Clinical Abstracts

July 2004

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

Identifying M. tuberculosis sputum contaminants by genetic fingerprinting
Prenatal exposure to bisphenol A
Y chromosome abnormalities
Serum cytokine levels in tuberculosis
Anti-HDL antibodies in lupus
Prognostic value of nucleated red blood cells
CD26/dipeptidyl peptidase IV activity in lupus
Significance of increased amniotic fluid monocyte chemotactic protein-1

Identifying M. tuberculosis sputum contaminants by genetic fingerprinting

The New York City Department of Health Tuberculosis Control Program uses DNA genotyping to investigate potential laboratory cross-contamination and transmission of Mycobacterium tuberculosis . The two primary methods of DNA genotyping are IS6110-based DNA fingerprint analysis and spoligotyping (spacer oligonucleotide typing). The latter test has increasingly been used as a preliminary screen because it is faster and has fewer material requirements. In August 2000, the mycobacteriology laboratory of the New York City Department of Health Public Health Laboratories identified a cluster of eight positive M. tuberculosis isolates among 36 specimens processed during one laboratory session. Five specimens were from induced sputa collected on the same day at one New York City Department of Health chest clinic (clinic A). This high positivity rate elicited an investigation of specimen-collection and processing procedures. An investigation was conducted at the mycobacteriology laboratory and at clinic A’s sputum induction collection site. In 2000, the number of confirmed tuberculosis cases in New York City declined for the eighth consecutive year to a total of 1,332, of which 1,066 (80 percent) cases were culture confirmed. The borough in which clinic A is located had 363 newly diagnosed cases for the year, whereas the specific area in which clinic A is located had approximately 100 cases, with a case rate of more than twice that of the city as a whole. Despite this high volume, a finding of positive cultures from specimens obtained from five patients on the same day at one facility seemed suspect and warranted further investigation. Consequently, spoligotyping was performed on M. tuberculosis isolates processed during one laboratory session. Laboratory and sputum induction protocols and records were reviewed, and sputum induction staff members were interviewed. An environmental assessment of the sputum induction booth was performed. Spoligotyping identified a unique strain of susceptible M. tuberculosis from five induced sputa collected at clinic A on the same day. Three specimens processed concurrently from other clinics had spoligotypes that were different from each other and from the cluster strain. A laboratory investigation revealed no procedural lapses. Sputum induction was overseen that day by a new employee who had limited training and no supervision. A review of the sputum induction protocol identified ambiguity regarding care of the ultrasonic nebulizer between patients, which may have led to reuse of the discarded nebulizer solution from the first patient, who had prior culture-confirmed tuberculosis. The authors concluded that a break in the sputum induction protocol may have contributed to contamination of patient specimens. Sputum induction is complicated and requires adequate staff training, supervision, and patient preparation. Spoligotyping identified a potential source of M. tuberculosis contamination.

Nivin B, O'Flaherty T, Leibert E, et al. Sputum induction problems identified through genetic fingerprinting. Infect Control Hosp Epidemiol. 2002;23:580-583.

Reprints: Beth Nivin, New York City Dept. of Health, 125 Worth St., Room 225, New York, NY 10013

Prenatal exposure to bisphenol A

Bisphenol A, an estrogenic endocrine-disrupting chemical, is produced at the rate of about 1.7 billion kg annually worldwide and used in the production of polycarbonate plastics and epoxy resins, which are used in dentistry, food packaging, and as lacquers to coat food cans, bottle tops, and water pipes. Thus, there is broad human exposure to bisphenol A (BPA), which can act at the very low doses detected in the environment. BPA administered to pregnant mice is transferred to their fetuses and alters postnatal development and sexual maturity at doses typically found in the environment (2.4 B5g/kg). The authors recently measured serum BPA concentrations by novel enzyme-linked immunosorbent assay and detected BPA in all human sera. They then extended those observations by using the same assay to measure contamination of BPA in various human biological fluids. Blood samples were obtained from healthy premenopausal women, women with early and full-term pregnancy, and the umbilical cord at full-term delivery. Ovarian follicular fluids obtained during in vitro fertilization procedures and amniotic fluids obtained at mid-term and full-term pregnancy were also subject to BPA measurements. The authors found that BPA was present in serum and follicular fluid at approximately 1 to 2 ng/mL, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. Surprisingly, a five-fold higher concentration (8.3 + 8.7 ng/mL) was revealed in amniotic fluid at 15 to 18 weeks' gestation compared with other fluids. These results suggest accumulation of BPA in early fetuses and significant exposure during the prenatal period, which must be considered in evaluating the potential for human exposure to endocrine-disrupting chemicals.

