College of American Pathologists
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  Clinical Abstracts





October 2011

Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.

False-negative PCR results in Neisseria meningitidis False-negative PCR results in Neisseria meningitidis

Bacterial meningitis is life threatening, and rapid treatment is mandatory. Numerous culture-negative meningococcal disease cases have been observed in many countries due to the increasing use of preadmission antibiotics. Therefore, detection of meningococcal DNA by polymerase chain reaction is widely used for patients with suspected meningococcal meningitis and negative cerebrospinal fluid (CSF) cultures. Multiple nucleotide substitutions in two isolates of Neisseria meningitidis serogroup C have been reported to cause false-negative detection, with a real-time PCR targeting the ctrA gene. The authors encountered a similar problem with a clinical isolate of N. meningitidisserogroup B not detected by their real-time PCR targeting the ctrA gene. For five years, the authors have been using meningococcal DNA detection by PCR for patients with suspected meningococcal meningitis and negative CSF cultures, as PCR increases the number of confirmed cases. Before introducing it in their diagnostic laboratory, this real-time PCR was evaluated on 37 CSF samples (11 positive for N. meningitidis,10 positive for Streptococcus pneumoniae, three positive for Listeria monocytogenes, one positive for Haemophilus influenzae, six positive for other bacteria, and six negative for any bacteria) with a specificity and sensitivity of 100 percent. Then, from 2005 to July 2010, the authors used PCR to examine 419 CSF samples that were received with a diagnosis of meningitis. During these five years, five samples were PCR and culture positive for N. meningitidis,and 17 were positive by PCR but negative by culture. Among the remaining 397 PCR-negative CSF samples, one specimen was documented as being falsely negative with the N. meningitidis ctrA PCR. The patient, a two-year-old boy, presented to the hospital with meningitis and petechiae. He was already being treated with amoxicillin per os. The patient underwent blood cultures, and ceftriaxone and dexamethasone were administered intravenously before CSF puncture. Gram-negative diplococci were seen on Gram staining of CSF, but the culture remained sterile. The real-time PCR was done on CSF and, surprisingly, was negative. Then a broad-spectrum 16S RNA PCR was performed, and N. meningitidisDNA was detected in the CSF. Meanwhile, the blood cultures became positive for N. meningitidisserogroup B susceptible tob-lactams. The clinical progression was favorable, and the child was sent home after five days of hospitalization. The authors concluded that isolates with polymorphism or closely related isolates may generate false-negative or false-positive PCR results, especially when a single gene is targeted. Targeting at least two different genes may eliminate this risk. Microbiologists should be aware of this problem and alert the scientific community when such events occur.

Jaton K, Ninet B, Bille J, et al. False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis. J Clin Microbiol. 2010;48:4590–4591.

Correspondence: Katia Jaton at

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Biochemistry of heart failure Biochemistry of heart failure

The failing heart is subject to elevated metabolic demands, adverse remodeling, chronic apoptosis, and ventricular dysfunction. The interplay among such pathologic changes is largely unknown. Several laboratories have identified a unique post-translational modification that may have significant effects on cardiovascular function. The O-linked b-N-ace-tylglucosamine (O-GlcNAc) post-translational modification (O-GlcNAcylation) integrates glucose metabolism with intracellular protein activity and localization. Because O-GlcNAc is derived from glucose, the authors hypothesized that altered O-GlcNA-cylation would occur during heart failure and figure prominently in its pathophysiology. They conducted a study in which they found that after five days of coronary ligation in WT mice, cardiac O-GlcNAc transferase (OGT; which adds O-GlcNAc to proteins) and levels of O-GlcNAcylation were significantly elevated (P<0.05) in the surviving remote myocardium. The authors used inducible, cardiac myocyte-specific Cre recombinase transgenic mice crossed with loxP-flanked OGT mice to genetically delete cardiomyocyte OGT (cmOGT KO) and ascertain its role in the failing heart. After tamoxifen induction, cardiac O-GlcNAcylation of proteins and OGT levels were significantly reduced compared with WT, but not in other tissues. WT and cardiomyocyte OGT KO mice underwent nonreperfused coronary ligation and were followed for four weeks. Although OGT deletion caused no functional change in mice that underwent sham surgery, OGT deletion in infarcted mice significantly exacerbated cardiac dysfunction compared with WT. The authors concluded that these data provide keen insights into the pathophysiology of the failing heart and illuminate a previously unrecognized point of integration between metabolism and cardiac function in the failing heart.

