College of American Pathologists
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  Clinical Abstracts





July 2010

Michael Bissell, MD, PhD, MPH

Histone acetylation in systemic lupus erythematosus Histone acetylation in systemic lupus erythematosus

The formation of autoantibodies against chromatin components is a major feature of systemic lupus erythematosus, an autoantigen-driven, T cell-dependent autoimmune disease that affects millions of people worldwide. The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE). Nucleosome- and histone-specific autoreactive T cells and B cells have been identified. Years before clinical manifestation of the disease, antinuclear antibodies (ANAs) can be detected in the circulation of future SLE patients. Antichromatin autoantibodies complexed with chromatin are targeted to basement membranes, including the glomerular basement membrane, which incites glomerular inflammation, one of the most serious clinical manifestations of SLE. Nucleosomes can be detected in the circulation during disease in lupus patients and lupus mice. Evidence indicates that in lupus, either aberrant apoptosis or reduced clearance of apoptotic cells, or a combination of both, leads to an overflow of the cleaning machinery and the presence of apoptotic chromatin in the circulation. The contribution of aberrant apoptosis to the development of SLE is supported by mouse models with abnormal function of factors involved in apoptosis, such as Fas (lpr), FasL (gld), Bcl-2/Bim, programmed cell death 1, phosphatase and tensin homolog, B lymphocyte stimulator, and TACI, leading to the formation of ANAs and glomerulonephritis. Impaired removal of apoptotic cells also predisposes to the development of SLE. Mice deficient in factors required for proper clearance of apoptotic cells, such as DNase I, serum amyloid P/C-reactive protein, Clq, IgM, and Mer, develop antinucleosome autoantibodies and glomerulonephritis. It has been demonstrated that the clearance of apoptotic material by phagocytes is impaired in lupus mice and patients with lupus. Autoantigens targeted in SLE are clustered in blebs at the surface of late apoptotic cells. Autoantigens may contain apoptosis-induced modifications that constitute epitopes that normally are not encountered by the immune system when clearance of apoptotic cells is adequate. Apoptosis-induced autoantigen modifications include cleavage by caspases, endonucleases, or granzyme B, and the addition or removal of covalently linked moieties through acetylation, methylation, phosphorylation, ubiquitination, citrullination, ADP ribosylation, or transglutamination. These covalently linked modifications have also been reported for nucleosomes/histones, although with a primary role in the context of regulation of transcription. Although it has been demonstrated that apoptotic cells may induce a lupus-like immune response, little is known about apoptosis-induced chromatin modifications as targets for autoantibodies in SLE. Therefore, the authors evaluated whether apoptosis-induced histone modifications are targets for the immune system in SLE. The epitope of KM-2, a monoclonal antihistone autoantibody derived from a lupus mouse, was mapped by random peptide phage display. The reactivity of KM-2 and plasma with acetylated histone H4 peptides and with nonapoptotic, apoptotic, and hyperacetylated histones was determined by immunofluorescence staining, ELISA, and Western blotting. The authors found that KM-2 recognized apoptosis-induced acetylation of H4 at lysines 8, 12, and 16. The majority of plasma samples from SLE patients and lupus mice showed higher reactivity with triacetylated H4 peptide (residues 1–22) and with hyperacetylated and apoptotic histones than with nonacetylated H4 peptide and normal histones. Importantly, administering triacetylated H4 peptide to lupusprone mice before disease onset accelerated the disease by enhancing mortality and aggravating proteinuria, skin lesions, and glomerular IgG deposition, while the nonacetylated H4 peptide had no pathogenic effect. The delayed-type hypersensitivity response in lupus mice against the triacetylated H4 peptide was higher than that against the nonacetylated H4 peptide. Bone marrow-derived dendritic cells cultured in the presence of hyperacetylated nucleosomes showed increased expression or production of CD40, CD86, interleukin-6, and tumor necrosis factor α compared with dendritic cells cultured in the presence of normal nucleosomes. Finally, dendritic cells cultured in the presence of hyperacetylated nucleosomes were able to activate syngeneic T cells because IL-2 production increased. The authors concluded that apoptosis-induced acetylation of nucleosomes may represent an important driving force in the development of lupus.