Ikezuki Y, Tsutsumi O, Takai Y, et al. Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod. 2002;17:2839-2841.

Reprints: Osamu Tsutsumi, Dept. of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; osamut-tky@umin.ac.jp

Y chromosome abnormalities

Many people with sex chromosome abnormalities remain undiagnosed because their phenotype usually falls within the limits of "normality," although the karyotypic change affects most nonmosaic cases. Even now, when many abnormalities of the sex chromosomes have been defined and their clinical consequences reported in detail, the precise role of the sex chromosomes in sex differentiation and in the genetic control of gametogenesis remains obscure. In recent years, fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) have been used to identify constitutional chromosomal aberrations. These techniques, combined with conventional G-banding, have a clear diagnostic and prognostic value and contribute to genetic counseling. The authors described three cases where de novo Y chromosome structural anomalies were detected by pre- and postnatal diagnosis and characterized by molecular cytogenetic analysis. This illustrates the usefulness of FISH, polymerase chain reaction (PCR), and CGH for identifying Y chromosome structural anomalies as well as establishing a better correlation with the clinical manifestations. Case one was a 46,X,+mar karyotype. FISH analysis revealed an entire marker chromosome highlighted after hybridization with the Y chromosome painting probe. The PCR study showed the presence of Y chromosome markers AMG and SY620 and the absence of SY143, SY254, and SY147. CGH results confirmed the loss of Yq11.2-qter. These results indicated the presence of the deletion del(Y)(q11.2). Case two was a 45,X[14]/46,XY[86] karyotype with a very small Y chromosome. The PCR study showed the presence of Y chromosome markers SY620 and AMG and the absence of SY143, SY254, and SY147. CGH results showed gain of Yq11.2-pter and loss of Yq11.2-q12. These results show the presence of a Yp isodicentric: idic(Y)(q11.2). Case three was a 45,X,inv(9)(p11q12)[30]/46,X,idic(Y)(p11.3?),inv(9)(p11q12)[70] karyotype. The FISH signal covered all the abnormal Y chromosome using a Y chromosome paint. The PCR study showed the presence of Y chromosome markers AMG, SY620, SY143, SY254, and SY147. CGH showed only gain of Yq11.2-qter. These results support the presence of an unbalanced (Y;Y) translocation. The authors concluded that the combined use of molecular and classical cytogenetic methods in clinical diagnosis may allow a better delineation of the chromosome regions implicated in specific clinical disorders.

Hernando C, Carrera M, Ribas I, et al. Prenatal and postnatal characterization of Y chromosome structural anomalies by molecular cytogenetic analysis. Prenat Diagn. 2002;22:802-805.

Reprints: C. Fuster, Unitat de Biologia, Dept. de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autónoma de Barcelona, E-08193, Bellaterra, Barcelona, Spain; carme.fuster@uab.es

Serum cytokine levels in tuberculosis

Interferon-gamma plays a fundamental role in the immune response to tuberculosis. Targeted deletion of the interferon-gamma (IFN-γ) gene makes mice highly susceptible to tuberculosis, and patients with defective receptors for IFN-g or IL-12 are highly susceptible to severe mycobacterial infections. Production of IFN-γ in response to Mycobacterium tuberculosis infection is driven by the monokines IL-12 and IL-18, and bacterial burdens in IL-18-deficient mice are greater than those in wild-type mice, suggesting that IL-18 contributes to immunity against tuberculosis. Studies of the systemic cytokine response in patients with tuberculosis have focused on serum cytokine levels or cytokine production by peripheral blood mononuclear cells (PBMCs), and these studies have yielded contradictory results. The authors measured concentrations of cytokines in serum and M. tuberculosis -stimulated cytokine production by PBMCs from persons infected with M. tuberculosis. Serum IFN-γ and IL-10 concentrations were elevated in patients with tuberculosis compared with healthy persons who had reactions to tuberculin skin tests, but IL-18 concentrations were not. In contrast, M. tuberculosis -stimulated PBMCs from patients with tuberculosis produced less IFN-γ and IL-18 but similar amounts of IL-10 compared with PBMCs from healthy subjects who had reactions to tuberculin skin tests. Pretreatment of PBMCs from healthy subjects with reaction to tuberculin with serum from patients with tuberculosis inhibited IFN-γ production in response to M. tuberculosis, and inhibition was blocked by anti-IL-10. Thus, serum concentrations of IFN-γ, IL-18, and IL-10 do not parallel M. tuberculosis -induced cytokine levels, and increased IL-10 serum levels in patients with tuberculosis inhibit IFN-γ production in response to mycobacterial antigens.

