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April 2006

Editors:
Michael Bissell, MD, PhD, MPH

Fatal avian influenza in a child
Hemoglobin F as a potential tumor marker
Spurious automated platelet counts
Effects of lowering plasma homocysteine in stroke patients
A novel PCR test for identifying oculopathogenic adenoviruses
Population screening for hemochromatosis and iron overload
Improving on sweat ­chlorides for cystic ­fibrosis screening
A new oligoclonal band test for diagnosing MS
Agreement among INR methods

bullet Fatal avian influenza in a child

Forty-five cases of influenza A (H5N1) were reported in humans during 2004, of which 33 were fatal. All of the subjects presented primarily with severe respiratory illnesses. The authors reported on an additional fatal case of influenza H5N1, in southern Vietnam, involving a four-year-old boy who presented with severe diarrhea but no apparent respiratory illness. This was followed by rapidly progressive coma, leading to a clinical diagnosis of acute encephalitis. The cerebrospinal fluid showed one white cell per cubic millimeter, normal glucose levels, and increased levels of protein (0.81 g/L). The diagnosis of influenza H5N1 was established by isolating the virus from cerebrospinal fluid and fecal, throat, and serum specimens. Two weeks earlier, the boy’s nine-year-old sister had died of a similar illness. Neither patient had respiratory symptoms at presentation. These cases suggest that the clinical spectrum of influenza H5N1 is wider than previously thought and thus has important implications for clinical and public health responses to avian influenza.

De Jong MD, Van Cam B, Qui PT, et al. Fatal avian influenza A (H5N1) in a child presenting with diarrhea followed by coma. N Engl J Med. 2005;352:686–691.

Reprint information not available.

bullet Hemoglobin F as a potential tumor marker

Fetal hemoglobin is the main hemoglobin of the late fetus and the newborn, constituting about 80 percent of total hemoglobin. In normal adults, fetal hemoglobin (HbF) comprises less than one percent of total hemoglobin. However, increased concentrations of HbF are common in some hemoglobinopathies and many neoplasms. Some early studies claimed that fetal hemoglobin is produced by the tumor cells of germ cell tumor rather than by red blood cells residing in the tumor. The authors were able to distinguish between whole blood HbF and plasma HbF as two independent indicators of cancer. The origin of whole blood HbF is the circulating red blood cell. As such, whole blood HbF is not an oncofetal protein produced by tumor cells but an inducible protein in many neoplasms. In contrast, the origin of plasma HbF was not clear, especially with regard to nonhematological solid tumors. Similarly, raised plasma HbF was very common in persistent trophoblastic diseases. In their preliminary immunohistochemical study, the authors found faint staining of tumor cells using hemoglobin A-absorbed anti-HbF antibody. However, it was still unclear whether plasma HbF is a classic oncofetal protein, produced by tumor cells, or a component of hemopoietic cells or RBC, concentrated inside the tissue of certain tumors. To clear up this confusion and re-evaluate their preliminary observations, the authors prepared a new batch of immunoaffinity-purified anti-HbF antibody and used it to study HbF immunohistochemically in germ cell tumor, trophoblastic disease, lymphoma, myelodysplastic syndrome, multiple myeloma, and ovarian adenocarcinoma. In germ cell tumor, a distinction was made between tumors substantially without HbF-positive red blood cells (F-RBC) and those with F-RBC. Those without F-RBC were nonmetastatic mature teratomas and dermoid cysts. Those containing F-RBC were nonmetastatic mature teratomas and dermoid cysts. Those containing F-RBC were mainly embryonal carcinomas and metastatic teratomas. HbF-positive myeloid cells (F-MLC), HbF-positive normoblasts (F-NBS), and F-RBC were common in the bone marrow and lymphoid tissues of lymphoma, myelodysplastic syndrome, and multiple myeloma. In trophoblastic disease, normal and nucleated F-RBC were seen in the trophoblastic villi in one case with incomplete molar pregnancy but not in other cases of incomplete and complete molar pregnancy. However, F-RBC and F-MLC were seen in the decidua of both types of trophoblastic disease. F-cells generally were observed within blood vessels or concentrated in certain areas of the neoplastic tissue. The authors concluded that the dual distribution of these cells circulating in the blood or concentrated in areas of the neoplastic tissues might reflect the two independent serological indicators of HbF—one in whole blood and the other in plasma of patients with cancer.

