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September 2004

Editors:
Michael Bissell, MD, PhD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus
Ronald Domen, MD, professor of pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pennsylvania.

Hemoglobin Q-India
Molecular epidemiologic predictors of tuberculosis clustering
Isolated, prolonged activated partial thromboplastin time in pregnancy
Cytokine measurements in cancer screening
Cleanliness of environmental surfaces and infection control through handwashing
Bone hormone genes and bone mineral density

Hemoglobin Q-India

Hemoglobin Q-India Non-enzymatic glycosylation is a post-synthetic modification of hemoglobin that leads to the formation of hemoglobin A1c. The estimation of Hb A1c is used extensively in the management of diabetes mellitus. Some systems use low-pressure liquid chromatography (LPLC), and, occasionally, previously undetected variants are discovered in patients. This report describes three patients with diabetes who were found to have the uncommon variant Hb Q-India (α64(E13)Asp→His). Two cases were initially revealed during routine Hb A1c estimation and a third during the investigation of anemia in a patient with diabetes. To identify the variant, it was necessary to do additional studies by high-performance liquid chromatography (HPLC) and isoelectric focusing (IEF) electrophoresis. The Hb variant was identified initially by determining the HPLC retention time of the abnormal peak, measuring the IEF position of the variant band, and comparing the data with that of known variants. However, the techniques of IEF and HPLC specific for Hb variants are not available in many laboratories, especially in developing countries, and thus another simple approach is required. To date, mutation analysis techniques have only been developed and applied to the diagnosis of the common Hb variants, such as Hb S, Hb C, and Hb E. In this case, the authors reported on the use of a novel DNA test based on the amplification refractory mutation system (ARMS). The presence of an Hb variant was confirmed by cellulose acetate electrophoresis at a pH of 8.6. The variant was characterized further by HPLC (BioRad Variant) and IEF electrophoresis. A novel DNA analysis test based on the ARMS and polymerase chain reaction (PCR) was developed to confirm the presence of the mutation for the uncommon variant. Comparison of the HPLC retention time and IEF band position determined the presence of the variant Hb Q-India in all three cases. ARMS-PCR specific primers were designed and used successfully to confirm the presence of the mutation of Hb Q-India. The authors concluded that the ARMS-PCR technique, developed for the diagnosis of β thalassemia mutations, can also be adapted to identify abnormal hemoglobins.

Abraham R, Thomas M, Britt R, et al. Hb Q-India: an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test. J Clin Pathol. 2003;56:296-299.

Reprints: Dr. J. Old, National Haemoglobinopathy Reference Laboratory, Oxford Haemophilia Centre, Churchill Hospital, Oxford OX3, 71J, United Kingdom; john.old@orh.nhs.uk

Molecular epidemiologic predictors of tuberculosis clustering

The combination of DNA fingerprinting of Mycobacterium tuberculosis and conventional epidemiologic methods has improved the medical world's understanding of tuberculosis transmission. The authors used DNA fingerprinting in a population-based study of tuberculosis to determine predictors associated with being in a cluster (having M. tuberculosis isolates with an identical DNA pattern) to better understand the transmission of tuberculosis in Greater Vancouver, British Columbia. They used the restriction fragment length polymorphism (RFLP) technique and, if necessary, spoligotyping to determine DNA patterns of M.tuberculosis isolates from all patients with newly diagnosed tuberculosis in Greater Vancouver reported to the Division of Tuberculosis Control from January 1995 to March 1999. Isolates were considered to be part of a cluster if they had an identical DNA pattern. The authors also collected demographic and epidemiologic data. Predictors associated with being in a cluster were analyzed in a multivariate logistic regression model. Isolates from 793 patients (430 men) were identified; 137 (7.3 percent) were considered to be in clusters. After adjusting for multiple potential predictors, the following patients were found to be more likely to be part of a cluster: Canadian-born Aboriginals (versus foreign-born patients) (adjusted odds ratio, 6.0, 95 percent confidence interval, 3.0-11.7), Canadian-born non-Aboriginals (versus foreign-born patients) (adjusted odds ratio, 3.6, 95 percent confidence interval, 2.1-6.3), and injection drug users (versus patients who did not inject drugs) (adjusted odds ratio, 3.9, 95 percent confidence interval, 1.9-8.1). Patients with a prior history of tuberculosis were less likely to be part of a cluster than were patients with no history of tuberculosis (adjusted odds ratio, 0.3, 95 percent confidence interval, 0.1-0.8). The findings indicate the need to target groups at high risk of tuberculosis more aggressively to prevent transmission and to treat latent infection. DNA fingerprinting may be a useful adjunct to conventional epidemiologic methods to monitor the transmission of tuberculosis in an inner-city setting.

Hern87dez-Gardu96o E, Kunimoto D, Wang L, et al. Predictors of clustering of tuberculosis in Greater Vancouver: a molecular epidemiologic study. CMAJ. 2002;167: 349-352.