Dieker JW, Fransen JH, van Bavel CC, et al. Apoptosis-induced acetylation of histones is pathogenic in systemic lupus erythematosus. Arthritis Rheum. 2007;56:1921–1933.

Correspondence: Dr. John van der Vlag at

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Scleroderma peptides and endothelial proliferation Scleroderma peptides and endothelial proliferation

Systemic sclerosis is an autoimmune disorder of the connective tissue characterized by widespread vascular lesions and fibrosis. Raynaud’s phenomenon, increased thickness of the vascular wall, devascularization, and thickness of the basement membrane are typical features of systemic sclerosis (SSc). The digital arteries of patients with SSc exhibit marked intimal proliferation, resulting in severe narrowing and occlusion of arterial lumen and limited vasodilative responses to vasodilator therapies. The contributions of many molecules to the pathogenesis and diagnosis of SSc have been reported. Investigation of these known molecules is of value for understanding the pathogenesis of SSc. However, it is also important to identify novel SSc-related molecules by hypothesis-free comprehensive surveillance. Genomic surveillance using single-nucleotide polymorphisms, DNA arrays for messenger RNA expression, and proteomic surveillance have been used in this regard. These techniques can detect various molecules that may be involved in the pathogenesis of SSc or that may be useful for diagnosis. However, the targets of surveillance have been limited to genes or relatively large proteins. Peptides with low molecular weights (smaller than approximately 3 kd) generally cannot be investigated by these techniques. Yet peptides with such low molecular weights often play a central role in biologic and pathologic processes. Typical of these peptides would be bradykinin, a peptide that is only nine amino acid residues in length and produced from kininogen by specific proteolysis. Another example would be substance P in a neuropeptide of 11 amino acid residues. Peptides with such low molecular weights are estimated to be produced in large amounts by proteolysis of large proteins, which generates various bioactive or diagnostically useful short peptides, or both, in addition to the known peptides in the body. However, there are only a few ways to survey such peptides efficiently. Mass spectrometry methods that directly detect and identify peptides with low molecular weights and with low concentrations, referred to as peptidomics analysis, have been developed. These offer a promising approach to the discovery of novel short peptides that could be useful for diagnosing and treating diseases. The authors conducted a study to identify low molecular weight peptides in the serum of patients with SSc in order to understand the role of these peptides in disease pathogenesis. By combining a microamount peptide-separating method with magnetic beads and matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry, the authors were able to comprehensively detect short peptides in the sera of patients with SSc, those with non-SSc rheumatic diseases, and healthy donors. In comparing the results between each group of samples, they found that the sera of patients with SSc carried complement C3f-desarginine (DRC3f) and its degraded smaller fragments at very high levels and with high frequency compared with the sera of patients with non-SSc rheumatic diseases or healthy donors. Furthermore, they demonstrated that these peptides promoted proliferation of human microvascular endothelial cells in vitro. The authors concluded that the results of this study will open a new field of investigation into the pathophysiologic processes of SSc.

Xiang Y, Matsui T, Matsuo K, et al. Comprehensive investigation of disease-specific short peptides in sera from patients with systemic sclerosis. Arthritis Rheum. 2007;56:2018–2030.

