College of American Pathologists
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  Clinical Abstracts




April 2008

Michael Bissell, MD, PhD, MPH

Effects of age and gender on glomerular filtration rate
Wiskott-Aldrich syndrome and T cell function
Relevance of fever after returning from the tropics
A biosensor for detecting anti-double-stranded DNA
Luteinizing hormone levels in IVF and live birth rates
DNA microarray for bloodstream infections
Nucleolar staining as a screening test for a scleroderma marker

Effects of age and gender on glomerular filtration rate Effects of age and gender on glomerular filtration rate

The best measure of renal function is the glomerular filtration rate. The assessment of GFR is important for characterizing various renal diseases, evaluating the effect of different therapies, adjusting the doses of various drugs, and ensuring that potential kidney donors have normal renal function. Previous studies of renal function have shown conflicting results about the effects of age and gender on renal function. Moreover, various methods have been used to determine GFR. In the author’s pediatric nephrology unit, 70 percent of pediatric kidney transplantations are performed using living donors, primarily parents, and occasionally grandparents. Most of the potential donors are investigated by means of renal function tests—namely, by determining GFR and effective renal plasma flow (ERPF) by clearances of inulin and para-aminohippurate (PAH). The donors are examined before donation, approximately three months after, and thereafter every other year at the same time as their kidney recipients. The author reported on investigations done before transplantation to give reference values for GFR and ERPF, as well as the effects of age and gender on these variables. Because most parents were quite young, only the influence of age between 20 and 50 years could be evaluated. A total of 122 potential kidney donors, 62 of whom were females aged 21 to 67 years, were investigated with the GFR and ERPF determined by clearances of inulin and PAH. The mean ± standard deviation GFR and ERPF were 105±13 and 545±108 mL/min./1.73 m2, respectively, and no difference was noted between male and female donors. When relating GFR and ERPF to age, however, a significant decline was found in GFR and ERPF in males, but not females, in the age range of 20 to 50 years. GFR fell by a mean of 8.7 mL/min./1.73 m2 and ERPF by 90 mL/min./1.73 m2 per decade in male donors. The author concluded that with adequate methods for determining GFR and ERPF, a clear difference in the effect of age was seen between the genders. Males showed a significant decrease between 20 and 50 years of age and females did not. Females seem to be protected in the premenopausal period, probably by oestrogens. These results confirm clinically those found in rats.

Berg UB. Differences in decline in GFR with age between males and females. Reference data on clearances of inulin and PAH in potential kidney donors. Nephrol Dial Transplant. 2006;21:2577–2582.

Reprints: Dr. U.B. Berg, Dept. of Pediatrics, Karolinska Institutet, Karolinska University Hospital Huddinge, S-14186 Stockholm, Sweden;
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Wiskott-Aldrich syndrome and T cell function Wiskott-Aldrich syndrome and T cell function

Wiskott-Aldrich syndrome protein is expressed in cells of hemopoietic origin and is involved in actin cytoskeleton remodeling, thereby playing a key role in chemotaxis, adhesion, phagocytosis, and traffic of various hemopoietic cells. The role of Wiskott-Aldrich syndrome protein (WASP) in controlling downstream signaling and, more generally, T cell functions is still largely unknown. Mutations in the Wiskott-Aldrich syndrome (WAS) gene in humans may lead to different clinical phenotypes, ranging from WAS, characterized by microthrombocytopenia, ecze­ma, immunodeficiency, autoimmune disorders, and high incidence of lymphoid malignancies, to X-linked thrombocytopenia, a milder form of the disease with minimal or no immunodeficiency. A strong correlation between expression of WASP in lymphocytes and clinical phenotype has been reported in patients carrying WAS gene mutations. Although WASP deficiency affects the functions of lymphoid and myeloid cells, results obtained from the followup of WAS patients who underwent HLA-matched hemopoietic stem cell transplant, and from analyzing WAS patients with spontaneous genetic reversion, indicate that WASP expression confers a selective growth advantage to T cells only. It has been shown consistently that WAS patients have a reduced number of circulating T lymphocytes, especially within the naive compartment. WAS patients suffer from recurrent infections caused by encapsulated bacteria, viruses, and fungi, suggesting an impairment in cellular-mediated immunity. Moreover, they have a high susceptibility to develop lymphomas, which could be due to poor immune surveillance. However, T cell functions are not completely abrogated in WAS patients, as illustrated by the high frequency of T cell-mediated autoimmune manifestations. T cells from WAS patients proliferate poorly, secrete low levels of IL-2 in response to anti-CD3 mAb, and display abnormal cell morphology. Defective secretion of IL-2, IFN-g, TNF-a, and IL-4 in response to TCR or TCR/ CD28-mediated stimulation has been shown in WASP knockout mice. Although proximal TCR signaling is not significantly affected in T cells from WASP knockout mice, WASP seems to be required for downstream events, including activation of NFAT-1 (NFAT-p), NFAT-2 (NFAT-c), and expression of Fos. Similar results have been obtained in NK cells from WAS patients, in which a time-restricted defect in the nuclear translocation of NFAT-2 and RelA (NF-kB) was observed in response to ligation of the NKp46R. In this study, the authors investigated the effector functions of WASP-deficient CD4+ and CD8+ T cell lines generated from WAS patients. They show that, in response to TCR/CD28-mediated activation, WAS CD4+ and CD8+ T cells have a selective impairment in Th1 cyto­kine production, which is due to defective gene transcription. This defect is associated, in WAS CD4+ T cells, with a reduction in T-bet mRNA expression and in the early nuclear recruitment of NFAT-1. In WAS CD8+ T cells, the block in cytokine production correlates with reduced nuclear levels of NFAT-1 and NFAT-2. These results suggest that WASP may play a predominant role in cellular-mediated immunity by controlling Th1 cytokine production at the transcriptional level.