Wolk M, Martin JE, Constantin R. Blood cells with fetal haemoglobin (F-cells) detected by immunohistochemistry as indicators of solid tumours. J Clin Pathol. 2004;57:740–745.

Reprints: Dr. M. Wolk, Central Laboratories, Israel Ministry of Health, Ya’akov Eliav St. 9, P.O. Box 34410, Jerusalem 91342, Israel; moshe.wolk@eliav.health.gov.il

bullet Spurious automated platelet counts

A 39-year-old woman was admitted to the coronary care unit of an Indian hospital with sudden onset of breathlessness. On examination, she was tachypnoeic and restless. Respiratory examination revealed bilateral crepitations. Prosthetic valve sounds could be well heard. The patient had tachycardia, but her heart rate was regular. She was diagnosed with acute pulmonary edema, and supportive treatment was initiated. The patient was being followed up after mitral valve replacement, the surgery having been performed 11 years earlier for chronic rheumatic valvular heart disease. Her presentation with pulmonary edema was attributed to her failure to increase the dosage of the prescribed diuretic (Furosemide) from 40 mg to 80 mg each day. As part of the initial investigation, a complete blood count was requested. The laboratory’s Advia-60 three-part differential automated analyzer revealed a hemoglobin of 128 X 109 g/L, total leukocyte count of 8.5 X 109/L with a normal differential leukocyte count, mean corpuscular volume of 78 fL, mean corpuscular hemoglobin of 26.1 pg, and mean corpuscular hemoglobin concentration of 33.6 percent. The platelet count was 1152 X 109/L. However, a review of the peripheral blood smear revealed a platelet count of approximately 130 X 109/L to 150 X 109/L, a discrepancy of nearly 1,000 X 109/L compared with the automated value. Numerous rod-shaped bacteria were present in clumps and were seen lying singly. A few intraneutrophilic bacteria were also present. The white blood cells showed pronounced artefactual changes, with nuclear distortion and cytoplasmic vacuolation. The bacteria seen in the peripheral smear were thought to be responsible for the spuriously raised platelet count. Medical personnel sought additional clinical details on the patient in view of the history of prosthetic valve surgery and thereby a high risk of bacteremia. The patient had no symptoms or signs of infective endocarditis, and valve function was satisfactory clinically and on echocardiography. No other obvious focus of infection could be identified. Medical personnel subsequently discovered that the sample was drawn early in the morning in the emergency room. It was then delayed eight hours before it reached the laboratory because a requisition form was not completed properly. The blood sample was kept at room temperature until this time. A repeat sample was taken, which revealed an automated platelet count of 158 X 109/L. The peripheral blood smear made from the repeat sample revealed no bacteria. It was concluded that bacteria seen in the blood smear of the patient’s initial sample were the result of bacterial overgrowth in the blood sample caused by a delay in analysis rather than bacteremia in the patient. Overgrowth was probably aided by the ambient temperature of approximately 35°C at the time of sampling. The patient was discharged in satisfactory condition after five days in the hospital. She was asymptomatic at the first outpatient followup.

Kakkar N. Spurious rise in the automated platelet count because of bacteria. J Clin Pathol. 2004;57:1096–1097.