Reprints: Dr. J. Mark FitzGerald, Center for Clinical Epidemiology and Evaluation, Vancouver General Hospital Research Pavilion, 703-828 W. 10th Ave., Vancouver, BC V5Z 1L8, Canada; markf@interchange.ubc.ca

Isolated, prolonged activated partial thromboplastin time in pregnancy

Isolated, prolonged activated partial thromboplastin time in pregnancy A prolonged activated partial thromboplastin time in pregnancy can be a normal laboratory finding or can be secondary to several obstetric complications, such as eclampsia, placental abruption, amniotic fluid embolism, consumption coagulopathy, or an acquired factor inhibitor. Because data on the causes of isolated prolonged activated partial thromboplastin time (aPTT) in pregnancy are limited, the authors retrospectively studied data from 36 pregnant women with elevated aPTT. Patients who had a history of prior coagulopathy were excluded. None of the 36 patients had a history of excessive bleeding or thromboembolism. The median elevation of aPTT from control value was 2.5 seconds (range, 0.4 to 36.5 seconds). The prothrombin time and international normalized ratio were normal. On repeat aPTT testing, 24 (67 percent) patients were found to be normal and 12 (33 percent) remained elevated. A mixing study was performed for all 12 patients with persistently elevated aPTT. The mixing study was corrected at zero hours and did not prolong after a two-hour incubation in nine patients. In three patients, the aPTT remained prolonged at zero hours, suggesting an inhibitor. Additional coagulation testing was performed in this group of 12 patients. No cause for the repeated prolonged aPTT was identified in four of 12 patients. Factor XI deficiency was found in five of 20 patients (median factor XI activity, 36 percent). Abnormal dilute Russell viper venom time was found in three, elevated anticardiolipin antibody in two, and low von Willebrand Factor level in one. Twenty-three of the 36 patients had full-term pregnancies and were delivered. No patients had excessive postpartum bleeding, and none developed deep vein thrombosis. (Three patients received prophylactic heparin therapy during pregnancy.) Two patients had spontaneous abortion in the first trimester (both had normal dilute Russell viper venom time and anticardiolipin antibody level). The authors concluded that factor XI deficiency and antiphospholipid antibody are two major abnormalities identified in pregnant patients with prolonged aPTT. Proper specimen collection and processing are important for coagulation assays to avoid erroneous clotting times.

Ahmed S, Russo LA, Siddiqui AK, et al. Prolonged activated partial thromboplastin time in pregnancy: a brief report. Am J Med Sci. 2004;327:123-126.

Correspondence: Dr. Linda Russo, Long Island Jewish Medical Center, Hematology-Oncology, 270-05 76th Ave., New Hyde Park, NY 11040; shahidahmed00@yahoo.com

Cytokine measurements in cancer screening

Cytokine measurements in cancer screening Human papillomavirus, Epstein-Barr virus, hepatitis B virus, and hepatitis C virus are common viral infections that may be associated with the development of neoplasia. Studies aimed at understanding the factors that lead a small fraction of infected individuals to progress to more severe disease have focused on evaluating the immune response to these viruses. Cancers caused by viruses may arise as a result of inadequate control of the viral infection by the immune system, but the exact nature of a successful immune response is poorly understood. The authors attempted to validate a new multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines—interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, and tumor necrosis factor-α. They obtained from 26 participants 750 supernatant specimens from peripheral blood mononuclear cell cultures stimulated with various mitogens and antigens, including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and medial alone. These were tested in replicate using the 8-plex LINCOplex assay in a blinded fashion. Spearman correlation coefficients for continuous results and exact agreement rates and weighted kappa statistics for quartiled variables were computed to evaluate intra- and interassay agreement. IL-4 levels were consistently below the detectable level of the assay (3 pg/mL), whereas IL-6 and IL-8 levels were consistently above the highest detectable level of the assay (10,000 pg/mL), and these three cytokines were, therefore, not evaluated further. Excellent intra-assay reproducibility was observed for the remaining five cytokines, with Spearman correlation coefficients consistently above 0.90 for all five. Exact agreement rates ranging from 77.6 to 90.3 percent and weighted kappas ranging from 0.81 to 0.92 were observed. Similar results were observed when analysis was restricted to supernatants obtained from cultures that had been stimulated with HPV16 peptides and when analysis was restricted to supernatants obtained from cultures containing no antigen or mitogen. This suggests that the LINCOplex assay is applicable under conditions where moderate or weak cytokine responses or levels are expected. For IL-2, exact agreement and weighted kappa values were 68.5 percent and 0.72 (95 percent confidence interval, 0.65 to 0.79), respectively. For IFN-γ, they were 67.3 percent and 0.73 (95 percent confidence interval, 0.67 to 0.80), respectively. These results indicate that the LINCOplex assay is applicable for the simultaneous measurement of multiple cytokines using small amounts of biological material.

Hildesheim A, Ryan RL, Rinehart E, et al. Simultaneous measurement of several cytokines using small volumes of biospecimens. Cancer Epidemiol Biomarkers Prev. 2002;11:1477-1484.