Trifari S, Sitia G, Aiuti A, et al. Defective Th1 cytokine gene transcription in CD4+ and CD8+ T cells from Wiskott-Aldrich syndrome patients. J Immunol. 2006; 177: 7451– 7461.

Reprints: Dr. Maria-Grazia Roncarolo, San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, 20132, Milan, Italy;
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Relevance of fever after returning from the tropics Relevance of fever after returning from the tropics

The differential diagnosis of fever in travelers returning from the tropics remains a challenge, particularly for physicians less familiar with exotic pathology. Clinicians who treat such patients must consider various tropical pathogens as well as infections with worldwide distribution. Most tropical infections have a nonspecific presentation, and some may evolve rapidly into severe disease if not treated properly. Therefore, optimal patient management focuses on promptly identifying the most serious conditions while minimizing unnecessary workup and treatment for milder diseases. Morbidity profiles for international travelers vary significantly based on the region of exposure. Diagnostic predictors of several tropical diseases have been investigated in endemic settings, but only incompletely in returning travelers, except for malaria. The authors conducted a study to identify in febrile travelers and migrants the clinical and laboratory features that could help in diagnosing the most frequent tropical conditions and to quantify their predictive contribution. From April 2000 to December 2005, the authors prospectively enrolled in their study all patients who presented at their referral centers with fever within one year of visiting a tropical or subtropical area. For clinical relevance, the diagnostic predictors of the leading tropical conditions were investigated in the febrile episodes occurring during travel or within one month after return, which was defined as early-onset fever. The study addressed 2,071 fever episodes in 1,962 patients. Most patients were Western travelers (60%) or expatriates (15%). Regions of exposure were mainly sub-Saharan Africa (68%) and southern Asia/Pacific (14%). Early-onset fever accounted for 1,619 episodes (78%). Most tropical infections were related to specific travel destinations. Malaria (mainly Plasmodium falciparum) was strongly predicted by enlarged spleen, thrombocytopenia (platelet count of less than 150 ∞ 103/µL), fever without localizing symptoms, and hyperbilirubinemia (total bilirubin level greater than 1.3 mg/dL). When malaria had been ruled out, the main predictors were skin rash and skin ulcer for rickettsial infection (mainly African tick bite fever); skin rash, thrombocytopenia, and leukocytopenia (leukocyte count of less than 4 ∞ 103/µL) for dengue; eosinophil count of greater than 0.5 ∞ 103/µL for acute schistosomiasis; and enlarged spleen and elevated alanine aminotransferase level (70 IU/L or more) for enteric fever. The authors concluded that initial clinical and laboratory assessment can help in selecting appropriate investigations and empiric treatments for patients with imported fever.

Bottieau E, Clerinx J, Van den Enden E, et al. Fever after a stay in the tropics: diagnostic predictors of the leading tropical conditions. Medicine. 2007;86:18–25.