Watson LJ, Facundo HT, Ngoh GA, et al. O-linked b-N-acetylglucosamine transferase is indispensable in the failing heart. Proc Natl Acad Sci USA. 2010;107:17797–17802.

Correspondence: Steven P. Jones at

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PSA doubling time after radical prostatectomy PSA doubling time after radical prostatectomy

Many reports in recent years have explored the correlation between post-radical prostatectomy prostate-specific antigen doubling time (PSADT) and various prostate cancer endpoints. These have shown that the PSADT predicts prostate cancer-specific mortality (PCSM). Other reports have found that a short post-radical prostatectomy PSADT portends a poor prognosis and response to salvage treatment. However, many of these observations were in populations of relatively young, racially homogenous men from tertiary care centers. Therefore, these men likely had fewer competing mortality causes and medical co-morbidities than men from equal-access centers, such as Veterans Affairs hospitals. Only one study has examined the correlation between PSADT and PCSM in an older, more racially diverse cohort. That study compared men with a PSADT of less than three months with men with a PSADT longer than three months and found that the men with the shorter PSADT had an increased risk of PCSM. However, the study did not examine the correlation between PSADT and PCSM among men with intermediate PSADT values — that is, three to 15 months. Moreover, despite widespread acceptance that PSADT after radical prostatectomy predicts PCSM, the degree to which it correlates with overall survival has been understudied. One study has examined this issue among men with intermediate PSADTs. Although it found that PSADT predicted overall survival, the cohort was a relatively young, healthy patient population. Therefore, it remains untested whether more intermediate PSADTs will predict overall survival in older, more racially diverse populations with more medical co-morbidities and greater competing mortality. The authors examined the correlation between PSADT and overall survival among men in the SEARCH database, a racially diverse cohort treated with radical prostatectomy at multiple VA hospitals nationwide. In a secondary analysis, they examined the correlation between PSADT and PCSM in the same cohort. The authors performed a Cox proportional hazards analysis to examine the correlation between postrecurrence PSADT and the interval from recurrence to overall survival and prostate cancer-specific mortality among 345 men in the SEARCH database who had undergone radical prostatectomy from 1988 to 2008. They examined the PSADT as a categorical variable using the clinically significant cutpoints of less than three months, three to 8.9 months, nine to 14.9 months, and longer than 15 months. The authors found that a PSADT of less than three months (hazard ratio, 5.48; P=0.002) was associated with poorer overall survival than a PSADT of 15 or more months. A trend was seen toward worse overall survival for the men with a PSADT of three to 8.9 months (hazard ratio, 1.70; P=0.07). PSADTs of less than three months (P<0.001) and three to 8.9 months (P=0.004) were associated with an increased risk of prostate cancer-specific mortality. The authors concluded that in an older, racially diverse cohort, recurrence with a PSADT of less than nine months was associated with worse all-cause mortality. The results of this study have validated previous findings that PSADT is a useful tool for identifying men at increased risk of all-cause mortality early in their disease course.

Teeter AE, Presti JC, Aronson WJ, et al. Does PSADT after radical prostatectomy correlate with overall survival? A report from the SEARCH database group. Urology. 2011;77:149–153.

Correspondence: Stephen J. Freedland at

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Monitoring creatinine and urinary gamma-glutamyl transpeptidase in kidney injury Monitoring creatinine and urinary gamma-glutamyl transpeptidase in kidney injury