Correspondence: Dr. Tomohiro Kato at

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3333333333333333333 ELISA and dipstick to detect Strongyloides stercoralis

Strongyloides stercoralis, the causative agent of strongyloidiasis, is an intestinal nematode that is endemic throughout tropical and temperate regions of the world. The infection is frequently imported by travelers and immigrants to areas where S. ster-coralis is not endemic. The vast majority of patients with strongyloidiasis are asymptomatic, although some have a variety of symptoms, mostly localized in the skin (larva currens) or gastrointestinal system. However, immunocompromised patients may experience accelerated autoinfection— the serious, and often fatal, hyperinfection syndrome. Consequently, reliable diagnosis of patients who are at risk is needed to accurately recognize and treat the infection before immunosuppressive therapy is used or in patients with HIV infection. Finding the larvae in stool, duodenal fluid, or, occasionally, other tissues or fluids by means of microscopy makes a diagnosis of strongyloidiasis definitive. The Baermann concentration method is one of the most sensitive tests available for determining the presence of larvae in fecal samples. However, because of low larval densities, a single examination with this test is insensitive. Multiple examinations increase the sensitivity, but these extensive parasitological investigations of individuals who, for the most part, are asymptomatic are not practical or cost-effective. An alternative to the Baermann test is culture of stool samples. Both tests require freshly produced stools, which, in routine practice, are difficult to obtain. Serological methods to detect Strongyloides infection have been studied previously. The most convenient and widely used method is an ELISA that detects serum immunoglobulin G (IgG) against a crude extract of infective larvae. However, the ELISA is somewhat cumbersome and labor intensive, and a certain level of laboratory infrastructure is necessary to perform the test and interpret the results, factors that have hampered the test’s applicability in areas where Strongyloides is endemic. In contrast to ELISA, dipstick assays are fast and easy to perform. Studies with other infections demonstrated a high degree of concordance between the results of ELISA and those of the dipstick assay. In general, serological assays are easier to perform and require more easily obtainable patient material (serum versus fresh stools) than parasitological assays. However, most assays are homemade and require advanced laboratory infrastructure in order to be performed adequately. Therefore, the availability of commercially available serological tests would be advantageous. The authors conducted a study in which they evaluated a homemade ELISA (Academic Medical Center ELISA [AMC-ELISA]) and an easy-to-perform dipstick assay based on S. stercoralis antigens for the serodiagnosis of strongyloidiasis. They also evaluated two commercial ELISAs—IVD-ELISA and Bordier-ELISA—that were recently introduced. The sensitivities of the assays were evaluated using sera from 90 patients with parasitologically proven intestinal strongyloidiasis and from nine patients with clinical larva currens. The sensitivities of the AMC-ELISA, dipstick assay, IVD-ELISA, and Bordier-ELISA for intestinal strong-yloidiasis were 93, 91, 89, and 83 percent, respectively. In all tests, eight of nine sera from patients with larva currens were positive. The specificity was assessed using a large serum bank of 220 sera from patients with various parasitic, bacterial, viral, and fungal infectious diseases, as well as sera containing autoimmune antibodies and sera from healthy blood donors. The specificities of AMC-ELISA, dipstick assay, IVD-ELISA, and Bordier-ELISA were 95, 97.7, 97.2, and 97.2 percent, respectively. The authors’ data suggest that all four assays are sensitive and specific tests for diagnosing intestinal and cutaneous strongyloidiasis.

Van Doorn HR, Koelewijn R, Hofwegen H, et al. Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans. J Clin Microbiol. 2007;45:438–442.

Correspondence: H. Rogier van Doorn at

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Troponin T in perinatal asphyxia Troponin T in perinatal asphyxia

