College of American Pathologists
Printable Version

  Clinical Abstracts





March 2010

Michael Bissell, MD, PhD, MPH

T-cell responses to live antigenic cells T-cell responses to live antigenic cells

Studies of endogenous T-cell responses to foreign antigens have focused on the rapid and extensive response of antigen-specific CD8+ T cells during acute bacterial and viral infections. Following an encounter with these pathogens, naive CD8+ T cells become activated and rapidly undergo 15 or more divisions to establish an effector cytotoxic T-lymphocyte (CTL) population seven to eight days post-infection. This extensive proliferation is followed by contraction over the next seven to 14 days, during which 95 percent of antigen (Ag)-specific cells undergo apoptosis, leaving a long-lived memory population. Similar to CD8+ T cells, Ag-specific CD4+ T cells under-go a coordinate response of activation, expan-sion, contraction, and establishment of Agspecific memory. The main differences observed were that CD4+ T cells had a roughly 20-fold lower expansion, peaked a day later than CD8+ T cells, had a protracted contraction phase, and had memory cell numbers decline over time. Follow-ing immunization with most pathogens, primary CD8+ T-cell responses are independent of CD4+ T-cell help. Despite CD4+ T-cell help being dispensable for primary CD8+ T-cell responses, it is necessary for the maintenance and functioning of memory T cells. This is in contrast to the priming of most CD8+ T-cell responses to cell-associated Ags where CD4+ T cells are necessary for the induction of optimal primary T-cell responses. The authors conducted a study in which they characterized murine CD4+ and CD8+ T-cell responses to male-specific minor histocom-patibility (HY) Ags following injection of live male cells into females of the same strain. Male cells are rejected 10 to 12 days after transfer, coin-ciding with the expansion and effector function of CD8+ CTLs to two H-2Db-restricted epitopes. Although anti-HY CD4+ T-cell responses can be readily detected on the fifth day post-transfer, CD8+ responses cannot be detected until day 10. The early CD4+ response does not depend on direct presentation of Ag by donor male cells but on presentation of the male cells by recipient Ag-presenting cells. The CD4+ T-cell response is required for priming CD8+ T-cell effector responses and rejecting HY-incompatible cells. Unexpectedly, HY-specific CD4+ T cells are also capable of lysing target cells in vivo. The delay in the CD8+ T-cell response can be largely abrogated by depleting T cells from the male inoculum. Donor male CD8+ T cells in particular suppress host anti-HY CD8+ responses. The authors concluded that these data demonstrate dramatic differences in host T-cell responses to noninflammatory Ags compared with responses to pathogens. They propose that the delayed CD8+ response is due to a balance between cross-presentation of Ag by helper cell-licensed dendritic cells and veto suppression by live male lymphocytes.

Tyznik AJ, Bevan MJ. The surprising kinetics of the T-cell response to live antigenic cells. J Immunol. 2007;179:4988–4995.

Correspondence: Dr. Michael J. Bevan at

[ Top ]

Evaluation of <em>N. gonorrhoeae</em> and <em>C. trachomatis</em> confirmatory testing Evaluation of N. gonorrhoeae and C. trachomatis confirmatory testing

