College of American Pathologists
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  Clinical Abstracts





March 2011

Michael Bissell, MD, PhD, MPH

Prospective study of blood culture-negative endocarditis Prospective study of blood culture-negative endocarditis

Blood culture-negative endocarditis—that is, endocarditis in which no causative microorganism can be grown in a culture taken from a blood sample using the usual laboratory methods—accounts for 2.5 to 31 percent of all cases of endocarditis. This variation in incidence may be explained by several factors, including differences in the diagnostic criteria used; specific epidemiological factors, such as fastidious zoo-notic agents; variations in the early use of antibiotics prior to blood sampling; differences in sampling strategies; and involvement of unknown pathogens. The authors have diversified the diagnostic tests used in their laboratory over the past few years for the diagnosis of blood culture-negative endocarditis (BCNE). In particular, they have demonstrated the usefulness of systematic serological testing for detecting fastidious agents, particularly Coxiella burnetii and Bartonella species, but also Brucella species, Legionella pneumophila, and Mycoplasma species. Two other methods that exhibited great potential for diagnosing BCNE were histological examination and broad-range polymerase chain reaction (PCR), particularly when applied to valvular biopsies. The few studies that use PCR for the diagnosis of BCNE have highlighted the importance of streptococci and fastidious bacteria, although their respective prevalences have not been estimated. In addition, among fastidious bacteria, the role of Chlamydia pneumoniae in BCNE remains uncertain, as is the case for viruses. Consequently, diagnosing BCNE remains a challenge, as highlighted by the 21 percent rate of cases without any identified causative agent in the largest series of BCNE cases reported. Since the authors’ previous publication of a large series of BCNE cases, their laboratory has received an increasing number of specimens from patients with BCNE. The authors prospectively used a multimodal strategy incorporating serological, molecular, and histopathological assays to investigate specimens from 819 patients suspected of having BCNE. Diagnosis of endocarditis was eliminated for 60 patients. Among 759 patients with BCNE, a causative microorganism was identified in 62.7 percent and a noninfective etiology in 2.5 percent. Blood was the most useful specimen, providing a diagnosis for 47.7 percent of patients by serological analysis (mainly Q fever and Bartonella infections). Broad-range PCR of blood and Bartonella-specific Western blot methods diagnosed seven additional cases. PCR of valvular biopsies identified 109 more etiologies, mostly streptococci, Tropheryma whipplei, Bartonella species, and fungi. Primer extension enrichment reaction and autoimmunohistochemistry identified a microorganism in five additional patients. The authors did not detect virus or Chlamydia species. A noninfective cause of endocarditis, in particular neoplastic or autoimmune disease, was determined by histological analysis or by searching for antinuclear antibodies in 19 (2.5 percent) of the patients. The authors’ diagnostic strategy proved useful and sensitive for BCNE workup. The authors concluded that their study highlighted the major role of zoonotic agents and the underestimated role of noninfective diseases in BCNE. They propose serological analysis for Coxiella burnetii and Bartonella species and detection of antinuclear antibodies and rheumatoid factor as first-line tests, followed by specific PCR assays for T. whipplei, Bartonella species, and fungi in blood. Broad-spectrum 16S and 18S ribosomal RNA PCR may be performed on valvular biopsies when available.

Fournier P-E, Thuny F, Richet H, et al. Comprehensive diagnostic strategy for blood culture-negative endocarditis: a prospective study of 819 new cases. Clin Infect Dis. 2010;51:131–140.

Correspondence: Dr. Didier Raoult at

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Vitamin D and androgen level Vitamin D and androgen level