The syndrome of hypoxemia-related myocardial dysfunction in the newborn neonate occurs in about 30 percent of asphyxiated infants. In preterm infants, cardiovascular compromise is even more frequent because of impaired myocardial contractility and reduced cardiac output as a consequence of perinatal asphyxia and respiratory distress syndrome. However, different clinical pictures are associated with neonatal myocardial ischemia, and electrocardiogram is not as helpful in neonates as in adults. T-wave changes are frequently observed in healthy newborns. Ischemia-induced ST segment shifts or abnormal Q waves are less common in newborns than in adults. For these reasons, echocardiography (EchoCG) plays an important role in the diagnosis of myocardial ischemic involvement in neonates. Transient tricuspid insufficiency is often associated with myocardial ische-mia in newborns with persistent pulmonary hypertension. In these infants, right ventricular function may be brisk or markedly diminished. On the EchoCG, ischemia is also manifested by regional wall motion abnormalities, papillary muscle hyperechogenicity, poor left ventricular function, and mitral or tricuspid valve incompetence. Cardiac troponin T (cTnT) and troponin I are biochemical markers for myocardial necrosis in adults, but their clinical significance in newborns is still questioned. Elevated cTnT levels have been detected in newborns and in different perinatal conditions, such as placental insufficiency, birth asphyxia, and respiratory distress syndrome. However, in the absence of direct comparison between cTnT levels and instrumental signs of myocardial damage, it is hard to define which cTnT level is a specific marker of my-ocardial ischemia in newborns. To this end, the authors conducted a prospective study of newborns with and without birth asphyxia, correlating cTnT levels with instrumental findings of myocardial ischemia. Electrocardiograms and echocardiograms were obtained from 29 asphyxiated and 30 control infants during the first 24 hours of life and correlated with cTnT concentrations. The echocardiographic parameters included systolic ventricular performance, preload, afterload, diastolic function, stroke volume, left ventricular output, hyperechogenicity of the papillary muscles, and insufficiency of the atrioventricular valves. The authors found that left ventricular output and stroke volume were lower, but cTnT was significantly higher in asphyxiated infants than in control infants: 0.15 (0.10–0.23) µg/L versus 0.05 (0.02–0.13) µg/L (P<.001). Asphyxiated infants with signs of myocardial damage had significantly higher cTnT than those without at 0.20 (0.11–0.28) µg/L and 0.11 (0.05–0.14) µg/L (P= .04). The authors concluded that cardiac troponin may prove to be valuable in evaluating myocardial damage in newborns with birth asphyxia. However, the degree of prematurity may complicate the assessment.

Costa S, Zecca E, De Rosa G, et al. Is serum troponin T a useful marker of myocardial damage in newborn infants with perinatal asphyxia? Acta Pediatrica. 2007;96:181–184.

Correspondence: Simonetta Costa at

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Comparison of automated nucleic acid testing systems Comparison of automated nucleic acid testing systems

In response to a regulatory requirement, the Australian Red Cross Blood Service commenced HIV and hepatitis C virus nucleic acid testing with an HIV-1 and multiplex HCV assay (Procleix, Chiron Corp. [now Novartis Diagnostics]) from June 2000. The duplex assay markedly reduced the window period, defined as the time between infection and first detectable viral marker, from approximately 22 to nine days for HIV and 66 to seven days for HCV. With the reduction in residual risk for HIV and HCV due to nucleic acid testing (NAT) screening, attention focused on whether hepatitis B virus NAT would provide a similar risk reduction for HBV. The development of NAT assays that incorporate the simultaneous detection of HBV DNA, along with the concurrent development of automated testing platforms that allow for high-throughput identification testing, has provided the opportunity to reconsider the benefits of HBV NAT screening. The authors performed a head-to-head evaluation of two multiplex NAT assays and their testing platforms: the Procleix Ultrio assay on the fully automated Procleix Tigris, and the Cobas TaqScreen multiplex (Cobas MPX) test on the modular automated Cobas s 201 (Roche Molecular Diagnostics). The study was also intended to determine the HIV-1, HCV, and HBV analytical sensitivity of these assays; compare the HBV clinical sensitivity of the two assays with blood donor samples from Hong Kong; and investigate the impact of pool size on HBV DNA detection by each testing system. The HBV NAT yield rate was estimated by testing 10,397 Hong Kong donor samples as individual samples concurrently on the Ultrio assay with the Tigris and on the Cobas MPX test with the Cobas s 201 in pools of six. The authors assessed analytical sensitivity by probit analysis of diluted international standards and compared operational performance. Each system detected two different HBV NAT yield samples for a combined rate of 0.04 percent. One additional sample was reactive on the Cobas MPX test but remained unresolved. The 95 percent detection limits for HIV-1, HBV, and HCV were 42.2, 12.2, and 2.0 IU/mL, respectively, for Ultrio, and 50.5, 8.4, and 6.0 IU/mL, respectively, for the MPX. The invalid test and failed run rates were 0.05 and 2.92 percent, respectively, for the Tigris and 2.39 and 5.53 percent for the Cobas s 201. The authors concluded that the clinical sensitivity for HBV in blood donors from Hong Kong was equivalent, as was the analytical sensitivity for HIV-1 and HBV. However, the Ultrio assay had a higher analytical sensitivity for HCV. Despite a shorter downtime and mean time of repair for the Cobas s 201, the Tigris demonstrated better overall operational performance.

