiratory tract is the site of virus release and that oral fluid is a rich source of the virus. Accordingly, saliva has been used to detect measles virus-specific antibodies and measles viruses by reverse transcriptase (RT)-PCR and their subsequent genetic characterization. The authors conducted a study in which they demonstrated that oral fluid dried onto filter paper can be used to detect and characterize measles virus strains. Using this approach, a measles virus-positive sample by RT-PCR could be obtained from 67 percent of serologically confirmed acute measles cases. Mimicking certain environmental conditions and duration of transportation established that measles virus RNA remained detectable and suitable for nucleic acid sequencing in oral fluid spots for at least one week. The authors concluded that in the context of a measles outbreak in a remote region of the world, where infrastructure is poor, oral fluid samples dried onto filter paper and sent to a specialized laboratory for testing will aid in identifying and characterizing the causative measles virus strain.

Chibo D, Riddell MA, Catton MG, et al. Applicability of oral fluid collected onto filter paper for detection and genetic characterization of measles virus strains.
J Clin Microbiol. 2005;43:3145–3149.

Reprints: Doris Chibo, 10 Wreckyn St., North Melbourne 3051, Victoria, Australia; doris.chibo@mh.org.au

Statins and vein graft intimal hyperplasia Statins and vein graft intimal hyperplasia

Studies suggest that statins can protect the vasculature in a manner that is independent of their lipid-lowering activity by inhibiting the small guanosine triphosphate-binding protein Rho and Rho-associated kinase. Little information is available on the inhibitory effect of statins on vein graft intimal hyperplasia, the main cause of late graft failure after bypass grafting. Therefore, the authors examined the effects of a hydrophilic statin on vein graft intimal hyperplasia in vivo and Rho-kinase activity in vitro. In the first experiment, they randomized rabbits to a control group (n=7) that was fed regular rabbit chow or to a pravastatin group (n=7) that was fed regular rabbit chow supplemented with 10 mg/kg pravastatin sodium. The branches of the jugular vein were ligated, and an approximately 3-cm segment of the jugular vein was taken for an autologous reversed-vein graft. The carotid artery was cut and replaced with the harvested autologous jugular vein. At two and four weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. In the second experiment, human umbilical vein endothelial cells and vascular smooth muscle cells were cultured and then treated with 1 µmol/L and 30 µmol/L pravastatin for 24 hours and harvested. Immunoblotting was performed on the resulting precipitates. Quantitative evaluation of phosphorylated myosin binding subunit and endothelial nitric oxide synthase was performed by densitometric analysis. The authors demonstrated that orally administering pravastatin to normocholesterolemic rabbits inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts four weeks after implantation (pravastatin group, 39.5±3.5 µm versus control group, 64.0±7.1 µm; n=7; P<0.05) and suppressed cell proliferation and apoptosis in the neointima two weeks after implantation. They also found that pravastatin inhibited Rho-kinase activity and accelerated endothelial nitric oxide synthase expression in human umbilical vein endothelial cells but did not inhibit Rho-kinase activity in vascular smooth muscle cells. The authors concluded that a hydrophilic statin can suppress intimal hyperplasia of the vein graft in vivo and show endothelial cell-tropic inhibition of Rho-kinase in vitro. Furthermore, these results strongly support using hydrophilic statins to prevent intimal hyperplasia of the vein graft after bypass grafting.

Yamanouchi D, Banno H, Nakayama M, et al. Hydrophilic statin suppresses vein graft intimal hyperplasia via endothelial cell-tropic Rho-kinase inhibition. J Vasc Surg. 2005;42:757–764.

Reprints: Dr. Dai Yamanouchi, Dept. of Vascular Surgery, Nagoya University Graduate School of Medical Sciences, 65 Tsurumai-chou, Show-ku, Nagoya 466-8550, Japan; daiy@med.nagoya-u.ac.jp

Interference of blood cell lysis on coagulation testing Interference of blood cell lysis on coagulation testing