Vankayalapati R, Wizel B, Weis SE, et al. Serum cytokine concentrations do not parallel Mycobacterium tuberculosis -induced cytokine production in patients with tuberculosis. Clin Infect Dis. 2003;36:24-28.

Reprints: Dr. Peter F. Barnes, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 U.S. Hwy. 271, Tyler, TX 75708-3154; peter.barnes@uthct.edu

Anti-HDL antibodies in lupus

Premature atherosclerosis recently has been recognized as an important cause of morbidity and mortality in systemic lupus erythematosus, accounting for up to 30 percent of all deaths in some reported series. Specific factors, such as steroid treatment, chronic inflammation, and renal disease, could account for enhanced atheroma formation. Dyslipoproteinemia is a major factor in the development of atherosclerosis in systemic lupus erythematosus (SLE). Low levels of high-density lipoproteins (HDL) and apolipo protein A-1 (Apo A-1) have been related to disease activity and the presence of anticardiolipin antibodies. These antibodies, a hallmark of the antiphospholipid antibody syndrome (APS), can also be found in a wide range of conditions and are present in 30 to 40 percent of patients with SLE. Cross-reactivity between these autoantibodies and plasma lipoproteins, particularly when the latter are oxidized, has been found to occur in SLE and APS and could contribute to the increased risk of atherosclerosis found in both conditions. Oxidation is a major process in atherosclerosis, and oxidative stress has been described in SLE and APS. Paraoxonase, an enzyme with antioxidant activity that circulates in plasma attached to HDL, prevents oxidation of low-density lipoprotein (LDL), accounting for the antioxidant effect of HDL and explaining why HDL has a protective effect against atherosclerosis. The authors conducted a study to explore whether the activity of paraoxonase is impaired in patients with SLE and primary APS (contributing to enhanced atherosclerotic progression in these conditions), and, if so, whether the impairment could be dependent on the presence of autoantibodies against cardiolipin, β₂protein I (anti-β₂-GPI), prothrombin, and HDL. The authors enrolled 32 patients with SLE, 36 with primary APS, and 20 age- and sex-matched controls in a cross-sectional study. Serum levels of IgG and IgM anticardiolipin antibodies, anti-β₂-GPI, and antiprothrombin antibodies and IgG anti-HDL were measured by enzyme-linked immunosorbent assay. Total cholesterol, HDL cholesterol, HDL₂, and HDL₃ were determined using standard enzymatic techniques. Paraoxonase activity was assessed by quantifying nitrophenol formation, and total antioxidant capacity was assessed by chemiluminescence. The authors found that levels of total HDL, HDL₂, and HDL₃ were reduced in patients with SLE compared with controls. Patients with SLE and primary APS had higher titers of anti-HDL antibodies and lower paraoxonase activity than controls. In the SLE population, paraoxonase activity was inversely correlated with IgG anti-HDL titers, whereas in the primary APS population, IgG anti-β₂-GPI was the only independent predictor of paraoxonase activity. In the SLE group, anti-HDL was inversely correlated with total antioxidant capacity, and paraoxonase activity was positively correlated with total antioxidant capacity.

Alves JD, Ames PRJ, Donohue S, et al. Antibodies to high-density lipoprotein and β₂-glycoprotein I are inversely correlated with paraoxonase activity in systemic lupus erythematosus and primary antiphospholipid syndrome. Arthritis Rheum. 2002:46: 2686-2694.