Margaritis AR, Brown SM, Seed CR, et al. Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepati-tis C virus RNA and hepatitis B virus DNA. Transfusion. 2007:47:1783–1793.

Correspondence: Angelo R. Margaritis at

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Study of serum CA-125 in patients with stage III ovarian cancer Study of serum CA-125 in patients with stage III ovarian cancer

Early normalization of CA-125 during adjuvant postoperative chemotherapy for patients with advanced stage ovarian cancer is associated with improved survival. Monitoring serum CA-125 levels during therapy is a predictor of survival and may allow for early identification of patients who are not responding to therapy and require a change in treatment. Studies have suggested survival advantages associated with intraperitoneal infusion of chemotherapy, which allows for high doses of chemotherapy to be infused because of the slow and incomplete transit of intraperitoneal agents into the circulation. The use of intraperitoneal chemotherapy with cisplatin and paclitaxel may irritate the peritoneal cavity and, theoretically, increase CA-125 levels. Previous reports focused on serum CA-125 levels in patients who had undergone surgery and were then treated with intraperitoneal 32P. These reports found that patients treated with intraperitoneal 32P had elevated serum CA-125 levels and that this tumor marker may not reflect recurrent or progressive ovarian cancer. Another single-institution review did not demonstrate any difference in the rate of decline of CA-125 levels when comparing patients treated with intravenous versus intraperitoneal chemotherapy for advanced ovarian cancer. The authors conducted a study using data from a large cooperative group clinical trial to evaluate the utility of CA-125 levels in patients treated with intraperitoneal versus intravenous chemotherapy. They retrospectively reviewed patient charts from a phase III clinical trial (GOG 172). Serum CA-125 levels prior to each cycle of therapy were collected and compared between the intravenous and intraperitoneal chemotherapy delivery. The authors estimated the association between CA-125 and progression-free survival or overall survival and assessed the homogeneity of the results between intraperitoneal and intravenous chemotherapy. A total of 177 patients were treated with intravenous chemotherapy and 165 patients with intraperitoneal chemotherapy, with CA-125 data available. The observed difference in median CA-125 levels between the intravenous and intraperitoneal arms at any time point was not significant (P>.05 for all). Following surgery and adjuvant chemotherapy, patients with an abnormal CA-125 greater than 35 U/mL were 2.45 times more likely to have disease progression (95 percent confidence interval, 1.52–3.95; P<.001) and 2.78 times more likely to die of disease (95 percent confidence interval, 1.66–4.65; P<.001) compared to those with a CA-125 of less than 35 U/mL. These results were consistent for intraperitoneal and intravenous chemotherapy. The authors concluded that serum CA-125 levels decrease in a similar manner during intraperitoneal chemotherapy when compared to intravenous chemotherapy. Serum CA-125 algorithms for monitoring treatment effect that have been established for intravenous chemotherapy may also be applied for patients receiving intraperitoneal chemo-therapy.

Krivak TC, Tian C, Rose GS, et al. A gynecologic oncology group study of serum CA-125 levels in patients with stage III optimally de-bulked ovarian cancer treated with intraperitoneal compared to intravenous chemotherapy: An analysis of patients enrolled in GOG 172. Gynecol Oncol. 2009;115:81–85.

Correspondence: Thomas C. Krivak at

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