Coagulation testing is a central aspect of the diagnostic approach to patients with hemostasis disturbances, and it is pivotal for monitoring antithrombotic therapies with heparins or oral anticoagulants. Among the major determinants of coagulation testing, the standardization of the preanalytical phase significantly influences result reliability. A standardized procedure for specimen collection has been found to be essential for achieving accurate and precise measurements that might provide appropriate clinical information. However, additional circumstances, besides specimen collection, might influence the results of coagulation testing, generate misleading results, and induce inappropriate diagnostic or therapeutic approaches to patient care. Problems arising from a cumbersome blood draw, such as unsatisfactory attempts to draw blood, difficulty locating easy venous accesses, and missing the vein, are known to interfere with routine coagulation assays, and the use of hemolyzed specimens has been discouraged to avoid unreliable results. However, little is known about how unsuitable samples caused by blood cell lysis influence routine coagulation testing. Therefore, the authors evaluated the interference of in vitro blood cell lysis on prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and dimerized plasmin fragment D (D-dimer) testing. They tested 12 aliquots for PT, aPTT, fibrinogen, and D-dimer. The aliquots were prepared by serial dilutions of homologous lysated samples collected from 10 subjects and displayed a final percentage of lysis ranging from zero to 9.1 percent. Lysis was achieved by subjecting citrated whole blood to a freeze-thaw cycle. The outcome measure was interference from blood cell lysis on routine coagulation testing. The authors observed statistically significant increases in PT and D-dimer in samples containing final lysate concentrations of 0.5 percent and 2.7 percent, respectively. Significant decreases were observed in aPTT and fibrinogen in samples containing a final lysate concentration of 0.9 percent. The analytical quality specifications for desirable bias are ±2.0 percent for PT, ±2.3 percent for aPTT, and ±4.8 percent for fibrinogen. Percent variations from the baseline values exceeding the analytical quality specifications for desirable bias were achieved for lysate concentrations of 0.9 percent (PT and aPTT) and 1.8 percent (fibrinogen), corresponding to average free plasma hemoglobin concentrations of 1.7 and 3.4 g/L, respectively. The authors concluded that although slightly hemolyzed specimens might still be analyzable, a moderate blood cell lysis, as low as 0.9 percent, influences the reliability of routine coagulation testing. Because interference in coagulation assays has a wide interindividual bias, the authors did not recommend correcting for lysis. They suggested that the most appropriate corrective measure is to quantify free hemoglobin and re-collect the sample.

Lippi G, Montagnana M, Salvagno GL, et al. Interference of blood cell lysis on routine coagulation testing. Arch Pathol Lab Med. 2006;130:181–184

Reprint information not available.

Structure of the tumor suppressor p53 Structure of the tumor suppressor p53

The tumor suppressor p53 performs an unusually large number of functions in the cell. It also binds to many proteins and ligands and has a complex architecture. Its core domain—residues 94 to 312—is the location of nearly all mutations that inactivate p53 in approximately half of human cancers. The core domain is naturally unstable, with a melting temperature of about 42°C to 44°C, and many oncogenic mutations inactivate the core by destabilizing or distorting it. Although the crystal structures of the wild-type human core domain and several mutants have been identified, it is not clear why the protein is so unstable. Nuclear magnetic resonance (NMR) spectroscopy can give important complementary information about local dynamics and does not suffer from constraints induced on mobile structures by crystal packing. Therefore, it may provide clues about the stability and function of p53. However, the size of the domain (~25 kDa) and its low solubility (~400 µM) and thermal and long-term stability render NMR experiments difficult. The authors solved the structure by using a combination of high-field magnets, ultrasensitive cryoprobes, and complex labeling patterns (15N, 13C, 2H and reverse protonation). They also determined amide proton exchange rates to study local and global unfolding at the level of individual residues. Direct observation of buried protons and dynamic processes provided structural information that is inaccessible by x-ray crystallography and identified structural reasons for instability that were confirmed by protein engineering. The authors found that the structure was very similar to that in the crystal but far more mobile than expected. Importantly, they were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. The authors mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. They also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to alternative hydrogen-bond patterns in the protein interior. The authors concluded that p53 appears to have evolved to be dynamic and unstable.

Canadillas JMP, Tidow H, Freund SMV, et al. Solution structure of p53 core domain: structural basis for its instability. Proc Soc Natl Acad Sci (USA). 2006;103:2109–2114

Reprints: Alan R. Fersht, Medical Research Council Centre for Protein Engineering, Medical Research Council Centre, Hills Rd., Cambridge CB2 2QH, United Kingdom; arf25@cam.ac.uk

Complement factor I and Staphylococcus aureus phagocytosis Complement factor I and Staphylococcus aureus phagocytosis