Reprints: Dr. J. Delgado Alves, Windeyer Institute of Medical Sciences, Room 118 - Centre for Rheumatology, 46 Cleveland St., London W1T 4JF, United Kingdom; j.alves@ucl.ac.uk

Prognostic value of nucleated red blood cells

The peripheral blood of healthy adults is generally free of nucleated red blood cells, but such cells are found in the blood of patients with a variety of severe diseases for whom the prognosis is relatively poor. A recent retrospective study of patients who underwent cardiothoracic surgery confirmed this poor prognosis. In these studies, nucleated red blood cell (NRBC) concentrations generally were determined microscopically in a stained peripheral blood smear. Using such a technique, it is difficult to detect NRBC concentrations of less than 200 x 10⁶/L. Using an automated hematology analyzer, one can routinely determine NRBC concentrations of less than 100 B4 10⁶/L. However, epidemiological data on the general clinical impact of such routine analyses are missing. The authors measured the NRBCs in the blood of more than 4,000 unselected hospitalized patients. The aim was to elucidate the overall prognostic power of NRBCs with regard to in-hospital mortality. Using a Sysmex XE-2100, the authors measured NRBC concentrations in 15,541 blood samples from 4,173 patients at a university clinic during 12 weeks. NRBCs were found at least once in 7.5 percent of all patients. The highest incidence (20 percent) was found in patients from the intensive care unit of the general and accident surgery. The incidence of NRBCs increased with age. The mortality of NRBC-positive patients (n=313) was 21.1 percent (n=66), which was significantly higher (P<.001) than the mortality of NRBC-negative patients (1.2 percent; n=3,860). Mortality increased with increasing NRBC concentration. With regard to in-hospital mortality, NRBCs in blood showed sensitivity and specificity of 57.9 percent and 93.9 percent, respectively. NRBCs were detected for the first time, on average, 21 days (median, 13 days) before death. The routine analysis of NRBCs in blood is of high prognostic power with regard to in-hospital mortality. This parameter may serve as an early indicator of patients at increased risk. These results suggest that the routine measurement of NRBCs would aid in the early identification of high-risk patients.

Stachon A, Sondermann N, Imohl M, et al. Nucleated red blood cells indicate high risk of in-hospital mortality. J Lab Clin Med. 2002;140:407-412.

Reprints: Axel Stachon, Institute of Clinical Chemistry, Transfusion, and Laboratory Medicine, Ruhr-University, B9Frkle-de-la-Camp-Platz 1, D-44789, Bochum, Germany

CD26/dipeptidyl peptidase IV activity in lupus

T cell activation antigen CD26 is a 110 kDa cell surface glycoprotein with diverse functions. Its expression level on T cells is tightly regulated, and its density is markedly enhanced after T cell activation. In the resting state, CD26 is expressed on a subset of CD4 memory T cells, and this CD4+ CD26^bright T cell population has been shown to respond maximally to recall antigens and has high migratory capacity. CD26 contains dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) enzyme activity in its extracellular domain that can cleave aminoterminal dipeptides with proline or alanine in the penultimate position. Abnormal immune activation, such as hyperactivity of T and B cells, may cause lymphocytes to infiltrate tissue, leading to increased serum cytokine levels and production of a variety of autoantibodies. In this regard, aberrant expression of immune co-stimulatory molecules may contribute to the pathophysiology of systemic lupus erythematosus (SLE). The authors investigated the levels of soluble CD26 (sCD26) and its specific DPPIV enzyme activity in the serum of patients with SLE and investigated their relationship with disease status and activity. They measured serum levels of sCD26 and its specific DPPIV activity by sandwich enzyme-linked immunosorbent assay in 53 patients with SLE and 54 healthy control subjects. Serum sCD26 was identified by immunoprecipitation and immunoblot analysis. Expression of CD26 on T cells was analyzed by flow cytometry. Serum levels of sCD26 and its specific DPPIV activity were significantly decreased in SLE and were inversely correlated with SLE disease activity index score but not with clinical variables or clinical subsets of SLE. Close correlation between sCD26/DPPIV and disease activity was observed in the longitudinal study. The authors concluded that serum levels of sCD26 may be involved in the pathophysiology of SLE and appear to be useful as a new disease activity measure for SLE.

