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January 2007

Editors:
Michael Cibull, MD
Subodh Lele, MD
Melissa Kesler, MD

Organochlorine contaminants in commercial fish oil preparations
Circulating adiponectin concentrations and lipid metabolism
Intron retention within the human kallikrein gene family
Standardization of serum creatinine measurements
Role of matrix metalloproteinases in rheumatoid arthritis
Prestorage leukoreduction and hemolysis of red cell concentrates
Laboratory-based surveillance of influenza virus in Taiwan
West Nile virus in the Canadian blood supply

bullet Organochlorine contaminants in commercial fish oil preparations

Fish possess antiatherogenic properties, presumably because of their high content of essential omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid. Several studies demonstrate that, as a group, patients with cardiac disease who consume fish regularly experience decreased mortality following myocardial infarction as well as other health benefits. Regular fish intake is also recommended to decrease the risk of coronary artery disease. On the other hand, many studies have illustrated that some types of fish contain high levels of environmental toxins, such as mercury, polychlorinated biphenyls (PCBs), organochlorine (OC) pesticides, and related compounds. Some of these toxins may negate the cardiovascular health advantages of eating fish. Organochlorines can be divided into pesticide OCs, or dichlorodiphenyltrichloroethane (DDT), and nonpesticide OCs, or PCBs. PCBs are unwanted byproducts of a variety of industrial processes and are found in transformers and capacitors that were manufactured before PCBs were banned in 1977. DDT is the best-known OC pesticide. Similar to PCBs, OC pesticides such as DDT are resistant to degradation and accumulate in fish and the environment. Exposure to OC pesticides may cause neurotoxicity and cardiac and pulmonary dysfunction. The levels of PCBs, OC pesticides, and related compounds in fish have received considerable attention in the press recently. Articles warn the public about the hazards of toxins in certain fish and discuss the use of fish oil as an alternative. Commercially available fish are either farm raised or wild. Researchers have addressed concerns about the source of fish and its associated contaminants. A Wall Street Journal article stated that farm-raised salmon have higher levels of certain toxins than wild salmon. Consequently, people who regularly consume farm-raised salmon may have an increased risk of cancer later in life. On the other hand, toxins such as mercury are detected at equal or higher concentrations in wild fish, so neither appears superior. In this study, the authors examined the levels of organochlorines in five over-the-counter fish oil preparations to further evaluate whether advocating fish oil supplements over eating fish is warranted. The contents of the five commercial fish oil brands were sent for OC analysis. The analysis found that the levels of polychlorinated biphenyls and OCs were below the detectable limit. The authors concluded that fish oil supplements are more healthful than consuming fish high in OCs. Fish oils provide the benefits of omega-3 fatty acids without the risk of toxicity. In addition, fish oil supplements have been helpful in treating a variety of diseases, including bipolar disorder and depression.

Melanson SF, Lewandrowski EL, Flood JG, et al. Measurement of organochlorines in commercial over-the-counter fish oil preparations: implications for dietary and therapeutic recommendations for omega-3 fatty acids and a review of the literature. Arch Pathol Lab Med. 2005;129:74–77.

Reprints: Dr. Kent B. Lewandrowski, Division of Laboratory Medicine, Massachusetts General Hospital, 55 Fruit St., GRJ 5, Boston, MA 02114; klewandrowski@partners.org

