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  Clinical Abstracts

 

 

 

 

June 2009

Editor:
Michael Bissell, MD, PhD, MPH

  • Evaluation of critical values for serum and whole blood sodium
  • Frequency and relevance of troponin T elevation in acute ischemic stroke
  • Fecal occult blood testing in chronic kidney disease
  • Antimyelin antibodies in multiple sclerosis
  • Plasma IGF-1 and cognitive function in elderly males
  • Mechanism of action of antithrombic ADAMTS13 metalloprotease
  • Diagnosis of variant Creutzfeldt-Jakob disease associated with transfusion
  • Hepatitis C virus and renal transplantation
  • Use of ELISA to monitor anti-thymoglobulin antibodies development
  • Hepatitis B virus genotyping and resistance testing

Evaluation of critical values for serum and whole blood sodium Evaluation of critical values for serum and whole blood sodium

As a patient safety measure, the reporting of critical laboratory results, sometimes called panic values, is a widespread practice required by accrediting bodies. Critical results indicate the patient is in imminent danger unless appropriate treatment is started promptly. However, data are not readily available to determine the effectiveness of including a given analyte on a critical value list or at what concentration a result should be considered critical. Changes in the critical limits used can lead to an increase in the number of required phone calls without concomi-tant improvement in patient care or, in contrast, can cause failure in notifying clinicians of potentially harmful situations. Most clinical laboratories include total serum sodium on their critical value lists, albeit at different concentrations. The low range for critical value limits for serum sodium has been reported as 110 to 130 mEq/L (110–130 mmol/L) and the range for high critical limits as 150 to 170 mEq/L (150–170 mmol/L). Critical values used for serum sodium recently were found to vary significantly among hospitals in the United States. Of laboratories reporting critical serum sodium results, 50 percent used values of 120 mEq/L (120 mmol/L) or less as the lower limit and 160 mEq/L (160 mmol/L) or more as the higher limit. The authors studied clinician responses and patient outcomes for serum and whole blood sodium results using 120 mEq/L (120 mmol/L) or less for the lower and 155 mEq/L (155 mmol/L) or more for the upper critical value limits. They analyzed all critical serum and whole blood sodium results called to clinicians during a six-month period. Patients’ electronic medical records were reviewed for clinical responses and patient outcomes. Of the 111,545 sodium results used in the study, 615 (0.6 percent) were critical. Employing the criteria of 120 mEq/L (120 mmol/L) or less and 155 mEq/L (155 mmol/L) or more, the authors found 166 critically low results and 447 critically high results. In hypernatremic and hyponatremic patients, the lengths of stay were increased above average, and clinicians responded to more than 50 percent of results within four hours. The mortality rates of hyponatremic and hypernatremic inpatients were 19 percent and 48 percent, respectively. The authors concluded that disease severity, as measured by length of stay and mortality, indicated these critical limits should not be broadened.

Howanitz JH, Howanitz PJ. Evaluation of serum and whole blood sodium critical values. Am J Clin Pathol. 2007;127:56–59.

Correspondence: Dr. Joan H. Howanitz, Dept. of Pathology, SUNY Health Science Center at Brooklyn, 450 Clarkson Ave., Box 25, Brooklyn, NY 11203

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Frequency and relevance of troponin T elevation in acute ischemic stroke Frequency and relevance of troponin T elevation in acute ischemic stroke