Vitamin D status is primarily determined by ultraviolet-B–induced vitamin D production in the skin, with vitamin D intake via food and supplements playing only a minor role. Vitamin D from either source is hydroxylated in the liver to 25-hydroxy-vitamin D (25[OH]D), which is used to determine a patient’s vitamin D status. The 25(OH)D is further hydroxylated to its active form, 1,25-dihydroxy-vitamin D (1,25[OH]2D). Serum levels of 1,25(OH)2D are mainly determined by renal 1,25(OH)2D production, which is catalyzed by 1α-hydroxylase. Biological actions of vitamin D are mediated through the vitamin D receptor (VDR), which regulates about three percent of the human genome. The VDR is almost ubiquitously expressed in human cells, which supports the clinical significance of the vitamin D endocrine system. There is great interest in vitamin D because poor vitamin D status is common and has been associated with an increased risk of various chronic diseases, including cancer, diabetes, hypertension, autoimmune diseases, musculoskeletal diseases, and cardiovascular diseases, as well as all-cause mortality. Data on the association of vitamin D and gonadal function are sparse, but VDR expression has been observed in reproductive tissue, such as ovary, uterus, prostate, testis, and sperm. VDR knockout mice have significant gonadal insufficiency, decreased sperm count and motility, and histological abnormalities of testis. Moreover, there is distinct seasonal variation in testosterone levels, which appears to resemble the seasonal variation in 25(OH)D levels. The latter is a consequence of seasonal differences in sunlight-induced vitamin D production in the skin. However, the association between vitamin D status and androgens has not been studied in detail. Therefore, it remains unclear whether similar seasonal distributions of 25(OH)D and androgen levels exist within a given study population. To this end, the authors conducted a cross-sectional study to investigate the association of 25(OH)D levels with testosterone, sex hormone-binding globulin (SHBG), and free androgen index (FAI) and to examine the seasonal variation of 25(OH)D, testosterone, FAI, and SHBG levels. They assessed 25(OH)D, testosterone, and SHBG levels by immunoassay in 2,299 men who were routinely referred for coronary angiography between 1997 and 2000. The main outcome measures were associations of 25(OH)D levels with testosterone, SHBG, and FAI. FAI was calculated as testosterone (nmol/L) divided by SHBG (nmol/L) times 100. The authors found that men with sufficient 25(OH)D levels (≥30 µg/L) had significantly higher levels of testosterone and FAI and significantly lower levels of SHBG when compared with 25 (OH)D-insufficient (20 to 29.9 µg/L) and 25(OH)D-deficient (<20 µg/L) men (P<0.05 for all). In linear regression analyses adjusted for possible confounders, the authors found significant associations of 25(OH)D levels with testosterone, FAI, and SHBG levels (P<0.05 for all). The 25(OH)D, testosterone, and FAI levels followed a similar seasonal pattern, with a nadir in March (12.2 µg/L, 15.9 nmol/L, and 40.8, respectively) and peak levels in August (23.4 µg/L, 18.7 nmol/L, and 49.7, respectively; P<0.05 for all). The authors concluded that androgen levels and 25(OH)D levels are associated in men and reveal a concordant seasonal variation. Randomized controlled trials are warranted to evaluate the effect of vitamin D supplementation on androgen levels.

Wehr E, Pilz S, Boehm BO, et al. Association of vitamin D status with serum androgen levels in men. Clin Endocrinol. 2010;73:243–248.

Correspondence: Dr. Winifred Marz at

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Decreased CD20 expression in B-cell lymphomas Decreased CD20 expression in B-cell lymphomas

Mature B-cell lymphomas with dual translocations involving c-MYC and, typically, BCL2 or, alternatively, BCL6 are a subset of lymphomas that commonly show aggressive clinical behavior. These tumors typically appear intermediate or high grade on morphologic review. However, in some cases, tumors with an appearance compatible with a low-grade lymphoma or plasmablastic myeloma have also been described. These neoplasms may show features of Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL) in immunohistochemical studies but may also occasionally show features intermediate between the two. Despite the variable morphologic appearance and immunophenotype of these lymphomas, a commonality of these neoplasms is the identification of dual translocations involving c-MYC and, typically, BCL2 or BCL6 in these lesions that, together, lead to dual oncogenic stimuli resulting in biologically aggressive behavior. These translocations are thought to lead to increased proliferation (c-MYC and BCL6) or reduced apoptosis (BCL2), thereby driving aggressive growth characteristics. Because dual-translocation tumors commonly are more aggressive than their morphologic and immunohistochemical counterparts that lack dual translocations, identifying these tumors may be important for patient prognostication and therapy. Flow cytometry is a rapid and routine method used in many clinical laboratories to characterize leukocyte antigen expression in complex mixtures, such as those derived from peripheral blood, bone marrow, and tissue samples. Recent data have suggested that a significant proportion of mature B-cell lymphomas with c-MYC translocations may show a common immunophenotype, including increased expression of CD38, as assessed by immunohistochemical analysis or flow cytometry. Therefore, the authors sought to determine whether there may be a common immunophenotype of dual-hit, aggressive B-cell lymphomas that might identify these cases for cytogenetic analysis because this testing might not otherwise be routinely performed. To this end, they conducted a study at the University of Washington Medical Center, Seattle. The authors identified a cohort of B-cell lymphomas in their databases and retrospectively reviewed corresponding in-house flow cytometric data to determine whether a common immunophenotype might be present. They reported findings on 10 lymphomas, each with translocations involving c-MYC and BCL2 or BCL6, and showed that these cases frequently exhibit a common immunophenotype that includes decreased expression of CD20. The authors concluded that because these lymphomas often show aggressive biologic behavior and poor clinical outcome, recognizing this relatively common immunophenotype may be useful for identifying cases for confirmatory cytogenetic studies since flow cytometry is often used first to assess these clinical specimens.

Wu D, Wood BL, Dorer R, et al. “Double-hit” mature B-cell lymphomas show a common immunophenotype by flow cytometry that includes decreased CD20 expression. Am J Clin Pathol. 2010;134:258–265.