Deficiencies in culture and antigen-detection assay sensitivities and result reproducibility have, in part, characterized past problems with laboratory testing for Chlamydia trachomatis. The sensitivities of culture assays for the fastidious pathogen Neisseria gonorrhoeae can be affected by the specimen collection device used, viability of organisms in certain transport systems, and various selective media used. Such factors contributed to the development of molecular diagnostic assays for these sexually transmitted agents. Along with the rapid generation of results, C. trachomatis-specific and N. gonorrhoeae-specific nucleic acid hybridization assays provide analytical sensitivities equivalent to that of culture. The commercial availability of nucleic acid amplification tests (NAATs) has greatly enhanced the detection of these agents in genital swab specimens submitted to clinical laboratories. The advent of these highly sensitive assays has raised the potential for generating false-positive results. Studies have documented the reactivity of a commercial N. gonorrhoeae-specific assay with nongonococcal Neisseria species. Recent data have questioned the validity of low-positive C. trachomatis and N. gonorrhoeae screens generated by a second commercial system. Furthermore, prob-lems with reproducibility have been described for C. trachomatis- and N. gonorrhoeae-specific assays. Prompted by reports challenging the validity of the low-positive N. gonorrhoeae and C. trachomatis results generated by the Aptima Combo 2 assay (Gen-Probe) and by a Centers for Dis-ease Control and Prevention recommendation to confirm N. gonorrhoeae- or C. trachomatis-positive screens using an alternative amplification target, the authors reported on a comparison of this means of confirmation with an in-house algorithm of repeat testing. Primary clinical speci-mens yielding N. gonorrhoeae- or C. trachomatis-specific luminescent values between 100,000 and 1,000,000 were repeat tested in duplicate. A subset of specimens was forwarded for confirmatory assays (Gen-Probe) in-dividualized for alternative N. gonorrhoeae or C. trachomatis targets. An 18-month audit revealed that 230 of 29,977 C. trachomatis screens (0.8 percent) and 41 of 29,064 N. gonorrhoeae assays (0.1 percent) yielded low-positive data. When a subset of 40 low-positive N. gonorrhoeae screens was repeat tested, 20 (50 percent) remained positive, and 22 (55 percent) of the screens remained positive after the confirmatory assay was performed. In contrast, repeat testing of 153 low-positive C. trachomatis screens yielded a positive result for fewer specimens (n=97; 63.4 percent) than when commercial confirmatory testing was used (n=124; 81 percent). However, confirmation of the results for additional C. trachomatis screens using an alternative target did not translate into significant differences in the calculated overall C. trachomatis-positive screen rates (7.39 percent by repeat testing versus 7.52 percent by confirmatory assay; P=.53). Furthermore, use of the confirmatory assay raised the positive predictive value only 1.8 percent over that of repeat testing. The authors concluded that molecular confirmatory testing did not significantly enhance the reliability of C. trachomatis- or N. gonorrhoeae-specific nucleic acid amplification testing in this metropolitan setting when compared with repeat testing.

Munson E, Boyd V, Czarnecka J, et al. Evaluation of Gen-Probe APTIMA-based Neisseria gonorrhoeae and Chlamydia trachomatis confirmatory testing in a metropolitan setting of high disease prevalence. J Clin Microbiol. 2007;45:2793–2797.

Correspondence: Erik Munson at

[ Top ]

Methanol-causing matrix effect in electrospray tandem mass spectrometry Methanol-causing matrix effect in electrospray tandem mass spectrometry

High-pressure liquid chromatography coupled with tandem mass spectrometry increasingly is being used in clinical laboratories to quantify testosterone, mycophenolic acid, 25-hydroxyvitamin D2 and D3, homocysteine, methylmalonic acid, acylcarnitines, plasma metanephrine and normetanephrine, purines and pyrimidines, and antiretroviral drugs. The author’s institution uses liquid chromatographytandem mass spec-trometry (LC-MS/MS) in assays to quantify immunosuppressants in whole blood and serum. Methanol is often used in sample preparation and chromatography for these assays because it is readily available and less costly than acetonitrile. The use of methanol as a mobile-phase component in mass spectrometry methods for immunosuppressants has been frequently reported. The author observed a slow loss of 32-desmethoxyrapamycin, the internal standard for sirolimus, if the 8 µg/L methanolic working solution was stored at ambient temperature. However, this solution was very stable if stored at -20°C, so it is now stored in the larger stock bottle at this temperature and a daily working volume is poured off. The author presumed that the loss resulted from degradation of 32-desmethoxyrapamycin in the Fisher Optima methanol he was using, an effect similar to that reported for the internal standard ascomycin when it was dissolved in some brands or grades of acetonitrile. The author investigated whether several sources and grades of methanol would correct the slow loss of 32-desmethoxyrapamycin at ambient temperature and be suitable for use in the mobile phase. The author used drug stability studies, analyses of biological extracts, mixing experiments, and postcolumn infusions to test nine commercial methanols for ionization differences in LC-MS/MS assays for immunosuppressants. Area responses for the drugs and internal standards were compared for mobile phases prepared with each selected methanol. Postcolumn infusion experiments were performed to confirm the degree of ionization differences occurring at the ion source and to evaluate the proportions of ammonium, sodium, and potassium adducts. The author found that the decrease in signal for the immunosuppressant drugs resulted from differential ionization associated with the selected methanols. Product ion intensity varied by 10-fold among the methanols tested. For sirolimus, tacrolimus, and mycophenolic acid, the percentage change in ionization was the same for the drug and its corresponding internal standard. Postcolumn sirolimus infusion evaluation revealed that a 1,000-fold analyte concentration difference did not affect ionization. The proportions of ammonium, sodium, and potassium adducts of sirolimus precursor ions differed in relation to the source of methanol. The authors concluded that organic solvents used in mobile phases and extract preparation of biological samples may be associated with ion suppression, affecting adduct formation and assay sensitivity.

Annesley TM. Methanol-associated matrix effects in electrospray ionization tandem mass spectrometry. Clin Chem. 2007;53:1827–1834.