bullet Circulating adiponectin concentrations and lipid metabolism

Adipose tissue secretes a myriad of proteins that are increasingly recognized as influencing the metabolism of carbohydrates, proteins, and lipids. Adiponectin (also called Acrp30, or adipoQ in mice) is a 244-amino acid protein synthesized and secreted exclusively by adipose tissue. Adiponectin circulates at relatively high concentrations and can have a shelf life of several hours. This rate of turnover would be consistent with a role for this protein as a regulator of metabolic processes, such as in modulating fat partitioning, among other actions. Knowing how dietary habits may influence circulating adiponectin concentrations could be helpful in delineating dietary measures aimed at preventing type 2 diabetes. One can monitor the type of fat in a diet by recording the fatty acid composition in plasma. The authors evaluated the association between dietary fat, as inferred from plasma fatty acid composition (gas–liquid chromatography), and circulating adiponectin in 116 healthy subjects. The proportion of saturated fatty acids in plasma was significantly associated with circulating adiponectin concentration (r=–0.24; P=0.01). Specifically, percentage of palmitic acid (C16:0) was significantly associated with lower adiponectin concentration (r=–0.28; P=0.002), particularly among women (r=–0.37; P=0.02) and nonsmokers (r=–0.30; P=0.007). Percentage of myristic acid (C14:0) was also significantly associated with lower adiponectin among nonsmokers (r=–0.26; P=0.02) and women (r=–0.39; P=0.01). The other fatty acids were not significantly associated with adiponectin, except for eicosanoic acid (C20:1 omega-9), which was significantly and positively associated with adiponectin in all individuals (r=0.23; P=0.01). This latter association was most significant in smokers (r=0.43; P=0.007). In a multivariate regression analysis for circulating adiponectin, after controlling for age, body mass index, waist-to-hip ratio, and the remaining individual fatty acids, the percentages of palmitic (P=0.005) and eicosanoic acid (P=0.03) contributed independently (six and three percent, respectively) to adiponectin variance. Among nonsmokers, the percentages of palmitic acid (P=0.01) and omega-3 fatty acids contributed eight and seven percent, respectively, to adiponectin variance. Among smokers, the percentage of eicosanoic acid (P=0.03) contributed to 10 percent of adiponectin variance, independently of body mass index, age, waist-to-hip ratio, and the remaining individual fatty acids. The authors concluded that saturated and omega-3 fatty acids of dietary origin (as inferred from plasma fatty acid concentration) are associated with circulating adiponectin concentrations in healthy humans. The proportion of eicosanoic acid also appears to be positively associated with circulating adiponectin. Knowing how these interactions occur may be helpful in planning dietary measures to modulate inflammatory activity.

Fernandez-Real J-M, Vendrell J, Ricart W. Circulating adiponectin and plasma fatty acid profile. Clin Chem. 2005;51:603–609.

Reprints: J-M Fernandez-Real, Unitat d’Endocrinologia, Diabetes i Nutricio, Hospital de Girona, Carretera de Francia s/n, 17007 Girona, Spain; uden.jmfernandezreal@htrueta.scs.es

bullet Intron retention within the human kallikrein gene family

Reports indicate that 10 to 30 percent of alternatively spliced human genes possess tissue-specific splice forms and at least 300 genes possess cancer-specific mRNA variants. Among the hallmarks of cancer are genomic mutations. It has been estimated that at least 15 percent of mutations that cause genetic diseases affect pre-mRNA splicing. The human kallikrein (KLK) genes are a family of 15 serine protease genes that have clinical utility. Human kallikrein 3 (prostate-specific antigen) is the best biomarker for prostate cancer screening, diagnosis, staging, and monitoring. Alternative pre-mRNA splicing is a common event among members of the KLK gene family. Approximately 70 KLK splice variants have been reported, and each KLK gene possesses at least one variant. The authors conducted a study to examine the frequency of a common type of splicing (intron retention) within the KLK gene family. Intron retention has been reported for several other genes. An earlier study examining intron retention in a set of 21,106 known human genes revealed that 14.8 percent retained at least one intron. The probability of intron retention increases as intron length decreases because introns of less than 100 bp in length are retained in 95 percent of cases. Among human kallikrein splice variants, Psa-rp2 for the KLK33gene (GenBank accession No. AJ310938) and KLK4 variant one for the KLK44 gene (GenBank accession No. AF148532) retain intron III, which is relatively short (143 and 83 bp, respectively). Because other kallikrein genes, such as KLK1, -2, -5, and -15, also possess a short intron III, the authors speculated that retention of intron III might be a common splicing event among members of the KLK family. The authors used variant-specific reverse transcription-PCRs (RT-PCRs) for KLK1, -2, -5, and -15 to identify and clone the full coding sequence of intron III-containing splice variants. In addition, variant-specific RT-PCRs for the cloned KLK3 and KLK4 variants, as well as for the classical forms of the six genes, were used to determine their expression profiles in healthy tissues, regulation by steroids, and differential expression in prostate cancer. The authors found that KLK1, -2, -3, -4, -5, and -15 showed a common type of splice variant in which intron III is retained. Expression profiling of these splice variants revealed expression profiles similar to those of the classical mRNA forms, although the pattern of hormonal regulation differed. The KLK15 splice variant was upregulated in eight of 12 cancerous prostate tissues. All encoded variant proteins were predicted to be truncated and catalytically inactive because of a lack of the serine residue of the catalytic triad. The authors concluded that the first six centromeric members of the KLK gene family have splice variants that retain intron III. Some variants show tissue-specific expression. The KLK15 splice variant appears to be a candidate biomarker for prostate cancer.