Reprints: Dr. Emmanuel Bottieau, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium;
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A biosensor for detecting anti-double-stranded DNA A biosensor for detecting anti-double-stranded DNA

Systemic lupus erythematosus, often considered the prototypic systemic autoimmune disease, is associated with the production of a set of different autoantibodies, predominantly against components of the cell nucleus. Both the detection of antinuclear antibodies by immunofluorescence and findings of “antibodies to native DNA in abnormal titer” (American College of Rheumatology criterion 10.b) are of high diagnostic significance and considered specific and early markers for systemic lupus erythematosus (SLE). Three clinical laboratory methods are commonly used for determining and quantifying anti-double-stranded DNA (anti-dsDNA): Crithidia luciliae indirect immunofluorescence, enzyme-linked immunosorbent assay, and radioimmunoassay (Farr assay). The main discrepancies between these methods depend on whether IgG, IgM, or the collection of isotypes is detected and whether low-avidity antibodies are detected. High-avidity anti-dsDNA IgG has been shown to be associated with glomerulonephritis. Avidity has been discussed repeatedly in the literature but is still controversial. The authors conducted a study to develop a novel biosensor device on the basis of surface plasmon resonance (SPR) that would allow label-free monitoring of the interaction between ­dsDNA and anti-dsDNA in real time. The change of mass concentration at the interface because of specific binding of anti-dsDNA to surface-­immobilized dsDNA would be detected as changes in the refractive index via the SPR effect. For the study, a synthetic oligonucleotide was coupled to biotinylated human transferrin, hybridized with the complementary antistrand, and ligated with a human recombinant dsDNA fragment 233 bp in length. After surface immobilization of this antigenic construct, diluted sera from SLE patients and healthy donors were analyzed with the resulting SPR bio­sensor system. The authors found that this SPR biosensor allowed specific detection of anti-­dsDNA. In pilot experiments, sera from SLE patients were distinguished from control sera. The authors also confirmed the specificity of this biosensor by supplementing anti-dsDNA-positive sera with salmon sperm DNA, which blocked the surface binding of anti-dsDNA in a concentration-dependent manner. The authors concluded that an SPR biosensor monitors interactions in real time under homogeneous conditions, providing information about binding kinetics and affinities. Its applicability depends on the design of the solid-state surface of the sensor chips. Covalently immobilizing dsDNA as the antigen to the surface in a flow-through cell assured maximal stability for multiple serum injections and regeneration cycles. This technique enhances existing methods and may be beneficial in the diagnosis and clinical monitoring of SLE.

Buhl A, Metzger JH, Heegaard NHH, et al. Novel biosensor-based analytic device for the detection of anti-double-stranded DNA antibodies. Clin Chem. 2007; 53: 334– 341.

Reprints: Peter B. Luppa, Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der Technischen Universitat Munchen, Ismaninger Str. 22, D-81675 Munchen, Germany;
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Luteinizing hormone levels in IVF and live birth rates Luteinizing hormone levels in IVF and live birth rates

With the introduction of recombinant luteinizing hormone, supplementation of LH in down-regulated in vitro fertilization/ intra­cyto­plasmic sperm injection (IVF/ ICSI) cycles using a GnRH analog and recombinant follicle-stimulating hormone has become more common. An unpublished survey performed by the authors in 2005 revealed that in Sydney, Australia, 82.4 percent of clinicians occasionally used LH supplementation (either recombinant LH or low-dose human chorionic gonadotropin). In addition, 64.7 percent routinely monitored LH concentrations during controlled ovarian hyperstimulation (COH) for in vitro fertilization. The authors conducted a large observational trial to address whether low LH concentrations result in adverse outcomes. A recent systematic review of studies investigating an association between endogenous LH levels during ovarian stimulation using GnRH agonists failed to detect a significant negative effect of low endogenous LH levels on pregnancies past 12 weeks. The same review suggested that high levels of endogenous LH were associated with a decreased probability of pregnancies beyond 12 weeks. Based on experience with GnRH antagonist cycles, it has been suggested that the dynamic changes in LH levels may be a cause for poorer pregnancy outcomes with in vitro fertilization cycles. Using IVF/ICSI treatment cycles that utilize GnRH analog down-regulation and COH with recombinant follicle-stimulating hormone (rFSH), the authors sought to explore possible associations between low mid-follicular LH values and changes between mid- to early follicular LH concentrations and their relationship with ovarian response and pregnancy rates. Blood samples were collected prospectively from 701 cycles (560 patients) of assisted reproduction and analyzed retrospectively. On the basis of LH concentrations on stimulation day 7/8, the patients were divided into two groups: LH of less than 1.2 IU/L (n=179) and LH of 1.2 IU/L (n=522) or greater. Cycle outcomes were also compared on the basis of a ratio of mid- to early follicular LH concentrations (0.5 or less, n=210; greater than 0.5, n=491). The authors found that patients with low LH concentrations have a significant reduction in late follicular estradiol concentrations (P<0.001), number of oocytes retrieved (P<0.01), and number of usable embryos (P<0.01). They also required significantly more rFSH (430 IU difference, P<0.01). These differences did not translate into a significant change in live birth rates. Conversely, a ratio of 0.5 or less mid- to early follicular LH concentrations (a reduction of 50% or more) was associated with a significant reduction in live birth rates per embryo transfer and per cycle started (27.3% versus 19%, P<0.05 and 22.2% versus 15.8%, P<0.05, respectively). The authors concluded that low mid-follicular levels of LH have a significant impact on ovarian response but not on live birth rates. A fall in LH level of 50 percent or more from the early to mid-follicular phase resulted in a lower live birth rate.