Correspondence: Thomas M. Annesley at

[ Top ]

Oxidant stress in children born small for gestational age Oxidant stress in children born small for gestational age

It has been proposed that events occurring before birth may influence the risk of developing many diseases, including type II diabetes, asthma, hypertension, and coronary heart disease. The fetal programming hypothesis proposes that these cardiovascular and related disorders derive from fetal adaptations, which increase the fetus’ chance of surviving in poor nutritional environments but permanently alter growth characteristics, postnatal metabolism, and physiology. Therefore, size at birth demands urgent attention not only because of the significantly increased risk it poses to infants and young children but also because it poses a risk of disease susceptibility in adulthood. Multifactorial events are involved in the pathogenesis of disorders linked to fetal programming, and identification of the mechanism is still being discussed. It has been established that an excessive or sustained increase in free radical production associated with diminished efficacy of the antioxidant defense systems results in oxidative stress, which occurs in many pathologic processes and contributes significantly to disease mechanisms. It is reasonable to suggest that oxidative stress would be the key link between an adverse prenatal environment and increased morbidity later in life. The authors conducted a study to analyze the parameters of lipoperoxidation and changes in the antioxidant defense system and to assess their relationship to birth weight. They measured concentrations of thiobarbituric acid-reactive substances and F2-isoprostanes, total antioxidant status, and the activity of superoxide dismutase and glutathione peroxidase in 65 children (33 boys, 32 girls; ages, eight to 13 years). The authors found that thiobarbituric acid-reactive substances and F2-isoprostane levels were significantly elevated in children born small for gestational age. Nevertheless, superoxide dismutase activity was significantly elevated in these children, and the levels of glutathione peroxidase activity and total antioxidant status were unchanged. Moreover, systolic blood pressure was positively associated with thiobarbituric acid-reactive substance levels in race- and gender-adjusted models but not in a multivariable regression model. The authors concluded that there is evidence of oxidative stress in children born small for gestational age, as supported by increased lipid peroxidation.

Franco MCP, Kawamoto EM, Gorjao R, et al. Biomarkers of oxidative stress and antioxidant status in children born small for gestational age: evidence of lipid peroxidation. Pediatric Res. 2007;62:204–208.

Correspondence: Dr. Maria C. P. Franco at

[ Top ]

A role for C-type natriuretic peptide in chondrogenesis A role for C-type natriuretic peptide in chondrogenesis

C-type natriuretic peptide is found in many tissues, including cartilage, and signals through natriuretic peptide receptor 2 (NPR2). Ectopic C-type natriuretic peptide (CNP) has been shown to rescue growth retardation in a mouse model of achondroplasia, suggesting significant therapeutic potential for CNP in treating dwarfism. Previous studies have demonstrated that CNP expression occurs in developing cartilage and acts locally as a positive regulator of endochondral ossification through the accumulation of cGMP by NPR2. Although CNP has been shown to promote chondrocyte proliferation and late (hypertrophic) differentiation, a role for CNP in early chondrogenesis has been suggested but not described. Identification of signaling pathways that regulate chondrocyte differentiation and chondrocyte matrix production are vital to understanding skeletal development and to advancing therapeutics for diseases such as osteoarthritis. The transcription factor Sox9 is expressed in condensed mesenchyme and is required for chondrogenic differentiation. Also necessary for chondrocyte differentiation are the transcription factors Sox5 and Sox6, which with Sox9 regulate expression of the matrix molecules collagen II and aggrecan. Collagens interact with other ma-trix components, such as the proteoglycan aggrecan. Aggrecan is the major core proteoglycan found in the chondrocyte extracellular matrix. It aggregates with other extracellular matrix components and is heavily modified posttranslationally by sulfated glycosaminoglycans (GAGs), giving the aggregate a highly negative charge detectable by the tetracationic dye Alcian blue. The attraction of numerous water molecules provides compressive strength to the matrix, and these aggregates also function to regulate and immobilize growth factors in the matrix. Link protein functions to stabilize the formation of the aggregates. GAGs are long unbranched polysaccharide chains made up of repeating disaccharide units that can be covalently linked to specific amino acids of aggrecan, beginning with the addition of xylose that is catalyzed by xylosyltransferases. Xylosyltransferases I and II are expressed in chondrocytes and are necessary for adding GAGs to the core protein. GAGs are then sul-fated by sulfotransferases to attach sulfate groups to specific sugar moieties. The signals and mechanisms that regulate the expression of genes involved in GAG synthesis during chondrogenesis are largely unknown. The authors demonstrated that CNP is an important regulator of chondrogenesis through the regulation of mesenchymal condensations and matrix biosynthesis. In particular, CNP treatment results in in-creased expression of transcripts for N-cadherin, link protein, xylosyltransferase I, Chst11, and Chst3.