For several years, increased activity of transaminase, creatine kinase, and creatine kinase-MB suggesting myocardial injury has been associated with stroke. Recently, it has been demonstrated that highly sensitive and specific markers for detecting myocardial necrosis, such as cardiac troponins, may be elevated in patients with ischemic stroke. The pathophysiologic mechanisms underlying these observations are not fully understood. In 2000, an international redefinition of myocardial infarction was introduced. The most striking clinical consequence of the redefi-nition was the increased importance of even minor elevations of troponin levels. The authors conducted a prospective study of patients with acute ischemic stroke to evaluate the prevalence of troponin T (TnT) elevation, study the characteristics of patients with elevated levels of TnT, and assess the clinical importance of markers of myocardial ischemia and necrosis early after ischemic stroke. They examined the prevalence and characteristics of troponin elevation in 244 patients with acute ischemic stroke but without overt ischemic heart disease. TnT and creatine kinase-MB (CK-MB) concentrations were measured and 12-lead electrocardiograms obtained on admission and during each of the following five days. Myocardial perfusion scintigraphy was performed in patients with TnT levels of 0.10 µg/L and in comparable control subjects with-out elevated TnT. Patients were followed for a mean of 19±7 months, with all-cause mortality as the clinical end point. Elevated levels of TnT (>0.03 µg/L) and CK-MB (≥10 µg/L) were observed in 10 percent and nine percent of patients, respectively. Patients with elevated TnT had higher frequencies of heart or renal failure, or both. Perfusion abnormalities on myocardial perfusion scintigraphy at rest were not more fre-quent or pronounced in patients with TnT levels of 0.10 µg/L or greater than in the control group. Only seven patients (three percent) had ele-vations of TnT or CK-MB and electrocardiographic changes suggesting acute myocardial infarction. According to univariate and multivariate analyses, elevation of TnT was significantly associated with mortality. The authors concluded that elevated levels of TnT are rare in patients presenting with ischemic stroke but without overt ischemic heart disease. Heart and renal failure rather than myocardial infarction are the most likely causes. When present, elevation of TnT seems to be useful in identifying patients who are at increased risk of dying within the following two years.

Jensen JK, Kristensen SR, Bak S, et al. Frequency and significance of troponin T elevation in acute ischemic stroke. Am J Cardiol. 2007;99:108–112.

Correspondence: Jesper K. Jensen at jesperkjensen@dadlnet.dk

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Fecal occult blood testing in chronic kidney disease Fecal occult blood testing in chronic kidney disease

Colorectal cancer is the second leading cause of cancer-related mortality in the United States. But several large, randomized, controlled clini-cal trials have shown that screening for colorectal cancer significantly decreases mortality. Annual fecal occult blood testing (FOBT) starting at 50 years of age is recommended as one of several accepted methods for colorectal cancer screening in asymptomatic average-risk patients. However, screening for colorectal cancer in patients with chronic kidney disease (CKD) may be problematic. Numerous studies have shown that patients with advanced CKD and those on dialysis therapy have a high prevalence of mucosal inflammation and other trivial lesions in the upper gastrointestinal tract. In the presence of superimposed factors, these lesions may bleed, resulting in a positive FOBT result in the absence of clinically important colonic lesions, which may lead to overutilization of colonoscopy. The use of heparin in hemodialysis patients can also contribute to a false-positive FOBT result. However, the impact of severity of CKD on performance characteristics of FOBT is unknown. Because CKD may be associated with gastrointestinal bleeding from trivial mucosal lesions, the authors hypothesized that the positive predictive value of FOBT for clinically important lesions of the colon would decrease as the stage of CKD worsened. The authors prospectively identified 1,225 consecutive asymptomatic average-risk patients who were referred for colonoscopy to evaluate a positive FOBT result. Using the Modification of Diet in Renal Disease equation, the authors estimated glomerular filtration rate (GFR) and staged the severity of CKD using standard criteria: normal/stage 1 (GFR, ≥90 mL/min/1.73 m2 [≥1.50 mL/s]), stage 2/3 (GFR, 30 to 89 mL/min/1.73 m2 [0.50 to 1.48 mL/s]), and stage 4/5 (GFR, <30 mL/min/1.73 m2 [<0.50 mL/s] or dialysis). The authors identified clinically important lesions in 23.9 percent of 531 patients with none/stage 1 CKD, 32.8 percent of 497 with stage 2/3 CKD, and 42.6 percent of 197 with stage 4/5 CKD (P<.001). Compared with patients with none/stage 1 CKD, the adjusted odds for identifying a clinically important lesion were 1.61 (95 percent confidence interval [CI], 1.21–2.15) for subjects with stage 2/3 CKD and 2.33 (95 percent CI, 1.62–3.36) for patients with stage 4/5 CKD. The prevalence of adenomas of 1 cm or greater (15.1 percent versus 20.1 percent versus 22.8 percent; P=.007), carcinomas (5.1 percent versus 10.1 percent versus 13.2 percent; P<.001), and vas-cular ectasias (1.7 percent versus 2.4 percent versus 6.1 percent; P=.003) rose with an increase in the severity of CKD. The authors concluded that, contrary to their initial hypothesis, the predictive value of a positive FOBT result for clinically important colonic lesions increased as the severity of CKD worsened.