Michael IP, Kurlender L, Memari N, et al. Intron retention: a common splicing event within the human kallikrein gene family. Clin Chem. 2005;51:506–515.

Reprints: Eleftherios P. Diamandis, Dept. of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5; ediamandis @ mtsinai. on.ca

bullet Standardization of serum creatinine measurements

The National Kidney Disease Education Program of the National Institutes of Health recommends that an estimated glomerular filtration rate be calculated from serum creatinine and reported to physicians to help identify patients with renal disease in the early stages. The National Kidney Disease Education Program recognizes the importance of standardized creatinine measurements to achieve this goal. Creatinine measurements need to be standardized among all laboratories so that a common equation can be used to estimate glomerular filtration rate (GFR) and to provide consistent interpretive guidelines. The authors reported results for creatinine measurement from proficiency testing involving 5,624 laboratories using 50 different instrument-method combinations, which was conducted by the CAP in October 2003. The Survey included one specimen that was a specially prepared fresh-frozen serum pool intended to be commutable among all methods and thus able to evaluate the state-of-the-art in harmonization of results. This specimen had creatinine values assigned by higher-order isotope dilution mass spectrometry so accuracy could be evaluated for individual laboratories and trueness for method groups. To evaluate the state-of-the-art in measuring serum creatinine, as well as the ability of a proficiency testing program to measure bias for individual laboratories and method peer groups, a fresh-frozen, off-the-clot pooled serum specimen, plus four conventional specimens, were sent to participants in the CAP Chemistry Survey for assay of creatinine. Creatinine concentrations were assigned by isotope dilution mass spectrometry reference measurement procedures. The participants were clinical laboratories with an acceptable result for all five Survey specimens. The authors found that the fresh-frozen serum specimen had a creatinine concentration of 0.902 mg/dL (79.7 µmol/L). Mean bias for 50 instrument-method peer groups varied from –0.06 to 0.31 mg/dL (–5.3 to 27.4 µmol/L), with 30 (60 percent) of 50 peer groups having significant bias (P<0.001). The bias variability was related to instrument manufacturer (P=0.001) rather than method type (P=0.02), with 24 (63 percent) of 38 alkaline picric acid methods and six (50 percent) of 12 enzymatic methods having significant biases. Two conventional specimens had creatinine concentrations of 0.795 and 2.205 mg/dL (70.3 and 194.9 µmol/L) and had apparent Survey biases that differed significantly (P<0.001) from that of the fresh-frozen serum specimen for 34 (68 percent) and 35 (70 percent) of 50 peer groups, respectively. The authors concluded that 30 of 50 peer groups had significant bias for creatinine. Bias was primarily associated with instrument manufacturer, rather than type of method used. Proficiency testing using a commutable specimen measured participant bias versus a reference measurement procedure and provided trueness surveillance of instrument-method peer groups.

Miller WG, Myers GL, Ashwood ER, et al. Creatinine measurement: state of the art in accuracy and interlaboratory harmonization. Arch Pathol Lab Med. 2005;129:297–304.