Woods A, Khan S, Beier F. C-type natriuretic peptide regulates cellular condensation and glycosaminoglycan synthesis during chondrogenesis. Endocrinol. 2007;148: 5030–5041.

Correspondence: Frank Beier at

[ Top ]

DHEAS levels in people treated with TNF antagonists for rheumatoid arthritis DHEAS levels in people treated with TNF antagonists for rheumatoid arthritis

Rheumatoid arthritis is a chronic inflammatory disease that primarily affects the joints, causing local inflammation and subsequent joint de-struction, and that exhibits systemic manifestations. The etiology of the dis-ease is unknown, but in established disease, activated macrophages in the inflamed synovial tissue produce proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α). This leads to a proliferation of synoviocytes and production of inflammatory mediators and proinflammatory cytokines in a paracrine way. In healthy people, proinflammatory cytokines are reported to influence the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis. IL-1 and IL-6 influence the adrenal axis at all levels, from stimulation of corticotropin-releasing factor in the hypothalamus, to secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland and stimulation of production of cortisol and dehydroepi-androsterone sulfate (DHEAS) from the adrenal cortex. The authors conducted a study to determine if a major reduction in inflammation due to long-term TNF antagonist treatment influences the adrenal and gonadal axes in rheumatoid arthritis patients. For the study, 48 patients with rheumatoid arthritis were treated with infliximab or etanercept for two years. Disease activity, clinical response, and physical function were evaluated. Serum levels of high sensitivity C-reactive protein and IL-6 were analyzed before treatment was started and after one and two years. ACTH, cortisol, and DHEAS were analyzed at the same time points. Luteinizing hormone, estradiol, and testosterone were analyzed as well in 18 male patients. The authors found that DHEAS increased (P<.05) after one and two years of treatment with TNF antagonists. No change in serum levels of ACTH, cortisol, luteinizing hormone, estradiol, or testosterone was recorded during the two years. The increased levels of DHEAS correlated with improved physical function measured by Health Assessment Questionnaire (P<.01). No correlation was found between hormone levels and clinical response or inflammatory markers. Longitudinal stability in individual hormone levels was found between baseline and two years, most markedly for DHEAS levels (rs=0.90; P<.01). A female subgroup characterized by low levels of DHEAS had a lower age at disease onset. The authors concluded that increased DHEAS levels may indicate improved adrenal function during two years of treatment with TNF antagonists. Improved physical function, correlated to increased DHEAS levels, may be an effect of better adrenal function during powerful antiinflammatory treatment. The stability in individual hormone levels suggests a stable hormonal homeostasis, independent of inflammatory activity.

Ernestam S, Hafstrom I, Werner S, et al. Increased DHEAS levels in patients with rheumatoid arthritis after treatment with tumor necrosis factor antagonists: evidence for improved adrenal function. J Rheumatol. 2007;34:1451–1458.

Correspondence: Dr. S. Ernestam at

[ Top ]

IGF-1 as a hepatic fibrosis marker in hepatitis C IGF-1 as a hepatic fibrosis marker in hepatitis C