Bini EJ, Kinkhabwala A, Goldfarb DS. Predictive value of a positive fecal occult blood test increases as the severity of CKD worsens. Am J Kidney Dis. 2006;48:580–586

Correspondence: Dr. Edmund J. Bini at edmund.bini@med.va.gov

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Antimyelin antibodies in multiple sclerosis Antimyelin antibodies in multiple sclerosis

After a first episode of central nervous system dysfunction suggestive of multiple sclerosis, known as a clinically isolated syndrome, it is difficult to predict individual risk of multiple sclerosis. Significantly increased risk of clinically definite multiple sclerosis has been reported among patients with a clinically isolated syndrome and serum antibodies against recombinant myelin oligodendrocyte glycoprotein (MOG) and purified myelin basic protein (MBP) detected by Western blot analysis. MOG is a minor component of myelin (0.01 to 0.05 percent of central myelin protein) that is found exclusively in the central nervous system. The N-terminal domain of MOG is expressed on the myelin surface and is easily accessible to antibodies. Several lines of evidence suggest that antibody-mediated demyelination plays a role in multiple sclerosis. The Betaferon in Newly Emerging Multiple Sclerosis for Initial Treatment (BENEFIT) trial was conducted to explore the effect of interferon β-1b (Betaseron, Schering), administered subcutaneously every other day, on the rate of progression to clinically definite multiple sclerosis in patients with a clinically isolated syndrome. The authors conducted their own trial in which they measured anti-MOG and anti-MBP antibodies in patients recruited for the BENEFIT trial and compared the results with the primary outcomes. They measured serum anti-MOG and anti-MBP IgG and IgM antibodies in 462 patients with a first clinical event suggestive of multiple sclerosis and with at least two clinically silent lesions on brain magnetic resonance imaging. Antibodies were assessed by Western blot analysis at baseline and the results compared with time and rate of progression to clinically definite multiple sclerosis or a diagnosis of multiple sclerosis as defined by an international panel (McDonald criteria). Regular visits were scheduled for assessing neurologic impairment and for MRI before treatment and at months three, six, nine, 12, 18, and 24. The authors found no associations between the presence of anti-MOG and anti-MBP IgM and IgG antibodies and progression to clinically definite multiple sclerosis or a diagnosis of multiple sclerosis according to the McDonald criteria in the entire cohort or in any subgroups of the study population. The authors concluded that serum antibodies against MOG and MBP, as detected by Western blot analysis, are not associated with an increased risk of progression to clinically definite multiple sclerosis in patients who have had a clinically isolated syndrome suggestive of multiple sclerosis.

Kuhle J, Pohl C, Mehling M, et al. Lack of association between antimyelin antibodies and progression to multiple sclerosis. N Engl J Med. 2007;356:371–378.