Correspondence: Dr. David Wu, University of Washington Medical Center, Seattle Cancer Care Alliance, Campus Box 358081, 825 Eastlake Ave. E, Seattle, WA 98109

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Twenty-four-hour urine collections for kidney stones Twenty-four-hour urine collections for kidney stones

Routine laboratory 24-hour urine collections are part of the metabolic evaluation for patients with urolithiasis. Several groups have reported that a single 24-hour urinalysis may be sufficient to assess the risk of nephrolithiasis. However, others have suggested that two 24-hour urinalyses may yield a greater number of specific diagnoses, so two are needed for metabolic evaluation. The issue of whether one or two 24-hour urinalyses are sufficient has important implications for patient care and experience since eliminating the repeat 24-hour collection could lead to significant cost savings and decrease the time spent by patient and physician. It would also increase confidence in the results of a single 24-hour urine sample. The authors evaluated the variability of repeat 24-hour urinalyses in a large cohort of patients who presented for metabolic evaluation of urolithiasis. They hypothesized that there is minimal variation between 24-hour urine samples collected within three days of each other and that a single 24-hour urinalysis is sufficient. The authors analyzed two 24-hour urine collections from 777 patients, which were obtained between 2001 and 2005. The samples were collected no more than three days apart and before pharmacological intervention. They were analyzed elsewhere for the routine stone risk profiles of urine calcium, oxalate, citrate, uric acid, sodium, potassium, magnesium, phosphorus, ammonium, chloride, urea nitrogen, and creatinine. The authors found that no parameters showed a statistically significant difference between 24-hour urine samples one and two when the mean values were compared (pairwise t test, P>0.05; range, 0.06–0.87). Using Pearson’s correlation, all parameters showed positive correlation coefficients (r=0.68–0.89; each, P<0.0001). The mean of individual patient differences in samples one and two were compared to zero, and no differences were noted when comparing 24-hour urinary calcium, citrate, potassium, phosphorus, ammonium, and creatinine (P>0.05). However, significant differences were noted when comparing urinary oxalate, uric acid, sodium, magnesium, chloride, and urea nitrogen (each, P<0.05). The difference was 0.5 to 4.19 percent for all urinary parameters. The authors concluded that one 24-hour urine sample is sufficient for metabolic evaluation of recurrent stone disease. There is no significant difference in 12 urinary parameters between 24-hour urine samples collected within three days of each other. This information may help providers and may decrease patient inconvenience and the overall cost of metabolic stone evaluation.

Castle SM, Cooperberg MR, Sadetsky N, et al. Adequacy of a single 24-hour urine collection for metabolic evaluation of recurrent nephrolithiasis. J Urol. 2010;184:579–583.

Correspondence: Marshall L. Stoller at

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Calcitonin serum concentration after thyroid surgery Calcitonin serum concentration after thyroid surgery

Medullary thyroid carcinoma originates from the parafollicular cells of the thyroid. These cells produce calcitonin, which is a highly sensitive and specific marker of medullary thyroid carcinoma (MTC) for diagnosis and followup. The definitive cure for the tumor depends on the completeness of the first surgical treatment. If total thyroidectomy and central neck lymph node dissection are performed in all cases of MTC, it is a matter of debate whether to perform lateral lymph node dissection in any case or to limit this radical approach to only patients with metastases in the central neck compartment or palpable lateral neck lymph nodes. For this purpose, one should consider that the risk of metastases in the contralateral neck compartment and the mediastinum depends on the size of the primary tumor, number of central lymph node metastases, and tumor multicentricity. If the first surgical treatment is so crucial for the final outcome of the patient, on the other hand, there are no clear criteria that can indicate to the surgeon whether the tumor has been completely removed after total thyroidectomy plus central lymph node dissection. In a recent study evaluating the usefulness of the intraoperative pentagastrin (PG) test in predicting lymph node involvement in patients with MTC who underwent total thyroidectomy plus central lymph node dissection, 80 percent of subjects were correctly recognized to have postoperative tumor remnants. The procedure failed to detect tumor remnant in 20 percent of uncured subjects. The authors conducted their own study to evaluate the reliability of this intraoperative procedure in predicting the completeness of surgery. They monitored intraoperative calcitonin levels in patients who were recommended for surgery based on the finding of high basal and PG-stimulated calcitonin levels consistent with a diagnosis of MTC. Twenty patients underwent total thyroidectomy and central lymph node dissection on the basis of a positive PG test. A preoperative diagnosis of MTC was achieved on cytological examination in six cases. During the surgical intervention, calcitonin was measured at the time of anesthesia, time of manipulation, and 10 and 30 minutes after surgical excision. At the histological examination, 10 patients had MTC and 10 had parafollicular cell (C cell) hyperplasia. The authors found that when compared with calcitonin levels before thyroidectomy, a decrease of calcitonin greater than 50 percent at 30 minutes after surgery was able to significantly distinguish patients who were cured from those who were not. It was not possible to find a similar result when considering the decrease in calcitonin 10 minutes after surgery. The authors concluded that a calcitonin decrease of less than 50 percent 30 minutes after thyroidectomy with central neck lymph node dissection suggests that tumor tissue persists in patients who underwent surgery for MTC. These results indicate that intraoperative calcitonin monitoring may be a useful tool to predict the completeness of surgery in patients with MTC.

Faggiano A, Milone F, Ramundo V, et al. A decrease of calcitonin serum concentrations less than 50 percent 30 minutes after thyroid surgery suggests incomplete C-cell tumor tissue removal. J Clin Endocrinol Metab. 2010;95:E32–E36.

Correspondence: Dr. Antongiulio Faggiano at or

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