Reprints: N. Kakkar, Dept. of Pathology, Christian Medical College and Hospital, Ludhiana – 141 008, Punjab, India; n_kakkar@satyam.net.in

bullet Effects of lowering plasma homocysteine in stroke patients

The Vitamin Intervention for Stroke Prevention trial was designed to determine whether best medical and surgical management, risk factor modification, and a multivitamin containing high-dose folic acid, pyridoxine, and cobalamin, given to lower total homocysteine levels, would reduce the incidence of recurrent cerebral infarction (primary outcome) as well as coronary heart disease (CHD) and death (secondary outcomes) in patients with a nondisabling cerebral infarction and fasting total homocysteine levels greater than the 25th percentile for stroke patients. The authors conducted a study to determine whether high doses of folic acid, pyridoxine (vitamin B6), and cobalamin (vitamin B12), given to lower total homocysteine levels, reduce the risk of recurrent stroke over a two-year period compared with low doses of these vitamins. They conducted a double-blind, randomized, controlled trial (September 1996 to May 2003) involving 3,680 adults with nondisabling cerebral infarction at 56 university-affiliated hospitals, community hospitals, private neurology practices, and Veterans Affairs medical centers across the United States, Canada, and Scotland. All participants received best medical and surgical care plus a daily multivitamin containing the Food and Drug Administration’s reference daily intakes of other vitamins. Patients were randomly assigned to receive once-daily doses of the high-dose formulation (n=1,827), which contained 25 mg of pyridoxine, 0.4 mg of cobalamin, and 2.5 mg of folic acid, or the low-dose formulation (n=1,853), which contained 200 µg of pyridoxine, 6 µg of cobalamin, and 20 µg of folic acid. Mean reduction of total homocysteine was 2 µmol/L greater in the high-dose group than in the low-dose group, but there was no treatment effect on any end point. The unadjusted risk ratio for stroke, CHD event, or death was 1.0 (95 percent confidence interval [CI], 0.8 to 1.1), with chances of an event within two years of 18 percent in the high-dose group and 18.6 percent in the low-dose group. The risk of ischemic stroke within two years was 9.2 percent for the high-dose and 8.8 percent for the low-dose groups (risk ratio, 1.0; 95 percent CI, 0.8 to 1.3; P=0.8 by log-rank test of the primary hypothesis of difference in ischemic stroke between treatment groups). There was a persistent and graded association between baseline total homocysteine level and outcomes. A 3 µmol/L lower total homocysteine level was associated with a 10 percent lower risk of stroke (P=.05), 26 percent lower risk of CHD events (P<.001), and 16 percent lower risk of death (P=.001) in the low-dose group and a nonsignificantly lower risk in the high-dose group by two percent for stroke, seven percent for CHD events, and seven percent for death. The authors concluded that consistent findings of an association between total homocysteine and vascular risk suggest the need for further exploration and longer trials in different populations with elevated total homocysteine levels.

Toole JF, Malinow MR, Chambless LE, et al. Lowering homocysteine in patients with ischemic stroke to prevent recurrent stroke, myocardial infarction, and death. The Vitamin Intervention for Stroke Prevention (VISP) randomized controlled trial. JAMA. 2004;291:565–575.

Reprints: Elizabeth G. Sides, Stroke Research Center, Dept. of Neurology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157; esides@wfubmc.edu