Correspondence: Dr. Ludwig Kappos at lkappos@uhbs.ch

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Plasma IGF-1 and cognitive function in elderly males Plasma IGF-1 and cognitive function in elderly males

As the proportion of older people continues to rise, reducing the burden of such age-related conditions as cognitive decline and Alzheimer’s disease becomes increasingly important. Prevention of cognitive decline requires understanding the mechanisms whereby changes in cognitive function may occur, especially those that are important at the earliest stages of this process and may be most amenable to intervention. The medical literature has addressed the role of growth factors in relation to a variety of neurological diseases, including Alzheimer’s disease. Insulinlike growth factor-1 (IGF-1), in particular, has been a focus of biological and epidemiological study regarding its potential protective role in neurodegenerative diseases. Epidemiological data, largely from small-scale studies, suggest that higher total IGF-1 levels may be associated with better cognitive performance and lower risk of cognitive decline in the elderly. Nevertheless, bioavailable IGF-1 seems most likely to influence cognition.(IGF-1 binds to brain IGF receptors in its free, or unbound, form.) Therefore, free IGF-1 levels, or perhaps the molar ratio of total IGF-1 to its principal binding protein (IGF binding protein [IGFBP]-3), likely are important in determining the full impact of IGF-1. To explore these issues further, the authors examined the relationship between IGF-1 levels, using measures of free IGF-1 as well as IGF-1 to IGFBP-3 molar ratio, and cognition in a cohort of community-dwelling male participants in the Physicians’ Health Study (PHS) II. Participants (mean age, 57 years) provided blood samples from 1982 to 1984. Using stored samples, the authors measured free IGF-1 in 376 men and total IGF-1 and IGFBP-3 in 460 men. Starting in 2001, they administered telephone-based tests (Telephone Interview of Cognitive Status [TICS]) of general cognition, verbal memory, and category fluency. They also estimated multivariable adjusted mean differences in cognitive performance across levels of free IGF-1 and IGF-1 to IGFBP-3 molar ratio. The authors measured the global score (averaging performance across all individual cognitive tests), TICS, and verbal memory score. They found that each standard deviation (SD) increment in free IGF-1 was associated with a multi-variable-adjusted increase of 0.08 units (P=.02) on the global score. This mean difference was equivalent to that observed between men two years apart in age—that is, each SD increase in free IGF-1 appeared cognitively equivalent to staying two years younger. No significant mean differences in TICS scores were observed across free IGF-1 levels. For verbal memory, each SD increment in free IGF-1 was associated with an adjusted mean difference of 0.08 units (P=.03). Results appeared consistent for the molar ratio but were not statistically significant. The authors concluded that higher midlife free IGF-1 may be associated with better late-life cognition.

Okereke OI, Kang JH, Ma J, et al. Midlife plasma insulin-like growth factor I and cognitive function in older men. J Clin Endocrinol Metab. 2006;91:4306–4312.

Correspondence: Dr. Olivia Okereke at ookereke@partners.org

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xMechanism of action of antithrombic ADAMTS13 metalloprotease Mechanism of action of antithrombic ADAMTS13 metalloprotease