Kobayashi H, Hosono O, Mimori T, et al. Reduction of serum soluble CD26/dipeptidyl peptidase IV enzyme activity and its correlation with disease activity in systemic lupus erythematosus. J Rheumatol. 2002;29:1858-1866.

Reprints: Dr. C. Morimoto, Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; morimoto@ims.u-tokyo.ac.jp

Significance of increased amniotic fluid monocyte chemotactic protein-1

Fetal death before 28 weeks' gestation remains a clinical challenge because a cause cannot be established in most cases. A growing body of evidence suggests that intrauterine infections or intrauterine inflammation, or both, may be involved in the pathogenesis of fetal death and pregnancy loss. Monocyte chemotactic protein-1 (MCP-1) belongs to a family of cellular chemoattractant chemokines first identified in neoplasms as a solute factor capable of attracting macrophages and other leukocytes into sites of inflammation. MCP-1 serves not only as a specific monocyte chemotactic agent but also stimulates the respiratory burst and Ca²⁺ uptake by monocytes and histamine release by basophils. Although MCP-1 was initially characterized as a product of activated monocytes, it has subsequently been identified in a variety of cell types, including endothelial cells, fibroblasts, selected tumor cell lines, and smooth muscle cells. Moreover, MCP-1 expression has been observed in maternal gestational tissues such as decidua and endometrium, as well as in embryonically derived tissues such as the chorion and placenta. Accumulating evidence also suggests that MCP-1 plays an integral role in the control and maintenance of normal pregnancy from implantation to parturition, as well as host response to intrauterine infection. The authors conducted a study to determine whether the concentration of MCP-1 in amniotic fluid collected at the time of genetic amniocentesis varies between patients with normal outcomes and patients with a mid-trimester pregnancy loss. The authors designed a retrospective case-control study of women who had a mid-trimester amniocentesis. Cases (n=10) consisted of patients who had a spontaneous pregnancy loss after the procedure, and the control group (n=84) consisted of patients who had a normal pregnancy outcome after mid-trimester amniocentesis. MCP-1 was measured by a specific enzyme immunoassay (sensitivity, 18.3 pg/mL). The Kolmogorov-Smirnov test was used to assess normal data distribution. Logarithmic transformation was applied to achieve normality. Statistical analysis was performed using Student's t test. A receiver operating characteristic (ROC) curve analysis was used to select a cut-off to dichotomize amniotic fluid concentrations of MCP-1. MCP-1 was detected in all amniotic fluid samples. Patients who had a mid-trimester amniocentesis and subsequent pregnancy loss had a higher mean amniotic fluid log MCP-1 concentration than those with a normal pregnancy outcome (pregnancy loss, mean, 2.95 + 1 0.19 pg/mL versus normal outcome, mean, 2.78 B1 0.19 pg/mL; P=0.01). A cutoff greater than 765 pg/mL was selected by ROC curve analysis (area under the curve, 0.74; P=0.01). An amniotic fluid concentration of MCP-1 above this level was strongly associated with pregnancy loss (odds ratio, 7.35; 95 percent confidence interval, 1.7 to 31.1), a sensitivity of 70 percent, and specificity of 76 percent. The authors concluded that a subset of women who had pregnancy loss after a mid-trimester amniocentesis had higher concentrations of the chemokine MCP-1 than those who had a normal pregnancy outcome. Subclinical intra-amniotic inflammation is a risk factor for pregnancy loss after mid-trimester amniocentesis. This observation may have medicolegal and clinical implications. An elevated MCP-1 concentration in the amniotic fluid of patients with pregnancy loss after mid-trimester amniocentesis indicates that a pathological condition was present at the time of the procedure.

Chaiworapongsa T, Romero R, Tolosa JE, et al. Elevated monocyte chemotactic protein-1 in amniotic fluid is a risk factor for pregnancy loss. J Matern Fet Neonat Med. 2002;12:159-164.

Reprints: Dr. R. Romero, Perinatology Research Branch, NICHD, Wayne State University/Hutzel Hospital, Dept. of Obstetrics and Gynecology, 4707 St. Antoine Blvd., Detroit, MI 48201