Acute kidney injury is a common condition in intensive care units and has been associated with increased mortality. Early detection of renal impairment may improve outcome. In clinical practice, renal dysfunction may be identified by plasma creatinine alterations. The glomerular filtration rate and plasma creatinine concentrations are linked according to a converse hyperbolic relationship. However, plasma creatinine is less than optimal and tends to focus attention on later stages of injury, when therapies may be less effective. Therefore, more specific and sensitive markers are required for detecting acute kidney injury (AKI) in the early stages. AKI can be attributed to acute tubular necrosis or glomerular dysfunction. Plasma creatinine is a global marker of renal impairment. In hypoxic or ischemic events, although proximal and distal tubule injuries occur, the proximal injury seems prevalent. Detecting specific proteins released from damaged tubular cells can provide the opportunity to better investigate the renal function of patients with AKI. Among these proteins, the excretion of g-glutamyl transpeptidase (g-GT) in urine may reflect injury to the brush border membrane with loss of microvillous structure. Increased excretion of enzyme in urine appears to be a good marker for tubular injury. This has been shown in aminoglycoside-induced acute tubular necrosis in animal models and humans. Urinary b-2-microglobulin and N-acetyl-b-D-glucosaminidase can detect early renal tubular dysfunction in sick neonates. The wide availability of automated assays and low cost of urinary enzymes are beneficial. A previous study showed that the combined assessment of urinary g-GT and urinary alkaline phosphatase is a reliable, easy, and low-cost method to detect critically ill patients at high risk for AKI. However, this finding was based on a study that included 26 patients, only four of whom developed AKI. The authors hypothesized that, as compared with conventional methods, determining urinary g-GT may improve the early detection of patients with AKI. In an observational study, they sought to determine how to detect AKI at ICU admission by combining plasma creatinine and urinary g-GT. To determine acute kidney injury, the authors subtracted measured creatinine clearance from theoretical creatinine clearance, with a 25 percent reduction signifying AKI. Its incidence in 100 consecutive patients was 36 percent. An indexed urinary g-glutamyl transpeptidase-to-urinary creatinine ratio was significantly increased in patients with AKI and did not correlate with plasma creatinine (P=0.3). Using a predefined threshold of indexed urinary g-glutamyl transpeptidase-to-urinary creatinine ratio (greater than 12.4 U/mmoL) and plasma creatinine (greater than 89 µmol/L), the ability to detect acute kidney injury was significantly improved, making it possible to detect 22 (22 percent) additional patients with AKI. This finding was confirmed in the validation group. The rates of false-positive results were 30 percent and 19 percent in the data development and internal validation cohorts, respectively. The authors concluded that the use of such low-cost, widely available markers as creatinine and urinary g-glutamyl transpeptidase increases detection of AKI. Additional studies are needed to determine the impact of these biomarkers on outcome.

Blasco V, Wiramus S, Textoris J, et al. Monitoring of plasma creatinine and urinary g-glutamyl transpeptidase improves detection of acute kidney injury by more than 20 percent. Crit Care Med. 2011;39:52–56.

Correspondence: Dr. Marc Leone at

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CA 125 during chemotherapy for endometrial cancer CA 125 during chemotherapy for endometrial cancer

The tumor marker CA 125 has been studied extensively as a prognostic/predictive indicator of newly diagnosed epithelial ovarian cancer. These studies consistently found that higher preoperative values predicted a worse outcome on univariate analysis, but this effect was lost on multivariate analysis, except, possibly, for stage one disease. In contrast, an independent predictive effect has been found for changes in CA 125 levels at various time points in the treatment phase, as well as for CA 125 half-life and nadir CA 125 levels. The utility of CA 125 in endometrial cancer has been less well studied, and its role is not fully defined. The studies consistently found that levels rose with increasing grade, stage, depth of myometrial invasion, presence of lymphovascular invasion, and lymph node involvement—that is, known adverse factors. Elevated preoperative levels, greater than the upper limit of normal of 35 kU/mL, were also predictors of decreased survival in univariate and multivariate analyses. However, these studies predominantly were conducted with women who had early stage disease, the vast majority of whom would be cured by surgery alone. The decision of whether to use additional nonsurgical therapy in such women is determined from surgical staging and pathologic review. Routine preoperative CA 125 evaluation then would only be worthwhile if elevated levels led to a more stringent surgical evaluation or if CA 125 elevation in the absence of the other known adverse factors were prognostic and, therefore, indicated a need for additional treatment. Only in two studies of apparent early stage cancers was a multivariate analysis performed, and there were conflicting results with regard to the independent value of preoperative CA 125. However, in those with extra uterine disease, CA 125 values may be prognostic preoperatively and postoperatively. Unlike with ovarian cancer, the role of CA 125 monitoring in patients with endometrial cancer receiving chemotherapy has not been rigorously studied. One study that reported on chemotherapy-treated stage III endometrial cancer patients showed that an elevated preoperative CA 125 value was a predictor of worse survival. However, the vast majority of patients did not receive chemotherapy in this study. Therefore, the authors conducted a retrospective study to analyze the role of CA 125 as an outcome predictor for women receiving carboplatin-paclitaxel for advanced endometrial cancer. The review focused on women receiving carboplatin and paclitaxel for advanced endometrial cancer at any of the institutions of the British Columbia Cancer Agency between September 1995 and September 2006. For the study, 185 women newly diagnosed with endometrial cancer were treated with carboplatin and paclitaxel every three to four weeks. Univariate analysis for progression-free survival identified adverse predictors: grade three, positive residual, age older than 60 years, deep myometrial invasion, increasing stage/substage, papillary serous subtype, presence of cervical involvement, Eastern Cooperative Oncology Group score of one or greater, CA 125 above 35 preoperatively or at the start of cycle one, and CA 125 greater than 24 at the start of cycle three. On multivariate analysis, CA 125 above 24 at cycle three, grade three, and positive residual remained independent predictors. The single most important factor identified by decision tree analysis was CA 125 level at cycle three. The authors concluded that, as with epithelial ovarian cancer, changes in CA 125 are highly predictive of outcome for advanced chemotherapy-treated endometrial cancer.