Staphylococcus aureus continues to be a frequent cause of severe hospital-acquired and community-acquired infections. Staphylococcal infections are addressed at significant medical expense, and disability and death are common outcomes, despite antibiotic treatment. Multiple virulence factors of S. aureus contribute to its ability to cause human disease, and antibiotic resistance is increasing. Immune-directed strategies are attractive as a new frontier for developing antistaphylococcal therapies in an era of increasing antibiotic resistance. Complement is a critical host-defense for controlling many bacterial pathogens and seems to play a vital role in controlling S. aureus infections. The authors described a mechanism by which C3b is cleaved to iC3b on the surface of S. aureus by the complement regulatory protein factor I. Factor I is a serum enzyme that, in the presence of a cofactor such as serum complement factor H, cleaves C3b. This activity is thought to limit host tissue damage from uncontrolled complement activation. The authors speculated that factor I-mediated cleavage of C3b would decrease the amount of C3b available on the surface of S. aureus to interact with CD35. This would be expected to decrease staphylococcal phagocytosis by phagocytic cells. If true, this effect would indicate that S. aureus benefits from, and possibly manipulates, factor I cleavage of C3b, which in turn enhances bacterial survival and likely contributes to human disease. The authors conducted a study in which they examined the relevance of their reasoning by determining whether factor I-mediated cleavage of S. aureus-bound C3b decreased phagocytosis of S. aureus by neutrophils. Compared with controls, anti-factor I antibody inhibited C3b cleavage on the surface of S. aureus by more than 83 percent (as measured by iC3b generation) and increased phagocytosis of S. aureus by more than 100 percent. Treatment of C3b-coated S. aureus with factor I increased generation of iC3b (75%), decreased the total amount of C3 fragments bound to the surface of S. aureus (58%), and decreased the number of bacteria phagocytosed (40%). Testing specifically for C3 fragments shed from the surface of S. aureus, the authors found that factor I increased shedding (43%). Notably, these factor I-mediated effects were of the same magnitude regardless of whether factor H, a known cofactor for factor I, was present. These findings indicate that S. aureus benefits from, and possibly manipulates, the normally host-protective activity of factor I cleavage of C3b, which results in bacterial escape from complement-mediated opsonophagocytosis. The authors concluded that because escaping opsonophagocytosis-mediated destruction is a necessary mechanism for bacterial survival resulting in human disease, preventing cleavage of C3b on the S. aureus surface, and thereby enhancing opsonophagocytosis, is a promising potential target for therapeutic intervention.

Cunnion KM, Buescher ES, Hair PS. Serum complement factor I decreases Staphylococcus aureus phagocytosis. J Lab Clin Med. 2005;146:279–286.

Reprints: Kenji M. Cunnion, Center for Pediatric Research, 855 W. Brambleton Ave., Norfolk, VA 23510; cunniok@chkd.org

Automated preparation of platelet concentrates Automated preparation of platelet concentrates

The authors conducted a study about automating the technique for preparing platelets from pooled whole blood-derived buffy coats for transfusion, which includes a sequence of manual steps. To standardize the platelet content in platelet concentrates, the authors evaluated the effects of combining buffy coats in the pools on the basis of the platelet concentration in donor blood. They assumed that the automatic technique could possibly standardize the process owing to a reproducible recovery of platelets. Buffy coat-derived platelet concentrates are widely used in Europe. In the mid-1990s, the use of platelet concentrates from buffy coats in Northern Europe and Italy was reported to be approximately 500,000 therapeutic doses, with an annual growth rate of 10 to 15 percent. Preparing platelet concentrates from buffy coats, however, is a laborious process and includes many manual steps, which makes automating the process attractive. For their study, the authors evaluated the performance of a novel automated system for preparing platelet concentrates from buffy coats derived from whole blood by studying the platelet counts, recovery of platelets, and storage effects. They described in vitro studies and experiences with routinely preparing platelet concentrates using the automated technique, as well as further development of the preparation technique with introduction of an integrated leukoreduction filter, optimization of the preparation process, and standardization of the platelet content of platelet concentrates. The authors’ first experiment was a paired in vitro study of platelet concentrates from six buffy coats prepared by automated and manual procedures. Their second and third experiments evaluated platelet concentrates from six buffy coats from the automated OrbiSac system (Gambro BCT). Experiment three included buffy coats selected based on donor data. Experiment four was a paired in vitro study of platelet concentrates from six buffy coats with an integrated white blood cell (WBC) filter and two different storage containers. Experiment five evaluated platelet concentrates from six buffy coats from the OrbiSac with an integrated WBC filter. Experiment six was similar to experiment five, with computer-selected pools of five buffy coats. The in vitro studies evaluated the effects of seven-day storage of platelets on platelet metabolism and disintegration. Experiments one and four had similar in vitro results. In experiment two, platelet content was 370 ∞ 109 ± 70 ∞ 109 per platelet concentrate and recovery from buffy coats was 76 ± 6 percent. In experiment three, the platelet content was 380 ∞ 109 ± 50 ∞ 109 per platelet concentrate and variation was reduced compared with randomly pooled buffy coats. In experiment five, increased platelet content was found (420 ∞ 109 ± 70 ∞ 109 per platelet concentrate and recovery from buffy coats of 80 ± 5%). In experiment six, five rather than six buffy coats gave 340 ∞ 109 ± 60 ∞ 109 platelets per platelet concentrate and recovery was 79 ± 5 percent. The authors concluded that these in vitro studies suggest that the OrbiSac technique is equivalent to the standard manual method regarding platelet in vitro characteristics during storage for seven days. The results of standardizing the platelet count in platelet concentrates by selecting the buffy coat pools on the basis of blood donor platelet concentration were encouraging.

Larsson S, Sandgren P, Sjödin A, et al. Automated preparation of platelet concentrates from pooled buffy coats: in vitro studies and experiences with the OrbiSac system. Transfusion. 2005;45:743–751.

Reprints: Dr. Stella Larsson, Transfusion Medicine C2 66, Karolinska University Hospital/Huddinge, S-141 86 Stockholm, Sweden; stella.larsson@kus.se


Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.