Lahoud R, Al-Jefout M, Tyler J, et al. A relative reduction in mid-follicular LH concentrations during GnRH agonist IVF/ICSI cycles leads to lower live birth rates. Hum Reprod. 2006;21:2645–2649.

Reprints: R. Lahoud, IVFAustralia, Level 1, 24 Thomas St., Chatswood, New South Wales 2067, Australia; or rjlahoud@optusnet.
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DNA microarray for bloodstream infections DNA microarray for bloodstream infections

DNA microarray technology offers promise for simultaneously identifying a wide variety of genes. DNA probes specific to selected genes are spotted on a solid substrate (usually glass) in a lattice pattern. The DNA targeted for analysis is then labeled with a reporter molecule—for example, a fluorescent dye—and hybridized to the array. Specific target-probe duplexes are detected by measuring the fluorescent signals associated with each spot. The authors of this study developed a prototype DNA microarray for identifying and characterizing Staphylococcus aureus, Esherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, three reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific for identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. The authors found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-­methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes blaTEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. The authors concluded that this novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or pre-identification of the pathogen.

Cleven BEE, Palka-Santini M, Gielen J, et al. Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray. J Clin Microbiol. 2006; 44: 2389– 2397.

Reprints: Oleg Krut, Institute for Medical Microbiology, Immunology and Hygiene, Medical Center, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany;
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Nucleolar staining as a screening test for a scleroderma marker Nucleolar staining as a screening test for a scleroderma marker

Autoantibodies associated with scleroderma (systemic sclerosis [SSc]), such as anti-topoisomerase I antibodies, anti-RNA polymerase I/III (anti-RNAP I/III) antibodies, and anticentromere antibodies, are helpful in establishing a diagnosis of scleroderma. They also serve as a clinically useful marker for predicting specific organ involvement and outcome. However, with the exception of anticentromere antibodies, which can be identified by immunofluorescent antinuclear antibodies (ANAs), and anti-topoisomerase I antibodies, which can be identified by commercially available enzyme-linked immunosorbent assays (ELISAs), tests for SSc antibodies are not available. Anti-RNAP I/III antibodies, which are found in approximately 20 percent of patients with SSc, have been established as a disease marker associated with diffuse scleroderma and an increased risk of cardiac and kidney involvement. An anti-RNAP III ELISA using a recombinant fragment as an antigen became commercially available in 2004. It is a clinically useful test with excellent sensitivity and specificity. Anti-RNAP III antibodies always co-exist with anti-RNAP I antibodies, and RNAP I localize to nucleoli. Therefore, in theory, it would make sense to order an anti-RNAP III ELISA after confirming nucleolar staining with immunofluorescent ANAs. However, despite early reports of punctate nucleolar staining by anti-RNAP I antibodies, other reports suggest that nucleolar staining is not as apparent in sera with anti-RNAP I/III antibodies. One potential explanation is that the punctate nucleolar staining associated with anti-RNAP I antibodies may be obscured by the co-existing nuclear speckled staining of anti-RNAP II and anti-RNAP III antibodies. The authors conducted a study in which they examined anti-RNAP I/III antibody-positive sera by double staining HEp-2 ANA slides with an antifibrillarin mouse monoclonal antibody. They evaluated whether nucleolar-positive ANAs can be used as a screening test to order an anti-RNAP III ELISA. They also examined the relationships between nucleolar staining, levels of anti-RNAP III antibodies versus anti-RNAP I antibodies by immunoprecipitation (IP) assay, and results of anti-RNAP III ELISA. Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides by anti-RNAP III ELISA and by IP assay using 35S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. The authors found that all 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44 percent of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. The authors concluded that although some sera positive for anti-RNAP I/III antibodies stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.

Yamasaki Y, Honkanen-Scott M, Hernandez L, et al. Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies. Arthritis Rheum. 2006;54:3051–3056.

Reprints: Dr. Minoru Satoh, Division of Rheumatology and Clinical Immunology, University of Florida, P.O. Box 100221, Gainesville, FL 32610-0221;
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Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.