After vascular injury, platelets adhere to von Willebrand factor, a multimeric blood protein that can exceed 20,000 kDa in mass. Von Wille-brand factor (VWF) mediates platelet adhesion to exposed connective tissue, endothelial cells, and other adherent platelets. To prevent throm-bosis, platelet adhesion is regulated by a unique feedback mechanism involving tension-induced proteolysis. Flowing blood exerts a force on the growing platelet-rich thrombus that stretches VWF and exposes a cleavage site for plasma ADAMTS13, a member of a disintegrin and met-alloprotease with thrombospondin type I repeat family. The metalloprotease domain (M domain) of ADAMTS13 cleaves the Tyr1605-Met1606 bond in the A2 domain of VWF (Pro1480- Gly1672, numbered from the initiation codon), severing the VWF multimer and releasing adherent platelets. ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura, a life-threatening disease characterized by disseminated microvascular thrombosis. Conversely, mutations in the VWF A2 domain that lead to excessive proteolysis cause the inherited bleeding disorder von Wille-brand disease type 2A. Achieving balanced regulation of platelet adhesion is a formidable challenge under the conditions prevailing in blood. VWF and ADAMTS13 occur at low concentrations of approximately 10 μg/mL and approximately 1 μg/mL, respectively, compared with the total plasma protein concentration of approximately 80,000 μg/mL. Nevertheless, VWF is resistant to ADAMTS13 until it is subjected to high fluid sheer stress, adsorbed onto a surface, or treated with such chaotropic agents as urea or guanidine hydrochloride. Several mechanisms contribute to the remarkable specificity of ADAMTS13. The activation of cleavage by tensile stress indicates that the sessile bond is inaccessible in native VWF. The modeled structure of the VWF A2 domain supports this conclusion, suggesting that the cleavage site is buried in a central β-sheet and exposed on unfolding. Furthermore, von Willebrand disease mutations in the A2 domain that cause increased proteolysis are pre-dicted to destabilize the folded structure. Binding of platelet glycoprotein Ibα (GPIbα) or heparin to the VWF A1 domain allosterically promotes cleavage of the adjacent A2 domain by ADAMTS13. Finally, VWF cleavage is markedly impaired by deletion of the ADAMTS13 spacer domain or by deletion of residues El660APDLVLQR1668 from the VWF A2 domain, suggesting that contacts between “exosites” on the enzyme and sub-strate, located some distance from the active site and scissile bond, facilitate substrate recognition. To date, these exosite interactions have not been characterized. The authors demonstrated by mutagenesis and kinetic analysis that the spacer domain of ADAMTS13 binds to an exosite at the C terminus of the VWF A2 domain that is separated by approximately 50 aa residues from the cleaved Tyr1605-Met1606 bond. This exosite appears to be cryptic in native VWF, and its interaction with the ADAMTS13 spacer domain is essential for feedback inhibition of VWF-dependent platelet adhesion. This mechanism appears to be unique in its use of tension-induced conformational changes to expose cryptic exosites and cleavage sites, activating the proteolysis of VWF within platelet-rich thrombi in flowing blood and preventing microvascular thrombosis.

Gao W, Anderson PJ, Majerus EM, et al. Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease. Proc Natl Acad Sci USA. 2006;103:19099–19104.

Correspondence: J. Evan Sadler at esadler@im.wustl.edu

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Diagnosis of variant Creutzfeldt-Jakob disease associated with transfusion Diagnosis of variant Creutzfeldt-Jakob disease associated with transfusion

Since the arrival of variant Creutzfeldt-Jakob disease (vCJD) and the experimental confirmation that it is caused by the same prion strain as that causing epidemic bovine spongiform encephalopathy, there have been concerns that vCJD might be transmissible by iatrogenic routes, such as via blood and blood products. Such concerns were heightened by the recog-nition that the pathogenesis of vCJD differed substantially from that of sporadic or classical CJD by showing prominent and uniform prion accumulation in lymphoreticular tissues akin to ovine scrapie, in which prionemia has since been demonstrated experimentally. All clinically affected patients with vCJD have been homozygous for methion-ine at polymorphic codon 129 of the prion protein (PrP) gene, PRNP, where methionine or valine may be encoded. A 1997 study was set up be-tween the United Kingdom’s National CJD Surveillance Unit and U.K. Blood Services to identify recipients of blood components from donors who subsequently developed vCJD—that is, vCJD-implicated components—and to seek evidence for possible transmission of vCJD by blood transfusion. Two transfusion-associated cases of vCJD prion infection were reported in 2004 in individuals who had been identified in this way. The cohort of identified recipients of blood transfusion from donors who subsequently developed vCJD totaled 66. Forty-two of these people died. The authors reported on the development of vCJD in an individual from this cohort and discussed the clinical, neuroradiological, and pathological findings and their implications. The patient was admitted for investigation, and details of blood transfusion history were obtained from the United Kingdom’s National Blood Service and Health Protection Agency. After diagnosis of vCJD, the patient was enrolled into the MRC PRION-l trial. When the patient died, brain and tonsil tissue were obtained at autopsy and assessed for disease-related PrP by im-munoblotting and immunohistochemistry. A clinical diagnosis of probable vCJD was made; tonsil biopsy was not performed. The patient received experimental therapy with quinacrine but deteriorated and died after a clinical course typical of vCJD. Autopsy confirmed the diagnosis and showed prion infection of the tonsils. This case of transfusion-associated vCJD infection, identified ante-mortem, is the third instance from a group of 23 known recipients who survived at least five years after receiving transfusions from donors who subsequently developed vCJD. The risk to the remaining recipients of such tranfusions is probably high, and these patients should be offered specialist followup and investigation. Tonsil biopsy will allow early and pre-symptomatic diagnosis in other iatrogenically exposed individuals at high risk, such as those with primary infection with bovine spongiform encephalopathy prions.