Reprints: Dr. Greg Miller, Virginia Commonwealth University, P.O. Box 980286, Richmond, VA 23298-0286; gmiller@vcu.edu

bullet Role of matrix metalloproteinases in rheumatoid arthritis

Matrix metalloproteinases are a group of Zn2+-dependent extracellular enzymes that play a key role in normal and pathological tissue remodeling. The whole group can be divided into subclasses, such as collagenases, gelatinases, stromelysins, and membrane-type matrix metalloproteinases (MMPs). MMPs are involved in excessive degradation of joint tissue in rheumatoid arthritis. MMP-3 (stromelysin-1) can degrade various components of joint tissue and activate proMMPs. It has been suggested that MMP-3 is a marker of synovitis derived from the joint because it is produced by synovial fibroblasts of the rheumatoid joints and not by normal synovial cells and because it correlates with inflammatory markers in the systemic circulation, such as C-reactive protein (CRP). Other MMP subclasses, such as gelatinases and collagenases, are expressed by cells of the pannus-cartilage or bone junction and by infiltrating inflammatory cells, such as neutrophils and macrophages. Increased levels of collagenases and gelatinases are found not only at the tissue level, but also in synovial fluid and the systemic circulation. Moreover, high tissue levels of MMP-2 and -9 and systemic levels of MMP-1 in patients with rheumatoid arthritis are associated with joint erosion. Few studies are available on multiple MMP subclasses or the relationship between local and systemic levels of the different MMP subclasses. The authors conducted a study to provide a broad analysis of the MMPs that play a part in rheumatoid arthritis pathology and to investigate the relationship between local and systemic levels of MMPs and their tissue inhibitor (TIMP). They also investigated the relationship between MMPs and systemic marker of inflammation, CRP, and joint collagen degradation product, hydroxyproline (OHPro), in patients with rheumatoid arthritis. ProMMP-1, -2, -3, -8, -9, TIMP-1, levels of MMP/a2-macroglobulin complexes, and collagen degradation products were measured by sandwich ELISA, activity assays, and high-pressure liquid chromatography in paired synovial fluid and serum samples from 15 patients with rheumatoid arthritis and 13 with osteoarthritis. MMPs were higher in the synovial fluid of patients with rheumatoid arthritis than in that of patients with osteoarthritis or controls. MMP levels in the synovial fluid of patients with osteoarthritis were higher than those in controls. In serum, levels of proMMP-3, -8, and -9 were higher in patients with rheumatoid arthritis than in patients with osteoarthritis or controls, whereas only proMMP-8 and -9 were higher in the serum of patients with osteoarthritis than in controls. A strong correlation was noted between the serum and synovial fluid levels of MMP-8 and -9 in rheumatoid arthritis. Increased levels of MMP/α2-macroglobulin complexes indicated an MMP/TIMP imbalance in serum and synovial fluid in rheumatoid arthritis. Synovial fluid hydroxyproline correlated significantly with synovial fluid levels of proMMP-9 in rheumatoid arthritis. The authors concluded that systemic MMP-8 and -9 levels represent the situation in the inflamed joint. MMP-9 is likely to be involved in degradation of joint collagen. This strengthens the hypothesis of MMP/TIMP imbalance in rheumatoid arthritis.

Tchetverikov I, Ronday HK, van El B, et al. MMP profile in paired serum and synovial fluid samples of patients with rheumatoid arthritis. Ann Rheum Dis. 2004;63:881–883.

Reprints: Dr. R. Hanemaaijer, TNO Prevention and Health, P.O. Box 2215, 2301 CE, Leiden, Netherlands; r.hanemaaijer@pg.tno.nl