Reprints: Allan Hildesheim, Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd., Room 7062, MSC 7234, Rockville, MD 20852; Hildesha@exchange.nih.gov

Cleanliness of environmental surfaces and infection control through handwashing

Effective handwashing, recognized as one of the best means for preventing the spread of infections in hospitals, is aimed at minimizing the potential for infection to patients directly or indirectly by hand contact to prevent health care personnel from becoming vectors of nosocomial pathogens. Proper handwashing has five interrelated components. First, the hands should be hygienic with short, clean nails, free of dermatologic disruption, and free of jewelry. Second is compliance—that is, getting health care workers to wash their hands at appropriate times in relation to task demand. Unfortunately, rates of handwashing compliance are far lower than desirable, and rates of hospital-acquired infections are believed, in part, to reflect these failures. Third is effective handwashing—that is, removal of organic debris and microbial load, especially transient flora and potential pathogens. Fourth is effective hand drying, which helps to remove soil, loose stratum corneum, and microorganisms by application of kinetic/frictional energy (greater with paper towels), along with removing remaining moisture. This is important because residual moisture can considerably enhance the transfer (pickup and deposition) of any remaining microorganisms present on the hands to other surfaces. Fifth is the prevention of hand contamination at any time during the whole process. The first four components have received considerable attention, and although the potential for contamination has been recognized, the exposure routes and levels of risk are less well-defined and quantified. This is especially critical because terminal or end-process contamination may negate the value of all of the previous components. The ability of the various stages of handwashing to decrease skin-surface microbial counts has been documented. However, an important element, environmental surface cleanliness, and the potential for contamination of hands during the process, has not been well studied or quantified. The authors reported on measurements of adenosine triphosphate (a measure of residual organic soil), bacterial, and staphylococcal load on ward handwash station surfaces, which could be touched during handwashing. Hand contact surfaces tested consisted of approximately 620 each of faucet handles, soap dispenser activator mechanisms, and folded paper-towel dispenser exits. Failure rates in excess of benchmark clean values were higher with adenosine triphosphate assays than microbial counts. This could indicate the presence of a higher level of general organic debris—for example, skin cells—as opposed to microbial contamination or could reflect greater assay sensitivity. Faucet handles were more likely to be contaminated and to be in excess of benchmark values than paper-towel dispenser exits. However, the latter is likely to be the final surface touched during the handwashing process, and, overall, nearly 20 percent were above microbiologic benchmark values. Many of the organisms isolated were staphylococci.

Griffith CJ, Malik R, Cooper RA, et al. Environmental surface cleanliness and the potential for contamination during handwashing. Am J Infect Control. 2003;31:93-96.

Reprints: Dr. C.J. Griffith, School of Applied Sciences, University of Wales Institute, Cardiff CF23 9XR, United Kingdom

Bone hormone genes and bone mineral density

Genetic linkage studies and candidate gene association studies have implicated several loci and candidate genes in the regulation of bone mass, as measured by bone mineral density, and the pathogenesis of osteoporotic fractures. Such candidate genes also include those for interleukin-6 (IL-6), a multifunctional cytokine that plays an important role in the development of postmenopausal osteoporosis, as well as osteocalcin and the vitamin D receptor. The authors conducted a large-scale population-based study to examine whether the -634C→G single nucleotide polymorphism (SNP) of the IL-6 gene, the 298C→T SNP of the osteocalcin gene, and the 2C→T SNP of the vitamin D receptor gene are associated with bone mineral density in women or men. They studied community-dwelling Japanese women (between 1,108 and 1,113) or men (between 1,116 and 1,130) aged 40 to 79 years. The authors' results suggest that the -634C→G polymorphism of the IL-6 gene and the 298C→T polymorphism of the osteocalcin gene are associated with bone mineral density in postmenopausal women, with the respective GG and TT genotypes representing risk factors for reduced bone mass. IL-6 and osteocalcin genotypes showed additive effects on bone mineral density for postmenopausal women. The 2C→T polymorphism of the vitamin D receptor gene was associated with bone mineral density in men, with the CT genotype contributing to reduced bone mineral density. These results suggest that IL-6 and osteocalcin genes are susceptibility loci for reduced bone mineral density in postmenopausal women and that the vitamin D receptor gene constitutes such a locus in men. The combined IL-6 and osteocalcin genotypes may be helpful in assessing osteoporosis in women.

Yamada Y, Ando F, Niino N, et al. Association of polymorphisms of interleukin-6, osteocalcin, and vitamin D receptor genes, alone or in combination, with bone mineral density in community-dwelling Japanese women and men. J Clin Endocrinol Metab. 2003;88:3372-3378.

Reprints: Dr. Yoshiji Yamada, Dept. of Gene Therapy, Gifu International Institute of Biotechnology, Yagi Memorial Park, Mitake, Gifu 505-0116, Japan; yoyamada@giib.or.jp