Wroe SJ, Pal S, Siddique D, et al. Clinical pre-sentation and pre-mortem diagnosis of variant Creutzfeldt-Jakob disease associated with blood transfusion: a case report. Lancet. 2006;368:2061–2067.

Correspondence: John Collinge at j.collinge@prion.ucl.ac.uk

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Hepatitis C virus and renal transplantation Hepatitis C virus and renal transplantation

Hepatitis C virus infection is relatively common among dialysis patients and is higher in that group than in the general population. The prevalence of hepatitis C virus (HCV) varies based on treatment center, country, geographic origin of the recipient, race, history of intravenous drug abuse, modality and duration of dialysis therapy, retransplantation, presence of anti-hepatitis B core antigen, and number of blood transfusions. In Mediterranean countries, the prevalence of HCV among dialysis patients is usually higher than 20 percent. With improvements in the results of organ transplantation, numerous studies have shown that liver dysfunction is a major cause of morbidity and mortality in immunosuppressed renal recipients. Liver disease is frequently associated with viral infections, usually hepatitis C virus. Liver failure is the cause of death in eight percent to 28 percent of long-term graft survivors. The effects of immunosuppressive therapy on the natural history of HCV infection are unknown, and impact on patient and allograft survival rates remains controversial. Some studies have detected significant differences in patient survival rates between recipients with and without anti-HCV antibodies. The authors analyzed the prevalence of HCV among renal transplant recipients at their center and its impact on graft and patient survival. They performed a retrospective study that included all renal transplant recipients who underwent the procedure between July 1983 and December 2004. They compared HCV-positive (n=155) versus HCV-negative (n=1,044) recipients for the prevalence of anti-HCV, patient/donor characteristics, and graft/patient survival. The prevalence of HCV-pos-itive patients was 12 percent. The anti-HCV positive recipients displayed a longer time on dialysis (P<.001), greater number of blood transfusions prior to transplant (P<.001), and higher number of previous transplants (P<.001). No differences were noted in the incidence of acute rejection between the two groups. Patient (P=.006) and graft survival (P=.012) were significantly lower in the HCV-positive than the HCV-negative group. Graft survival censored for patient death with a functioning kidney did not differ significantly between HCV-positive and HCV-negative recipients (P=.083). Death from infectious causes was significantly higher among the HCV-positive group (P=.014). The authors concluded that HCV infection had a significant detrimental impact on patient and renal allograft prognosis. Death from infectious causes was significantly more frequent for the HCV-positive group than the non-HCV population.

Pedroso S, Martins L, Fonseca I, et al. Impact of hepatitis C virus on renal transplantation: association with poor survival. Transplant Proc. 2006;38:1890–1894.