bullet A novel PCR test for identifying oculopathogenic adenoviruses

Human adenovirus comprises 51 serotypes that are divided into six species (formerly subgenera A to F) and is associated with a variety of diseases that primarily affect the respiratory, ocular, and gastrointestinal systems. The eye infections caused by human adenoviruses (Ads) include epidemic keratoconjunctivitis (EKC), pharyngoconjunctival fever, and nonspecific follicular conjunctivitis. Six serotypes—Ad3, Ad4, Ad7, Ad8, Ad19, and Ad37—account for as many as 89 percent of cases of adenoviral conjunctivitis. No antiviral agent effective against strains responsible for EKC is available. Therefore, rapid and accurate methods for confirming the clinical diagnosis and identifying the strains that cause EKC are needed so that measures against the spread of the disease can be taken early in the course of infection. Culture of Ads in various cell lines and observation of a cytopathic effect are routinely used to detect the virus, and the neutralization test is routinely performed for identifying the serotype. In some cases, the hemagglutination inhibition assay is performed in addition to the neutralization test. Although the aforementioned methods are sensitive, a lack of rapidity makes them impractical in clinical situations. With the advent of PCR, a variety of Ad serotype identification methods have been developed, such as type-specific PCR, a combination of PCR and restriction enzyme analysis, and typing by sequencing of the PCR product. None of these methods is simple and rapid. In all cases, nested PCR is necessary to increase the sensitivity of the type-specific PCR and to obtain a sufficient amount of DNA, required for the cycle sequencing reaction in DNA sequencing analysis and for restriction endonuclease analysis by PCR-restriction fragment length polymorphism (RFLP) analysis. The hexon and fiber proteins are the major capsid proteins of Ad. Hypervariable regions of the hexon protein carry neutralization epitopes that react with neutralizing antibody in the neutralization test, and the fiber protein is responsible for hemagglutination. Together, they determine the serotype specificity of Ad. Therefore, the genes encoding the hexon and fiber proteins are genomic sites suitable for analysis for the genetic discrimination of Ad serotypes. The authors used a combination of primers that specifically amplify the fiber and hexon genes to discriminate common EKC-causing strains of Ads by multiplex PCR. The accuracy and reproducibility of the test were confirmed with field isolates and directly with clinical samples that were previously tested by culture-NT or PCR-RFLP analysis. Ad serotypes could be distinguished by the different amplicon sizes. The assay correctly identified prototype strains as well as isolates in clinical specimens. The authors concluded that their multiplex PCR assay has the potential to serve as a rapid and cost-effective tool for typing important ocular Ads.

Banik U, Adhikary AK, Suzuki E, et al. Multiplex PCR assay for rapid identification of oculopathogenic adenoviruses by amplification of the fiber and hexon genes. J Clin Microbiol. 2005;43:1064–1068.

Reprints: Arun Kumar Adhikary, Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; arun_ad hikary@msn.com

bullet Population screening for hemochromatosis and iron overload

Iron overload is associated with a wide range of genetic and environmental factors and can lead to parenchymal damage of organs. Homozygosity for the C282Y mutation of the HFE gene is associated with susceptibility to iron overload and is a common genetic mutation, occurring in 0.3 percent to 0.5 percent of white people of northern European descent. Phlebotomy treatment can prevent some of the major complications of iron overload, and patients have normal life expectancy if they are treated before organ damage occurs. Iron overload can occur in nonwhites and may be related to as-yet-undiscovered genetic mutations, environmental factors, or both. The Hemochromatosis and Iron Overload Screening (HEIRS) study was designed to evaluate the prevalence, genetic and environmental determinants, and potential clinical, personal, and societal effects of iron overload and hemochromatosis in a multicenter, multiethnic sample of 101,168 primary care adults 25 years of age or older. The authors reported findings from the first, or screening, stage of the study. Participants were recruited from primary care practices and blood-drawing laboratories. Blood samples were tested for transferrin saturation, serum ferritin, and C282Y and H63D mutations of the HFE gene. Before genetic screening, participants were asked whether they had a history of medical conditions related to iron overload. Of the 99,711 participants, 299 were homozygous for the C282Y mutation. The estimated prevalence of C282Y homozygotes was higher in nonHispanic whites (0.44 percent) than in Native Americans (0.11 percent), Hispanics (0.027 percent), blacks (0.014 percent), Pacific Islanders (0.012 percent), and Asians (0.000039 percent). Among participants who were homozygous for the C282Y mutation but in whom iron overload had not been diagnosed (227 participants), serum ferritin levels were greater than 300 µg/L in 78 of 89 men (88 percent) and greater than 200 µg/L in 79 of 138 women (57 percent). Pacific Islanders and Asians had the highest geometric mean levels of serum ferritin and mean transferrin saturation despite having the lowest prevalence of C282Y homozygotes. There were 364 participants in whom iron overload had not been diagnosed (29 C282Y homozygotes) who had a serum ferritin level greater than 1,000 µg/L. Among men, C282Y homozygotes and compound heterozygotes were more likely to report a history of liver disease than were participants without HFE mutations. The authors concluded that the C282Y mutation is most common in whites, and most C282Y homozygotes have elevations in serum ferritin levels and transferrin saturation. The C282Y mutation does not account for high mean serum ferritin levels and transferrin saturation values in nonwhites.