bullet Prestorage leukoreduction and hemolysis of red cell concentrates

Universal leukoreduction of the Canadian blood supply was implemented in 1998. Subsequent to this, Canadian Blood Services received reports of red blood cells (RBCs) that contained unacceptable levels of hemolysis by visual inspection. Such reports created a perception that the new, leukoreduced product may be more hemolyzed than its nonleukoreduced (NLR) predecessor. Several studies performed since 1989 have shown that leukoreduction can reduce the hemolysis seen during RBC storage for 42 days. Most of these studies compared two groups of donors—those whose donation was leukoreduced and those whose donation was not. The studies also addressed the effects of temperature and methodologic differences in different laboratories. In some cases, the same donors were used in crossover studies on two occasions, the blood being leukoreduced on one occasion and not the other. None of these studies, however, used a “pool-and-split” design to directly compare the same blood with and without leukoreduction. The authors conducted a pool-and-split study designed to remove all variables except leukoreduction. For the study, two ABO-matched units were pooled, divided, and processed into leukoreduced and nonleukoreduced units with Pall’s whole blood in-line leukoreduction filter (LT-WB) or platelet production system (RC-PL). They were then sampled during standard processing and storage for testing of sterility, counts, hemolysis, and osmotic fragility. Room temperature filtration of 10 pairs of LT-WB-LR and -NLR units showed significantly different percentages of hemolysis (0.39 percent) and osmotic fragility (0.643 percent) at 42 days. Cold-stored and -filtered units (two days at 4°C before processing) were less hemolyzed but showed a similar proportional decrease of hemolysis in leukoreduced units (0.13 versus 0.25 percent at 42 days). Red blood cells from RC-PL systems showed the lowest hemolysis, although there was a filtration effect (0.05 versus 0.12 percent at 42 days). Osmotic fragility paralleled hemolysis. Segment samples gave inaccurate results. Two-day prefiltration cold storage reduced hemolysis from 0.36 to 0.07 percent (P<0.001 at 42 days). RT-LR hemolysis became significantly higher by day 10 and 4°C leukoreduced by day 12. Nonleukoreduced units showed hemolysis by day seven. Leukoreduced units filtered cold were less hemolyzed (P<0.05) than RT-LR, but osmotic fragility was unchanged. The authors concluded that LR-RBCs prepared by any of three methods (LT-WB, room temperature or cold, RC-PL) and filtered at 4°C were less hemolyzed during storage than nonfiltered concentrates. Furthermore, 4°C leukoreduction is beneficial for RBCs and does not cause hemolysis or enhance fragility.

Gyongyossy-Issa MIC, Weiss SL, Sowemimo-Coker SO, et al. Prestorage leukoreduction and low-temperature filtration reduce hemolysis of stored red cell concentrates. Transfusion. 2005;45:90–96.

Reprints: Maria I.C. Gyongyossy-Issa, Canadian Blood Services, Dept. of Pathology, University of British Columbia, 2211 Wesbrook Mall, Room GF401; missa@interchange.ubc.ca or mariaissa@bloodservices.ca

bullet Laboratory-based surveillance of influenza virus in Taiwan

Influenza virus belongs to the family Orthomyxoviridae, which is characterized by a segmented, minus-stranded RNA genome. Influenza epidemics occur annually in many parts of the world and cause high mortality and morbidity. The low fidelity of the viral polymerase complex, as well as host immune selection, account for the accumulation of point mutations in hemagglutinin and neuraminidase genes, producing the antigenic drift on these surface glycoproteins. The antigenic drift plays an important role in influenza epidemics. Gene reassortment is also attributed to influenza virus mutation. An antigenic shift occurs in the event of a reassortment between two viruses of different surface antigenic subtypes, leading to a novel subtype composition. For example, the pandemic influenza A virus subtype H3N2 in 1968 and the subtype H2N2 in 1957 resulted from reassortment between avian and human viruses. Because of the high mutation rate of influenza virus, it is necessary to continuously monitor viral genetic change. The influenza virus surveillance system is well-established in many countries and has helped control influenza virus infections. The influenza surveillance work in Taiwan, however, was not well-organized until a laboratory-based surveillance network of influenza viruses was established in 2000 under the coordination of the Centers for Disease Control and Prevention Department of Health, Taiwan. This network includes 11 clinical virology laboratories geographically distributed in northern, central, southern, and eastern Taiwan. From October 2000 until March 2004, 3,244 influenza viruses were isolated, including 1,969 influenza A and 1,275 influenza B viruses. The influenza infections usually occurred in winter in the Northern Hemisphere. However, the influenza seasonality in Taiwan was not clear during the four seasons under investigation. For example, the influenza A viruses peaked during the winters of 2001, 2002, and 2003. However, some isolated peaks were also found in the summer and fall (June to November) of 2001 and 2002. An unusual peak of influenza B occurred in the summer of 2002 (June to August). Phylogenetic analysis shows that influenza A isolates from the same year were often grouped together. However, influenza B isolates from 2002 clustered into different groups, and the data indicated that B/Victoria/2/87-like and B/Yamagata/16/88-like lineages of influenza B viruses were co-circulating. The authors concluded that sequence comparison of epidemic strains versus vaccine strains shows that many vaccine-like Taiwanese strains were circulating at least two years before the vaccine strains were introduced. No clear reports of seasonality of influenza in Taiwan occurred, in contrast to reports in other, more continental, regions, probably because Taiwan is a subtropical island with insignificant temperature change between seasons.