Chronic hepatitis C is one of the most common causes of chronic liver disease in the Western world and is complicated by cirrhosis or hepatocellular carcinoma in approximately 20 percent and five percent of patients, respectively. Antiviral treatment with pegylated interferon-α plus ribavirin clears hepatitis C virus (HCV) infection in approximately 40 percent to 90 percent of patients, depending on the HCV genotype. Liver biopsy is recommended to aid in the decision to treat patients infected with HCV genotype 1, but it is an expensive procedure with occasional complications and poor patient acceptance. The accuracy of liver biopsy for assessing fibrosis also has been questioned because of sampling errors and intra- and interobserver variability that may lead to overstaging or understaging of fibrosis. Therefore, there is a need for accurate non-invasive methods for measuring the degree of liver fibrosis. Proposed approaches include physical examination, routine biochemical and hematologic tests, surrogate serum fibrosis markers, glycomics, and transient elastography. Insulin-like growth factor-1 (IGF-1) is a powerful anabolic hormone with diverse endocrine, paracrine, and autocrine effects. It is produced in various tissues, although the liver generates 90 percent of the circulating hormone, which is synthesized in response to growth hormone stimulation. The liver also synthesizes IGF-1 binding proteins, which determine the bioavailability of IGF-1. IGF-1 synthesis is disturbed in liver cirrhosis and reflects the severity of the clinical stage, but the clinical impact of impaired IGF-1 production in advanced cirrhosis is largely unknown. The authors conducted a study to investigate the corre-lation between serum IGF-1 levels and liver fibrosis stage in patients with chronic hepatitis C. Moreover, they evaluated the influence of com-bined therapy on IGF-1 values in these patients. Forty patients with chronic hepatitis C and persistently abnormal alanine amino-transferase values were enrolled in the study and treated with peginterferon α-2a 180 µg per week plus ribavirin for 24 (n=20) or 48 (n=20) weeks. All patients underwent liver biopsy before treatment (METAVIR fibrosis stage F0, n=13; F1–F2, n=14; F3, n=7; F4, n=6). Serum IGF-1 was measured at baseline, the end of the treatment period, and 24 weeks after finishing treatment. The authors found that mean IGF-1 values were significantly lower in patients with advanced fibrosis (F4, 65.9±17.9 ng/mL) than in the others (F0, 145.2±47.1; F1–F2, 150.3±89.6; and F3, 121.4±35.2 ng/mL; P<.05). Serum IGF-1 levels increased during combined therapy, being this increment is markedly higher in patients with sustained virologic response. The authors concluded that IGF-1 synthesis is disturbed in chronic hepatitis C and reflects the severity of liver fibrosis. Combined ther-apy improves serum IGF-1 levels. IGF-1 could represent a good, noninvasive marker of liver fibrosis.

Lorenzo-Zuniga V, Bartoli R, Masnou H, et al. Serum concentrations of insulin-like growth factor-I (IGF-1) as a marker of liver fibrosis in patients with chronic hepatitis C. Dig Dis Sci. 2007;52:3245–3250.

Correspondence: V. Lorenzo-Zuniga at

[ Top ]

Interferon-gamma and wheezing in childhood Interferon-gamma and wheezing in childhood

Evidence suggests that events occurring during the first years of life play an important role in the inception of many cases of childhood asthma. Longitudinal studies in which followup was started at birth have suggested that children who go on to develop asthma have alterations in the developmental processes that characterize the maturation of the immune system and airways. These processes appear to be regulated by environmental exposures and hereditary factors. Production of interferon-y (IFN-y) by peripheral blood mononuclear cells (PBMCs) has been used as a potential marker for the postnatal immune maturation processes associated with subsequent risk of developing asthma and allergies. It has been shown that exposure to bacterial products, such as endotoxin, stimulates the development of IFN-y responses in infants. Therefore, it has been speculated that IFN-y responses could be one pathway by which exposure to other children in day care, pets, or a farming environment might alter the risk of developing asthma and allergies. However, it is not known whether production of IFN-y in early life is associated with subsequent development of asthma and asthma-like symptoms after the first years of life. The authors conducted a study to assess the association between mitogen-stimulated IFN-y and IL-2 production by PBMCs at nine months of age and the subsequent development of wheeze up to 13 years of age. Cytokine production (IFN-y and IL-2) by mitogen-stimulated mononuclear cells was determined from peripheral blood samples (9.4 months; n=118) in a subset of healthy infants enrolled in the Tucson Children’s Respiratory Study. The occurrence of wheeze during the previous year was ascertained at ages 2, 3, 6, 8, 11, and 13 years by means of a questionnaire. Relative risk for wheeze was computed with generalized estimating equations. The authors found that the risk of wheezing between two and 13 years was significantly higher for subjects with low nine-month IFN-y production (relative risk, 2.29; 95 percent confidence interval [CI], 1.35–3.89) and borderline significant for those with intermediate IFN-y production (relative risk, 1.59; 95 percent CI, 0.95–2.68) compared with those who produced high levels of IFN-y (P=.002). Nine-month IL-2 production was unrelated to wheeze. In relation to complex wheezing phenotypes, nine-month IFN-y production was inversely related to toddler wheeze (occurring only before age six years; P=.03) and chronic wheeze (occurring before and after age six years; P=.007) but not school-age wheeze (occurring only after age six years; P=.06). The results suggest that characteristics of the immune system present during the first year of life can anticipate the likelihood of developing episodes of airway obstruction characterized by wheezing. They also suggest that immune susceptibility to asthma is established very early in postnatal life.

Stern DA, Guerra S, Halonen M, et al. Low IFN-y production in the first year of life as a predictor of wheeze during childhood. J Allergy Clin Immunol. 2007;120:835–841.

Correspondence: Dr. Fernando D. Martinez at

[ Top ]

Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.