Correspondence: Sofia de Lima Pedroso at sofiapedroso@sapo.pt

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Use of ELISA to monitor anti-thymoglobulin antibodies development Use of ELISA to monitor anti-thymoglobulin antibodies development

Monoclonal antibodies against human T cells, such as murine anti-human CD3 or such polyclonal antibodies to human thymocytes as horse or rabbit anti-human thymocytglobulin, are effective immunosuppressive agents for prophylaxis and treatment of allograft rejection. The anti-body-mediated immune response that develops against these infused foreign proteins may limit their prolonged or repeated use. Human anti-mouse antibodies are rarely pre-existent but do occur. During or after treatment, antibodies occur at an incidence rate of between 17 percent and 63 percent and may decrease the efficacy of therapy with Orthoclone OKT3, the murine anti-human CD3 from Ortho Biotech. Anti-ATGAM an-tibodies (anti-horse; Pharmacia) develop in about 40 percent of subjects and have been associated with early graft failure and decreased levels of circulating equine immunoglobulin. In contrast, early studies showed infrequent immune response to rabbit anti-lymphocyte serum, with only four of 66 (six percent) patients developing anti-rabbit immunoglobulin antibodies. Serum sickness was seen in those few patients who did develop antibodies, although another study showed a rate of sensitization substantially higher (83 to 87 percent). These anti-rabbit antibodies in-terfere with a variety of immunoassays. Solid-phase ELISAs and flow cytometric assays have been used to detect human antibodies to OKT3 and ATGAM. While ELISA technology can monitor the development of human anti-rabbit antibodies, no standardized assay is commercially available. The authors developed an ELISA to study posttreatment development of anti-thymoglobulin (rATG). In an institutional review board-approved trial, they tested 101 allograft recipients for anti-rATG antibodies. Patients received rATG intravenously at 1.25 to 2.0 mg/kg/day for two to 14 days. Serum samples were obtained pretreatment and at weeks one, two, four, and six, and months three and six post-rATG. ELISA plates were coated with rATG (10 µg/mL). Samples were diluted 1:100 and tested in quadruplicate. Positive samples were titrated. Horseradish peroxidase-conjugated (HRPO) affinity-purified goat anti-human immunoglobulin G antibody reacted with bound human antibody. A chromagenic substrate for HRPO was added and optical density (OD, 490 nm) read. Optical density of twice the negative control was considered positive. Mean optical densities of negative and positive controls were 0.113±0.030 and 1.042±0.196, respectively. Ten patients had detectable anti-rATG before rATG administration (1:100). Thirty-five of 101 (35 percent) patients developed anti-rATG antibody. Patients showed an initial positive anti-rATG antibody from days eight to 59 after infusion and titers from 1:100 to 1:4000. The authors concluded that despite the postulated anti-B-cell activity of rATG, this study confirms that rATG induces sensitization at a frequency and titer seen with other xenogeneic antilymphocyte antibodies. Formation of such anti-xenoantibodies can have a negative effect on treatment response and therefore warrants monitoring.

Book BK, Pescovitz MD, Agarwal A, et al. In vitro monitoring of in vivo development of human anti-thymoglobulin antibodies by ELISA. Transplant Proc. 2006;38:2869–2871.

Correspondence: Benita K. Book, Indiana University School of Medicine, Dept. of Surgery, 545 Barnhill Drive, Emerson Hall 203, Indianapolis, IN 46202

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Hepatitis B virus genotyping and resistance testing Hepatitis B virus genotyping and resistance testing

Although an effective vaccine exists for hepatitis B virus, the number of new cases around the world has not dramatically decreased. Yet virologic monitoring of hepatitis B virus (HBV) infection and its treatment is critical for controlling the disease and improving patient management. Genotypic assays focus on the major mutations in the region of the Pol gene coding for the virus reverse transcriptase that confer resistance to antiviral treatment. The authors have designed a prototype genotypic assay based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and two insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90 percent) could be amplified and analyzed by the chip. Only two samples with more than 103 genome copies/mL could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Both techniques were used to analyze 12,161 codons. Only 89.4 percent could be determined by sequencing, and among the remaining 11,335 codons, 92.8 percent were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for analyzing the HBV genome is ongoing. This evaluation showed that DNA chip technology can provide important information for clinically managing chronic hepatitis B.

Tran N, Berne R, Chann R, et al. European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes. J Clin Microbiol. 2006;44:2792–2800.

Correspondence: G. Vernet at guy.vernet@eu.biomerieux.com

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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.