Shih S-R, Chen G-W, Yang C-C, et al. Laboratory-based surveillance and molecular epidemiology of influenza virus in Taiwan. J Clin Microbiol. 2005;43:1651–1661.

Reprints: Ih-Jen Su, Division of Clinical Research, NHRI, 138, Shen-Li Rd., Tainan, Taiwan; suihjen@nhri.org.tw

bullet West Nile virus in the Canadian blood supply

West Nile virus, a member of the genus flavivirus, was first isolated in Uganda in 1937 and has been associated with sporadic cases and outbreaks in Africa, the Middle East, and Asia. Birds are the primary vertebrate host for West Nile virus (WNV), but humans and other mammals are also infected. The first incidence of WNV activity in birds and mosquitos in Canada was reported in August 2001, with the first confirmed human cases reported in 2002. Testing for WNV RNA in Canadian blood donors began in the summer of 2003. The commercial WNV TaqScreen assay (Roche Molecular Systems) was used for routine donor screening, which was done on minipools of six donations under a Health Canada investigational testing authorization. Because this assay was developed rapidly for use in experimental screening by summer 2003 under the investigational testing authorization, an in-house, real-time, quantitative WNV nucleic acid testing (NAT) assay that was specific for WNV was also used by the Canadian Blood Services to confirm positive results as WNV positive. An additional advantage of this assay was the ability to quantitate the levels of viremia. In addition, follow-up samples, where possible, were obtained from the donors and tested for WNV NAT with the in-house assay and for antibodies to WNV—immunoglobulin M (IgM) and immunoglobulin G (IgG). The seroconversion data were particularly valuable to classify the samples as true WNV-positive samples, whereas the NAT results were intended to provide data on the duration of WNV viremia. The original 14 samples and follow-up samples (two to 35 days after donation) available from 13 of the 14 donors were tested with the in-house, real-time, quantitative WNV NAT assay. All donors tested positive for WNV with the in-house assay. Donors 18 and 19, identified by single-donor testing, had extremely low levels of viremia that could only be detected in 1:38 or 1:39 replicate tests. The titers of the remaining index samples ranged from below log2.8 (the limit of quantitation) to log4.7 NAT detectable units per milliliter. Samples from donors 17, 18, and 19 were IgM positive, and samples from donors 18 and 19 were also IgG positive. The remaining 10 donors with follow-up samples seroconverted. The authors concluded that the 14 WNV donor samples detected by routine screening were confirmed as WNV RNA positive by a WNV RNA-specific in-house assay and by seroconversion in 13 of the 14 donors.

Cameron C, Reeves J, Antonishyn N, et al. West Nile virus in Canadian blood donors. Transfusion. 2005; 45:487–491.

Reprints: Dr. John Saldanha, Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, CA 94588; john.saldanha@roche.com


Dr. Cibull is professor of pathology and laboratory medicine and direct of surgical pathology, University of Kentucky Medical Center, Lexington. Dr. Lele is assistant professor of pathology and laboratory medicine, University of Kentucky Medical Center. Dr. Kesler is hematopathology fellow, University of Texas Southwestern Medical Center at Dallas.