Adams PC, Reboussin DM, Barton JC, et al. Hemochromatosis and iron-overload screening in a racially diverse population. N Engl J Med. 2005;352:1769–1778.

Reprints: Dr. Paul C. Adams, Dept. of Medicine, London Health Sciences Centre, 339 Windermere Rd., London, ON N6A 5A5, Canada; padams@uwo.ca

bullet Improving on sweat chlorides for cystic fibrosis screening

Determining sweat chloride concentration is still the gold standard test for making the diagnosis of cystic fibrosis. Since the inception of the sweat test 50 years ago, the pad method, using quantitative pilocarpine iontophoresis (QPI), is believed to be the most accurate test. This method is well established but time consuming and open to many sources of error. While it has been shown to be reliable even for infants being tested by laboratories with trained, experienced technicians, in the authors’ hands, difficulties arose in newborn infants in the first month of life because at least 75 mg of sweat should be obtained on gauze or filter paper in 30 minutes for an accurate result. The Macroduct coil system is widely used and internationally accepted for collecting sweat. This macro-collection system collects sweat within a coil of fine plastic tubing, from which the sweat can be taken and analyzed immediately for ionic composition or conductivity, minimizing evaporation and condensation. However, as with traditional QPI, it can be difficult to collect a sufficient amount of sweat (15 µL in 30 minutes), especially in very young children. In addition, measuring quantitative chloride concentration is time consuming, and the result is often not immediately available. Many studies have been performed to evaluate the use of osmometry and conductivity in sweat, and they suggested that both methods are as effective as QPI at discriminating for diagnosis of cystic fibrosis (CF). Furthermore, these methods are simpler and their results are available more quickly. Nanoduct is a novel, simple, practical sweat analysis system to measure sweat conductivity in situ. It requires only 3 µL of sweat, and results are available within 30 minutes. In this study, the authors wanted to determine if the Nanoduct system could reliably identify patients with CF and differentiate them from healthy subjects. On the same day and on the same patient, the new system was tested in comparison with the Macroduct sweat collection system measuring chloride concentration and osmolality. The authors studied subjects (n=111) who were three weeks to 60 years of age. Three children had no sweat production, and in 14 children, only conductivity could be measured. In the remaining 94 subjects, the new system identified all patients with classic CF (mean conductivity, 115 mmol/L; range, 92–137) and differentiated them from healthy subjects (mean conductivity, 36 mmol/L; range, 17–59) within a mean time of 20 minutes. The authors concluded that measuring sweat conductivity using the new test system reliably differentiated between patients with and without CF. This suggests that the new system could be used as a diagnostic test as well as for screening.

Barben J, Ammann RA, Metlagel A, et al. Conductivity determined by a new sweat analyzer compared with chloride concentrations for the diagnosis of cystic fibrosis. J Pediatr. 2005;146:183–188.

Reprints: Dr. J. Barben, Pediatric Pulmonology, Children’s Hospital, Ch-9006 St. Gallen, Switzerland; juerg.barben@gd-kispi.sg.ch

bullet A new oligoclonal band test for diagnosing MS

Multiple sclerosis is the most frequent demyelinating disease of the central nervous system. The rationale for treating multiple sclerosis (MS) is to reduce the disease activity and delay the progression of disability. Therefore, an early and accurate diagnosis of MS is important. Because no single test provides a definitive diagnosis, different criteria have been used. Different paraclinical tests, such as detection of intrathecal IgG synthesis (present in most patients with MS), evoked potentials, and imaging techniques, are used to support the diagnosis when necessary. Intrathecal IgG synthesis determined by oligoclonal IgG band (OCGB) detection is an important tool for MS diagnosis. The authors developed a novel OCGB assay that consists of isoelectric focusing (IEF) and IgG immunodetection by an alkaline phosphatase-labeled anti-IgG antibody. It yields high sensitivity and a sharp and strong pattern of bands that is easy to interpret. Alkaline phosphatase has proved to be useful in detecting oligoclonal IgM bands that play an important role as prognostic markers in MS. The authors analyzed the presence of OCGBs using the alkaline phosphatase method in 385 patients with different neurologic diseases to assess the validity of this test for diagnosing MS. Intrathecal IgG synthesis was found in 127 patients with MS (96.2 percent), 18 (35.3 percent) with central nervous system infections, and one with motor neuron disease. Two patterns reflected intrathecal IgG synthesis. One pattern, showing OCGBs restricted to cerebrospinal fluid, was predominantly found in MS. The other pattern, with OCGBs in serum and additional bands in cerebrospinal fluid, was mostly found in central nervous system infections. No patients with other inflammatory neurologic diseases showed intrathecal IgG synthesis. These patients frequently displayed a mirror pattern, with identical bands in serum and cerebrospinal fluid. Considering all patients, the sensitivity for the diagnosis of MS was 96.2 percent and the specificity was 92.5 percent. Excluding infections, which usually do not present a differential diagnosis problem with MS, the sensitivity was still 96.2 percent and the specificity increased to 99.5 percent. The authors concluded that the accuracy of the OCGB method reinforces the value of cerebrospinal fluid studies in the early differential diagnosis of MS.

Villar LM, Masjuan J, Sádaba MC, et al. Early differential diagnosis of multiple sclerosis using a new oligoclonal band test. Arch Neurol. 2005;62:574–577.

Reprints: Dr. Luisa M. Villar, Depts. of Immunology & Neurology, Hospital Ramón y Cajal, Carretera de Colmenar Km 9.100, 28034 Madrid, Spain; lvillar.hrc@salud.madrid.org

bullet Agreement among INR methods

It is difficult to harmonize the results of coagulation analyses in different laboratories, even those using the same method, because of a lack of a natural primary standard. This obstacle has been circumvented by the International Normalized Ratio (INR). A prerequisite to such harmonization has been adoption by the World Health Organization (WHO) of an international reference thromboplastin preparation. Each new commercial thromboplastin is calibrated against the primary WHO reference preparation. The results are used to calculate the relative sensitivity of the unknown preparation compared with the WHO standard International Sensitivity Index (ISI). The INR is calculated according to the formula INR=[PT ratio]ISI. Increasing use of the INR format has led to appreciation of its limitations. These include poorer accuracy and precision when insensitive (high ISI value) thromboplastins are used, incorrect assignment of an ISI value by the manufacturer, use of a reagent-instrument combination different from that used by the manufacturer, and acceptance of an incorrect prothrombin time (PT) value for the reference plasma used for calibration and quality control. Preliminary data from the authors’ laboratory point to the possibility that the Quick and Owren methods may produce different results. Moreover, some of the results may be systematically biased and clinically unacceptable. In this study, the authors sought to assess these problems by comparing the INR results for 150 blood samples and determining the INR values obtained with seven commercial INR methods—four Quick and three Owren-based methods. The authors took blood samples from 150 patients for whom oral anticoagulation had been prescribed. Plasmas were separated and PTs determined using seven commercial reagents and four calibrator sets. The differences in results were assessed by plotting, for each possible pair of methods, the differences in INR values for each sample against the mean INR value (Bland-Altman difference plots). Mean results differed significantly (P<0.001) for 17 of 21 possible paired comparisons of methods. Only two pairs of methods produced very similar results when assessed for problems of substantial differences in INR values; a significant, systematic increase in the difference with INR; and a significant, systematic increase in the variation in difference with increasing INR values. The authors concluded that the agreement among several, and perhaps most, commercial INR methods is poor. The failure of current calibration strategies may severely compromise the monitoring of individual patients and the application of oral anticoagulation guidelines and trial results to clinical practice.

Horsti J, Uppa H, Vilpo JA. Poor agreement among prothrombin time International Normalized Ratio methods: comparison of seven commercial reagents. Clin Chem. 2005;51:553–560.

Reprints: Juha Horsti, University Hospital of Tampere, Laboratory Centre, 33521 Tampere, Finland; juha.horsti@pshp.fi


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