Hoskins PJ, Le N, Correa R. CA 125 normalization with chemotherapy is independently predictive of survival in advanced endometrial cancer. Gyn Oncol. 2011;120:52–55.

Correspondence: P. J. Hoskins at

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Tacrolimus monitoring after liver transplantation Tacrolimus monitoring after liver transplantation

Elevated blood concentrations of tacrolimus are associated with renal toxicity among organ transplantation patients. Because tacrolimus shows strong interactions with other drugs metabolized by cytochrome P450, therapeutic drug monitoring (TDM) is important, especially in liver transplant patients. The microparticle enzyme immunoassay (MEIA, Abbott Laboratories) is popular, but it requires a manual pretreatment step consisting of precipitation of erythrocytes and proteins using methanol, followed by centrifugation. Problems reported with the MEIA include interference with tacrolimus metabolites and impact of hematocrit. Dade Behring recently developed a no-pretreatment monoclonal antibody-based immunoassay, the affinity column-mediated immunoassay (ACMIA), which uses the Dimension RxL HM module to measure tacrolimus. In the early post-liver transplantation period, many recipients exhibit hematologic, renal, or hepatic instability that might affect tacrolimus concentrations. Therefore, the authors compared the results of these two immunoassay systems relative to hematologic and biochemical renal and hepatic function values at two weeks after liver transplantation. They obtained 256 blood samples from 35 patients at two weeks after liver transplantation, excluding those samples from patients who were treated with interacting medications or renal replacement therapy. The authors also excluded mortality cases within two weeks of liver transplantation. A Dimension RxL HM with the tacrolimus Flex reagent cartilage was used for the ACMIA, and the IMx tacrolimus II was used for the MEIA method. The authors found that the tacrolimus concentrations measured by the ACMIA method correlated closely with those measured by the MEIA method (r=0.953). However, the weighted concordance correlation coefficient for the repeated-measurement design was 0.74 (95 percent confidence interval, 0.66–0.85). The discrepancies in tacrolimus level between the two methods was large among samples with low tacrolimus concentrations, especially at less than 5 ng/mL. When the difference ratio of the two methods ([ACMIA-MEIA]/ACMIA) was analyzed with a linear mixed-effects model to identify significant laboratory findings, no significant differences were found based on hematocrit, renal function, or hepatic function. However, the serum potassium level correlated with the difference ratio of the two methods (estimated slope, 10.173; P=0.02). The authors concluded that both the ACMIA and the MEIA methods are precise. However, the ACMIA method has the advantage of fewer pretreatment procedures. In the early liver transplant period, a difference was noted between the serum tacrolimus concentrations measured by the two methods, especially at low drug concentrations.

Joo DJ, Jung I, Kim MS, et al. Comparison of the affinity column-mediated immunoassay and microparticle enzyme immunoassay methods as a tacrolimus concentration assay in the early period after liver transplantation. Transplant Proceedings. 2010;42:4137–4140.

Correspondence: Myoung Soo Kim at

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Diagnosing neonatal sepsis Diagnosing neonatal sepsis

Neonatal sepsis is a chief cause of neonatal mortality. The need for early diagnosis has led many investigators to discover rapid diagnostic tests, and combinations of these tests have been described in the literature as sepsis screens. A large number of studies have been conducted over the last three decades to evaluate the diagnostic value of sepsis screens, but doubts still remain regarding their clinical utility. One of the most commonly used sepsis screens consists of C-reactive protein (CRP), various hematologic parameters, and microerythrocyte sedimentation rate (mESR), which are referred to here as the standard sepsis screen. Among the reasons for widespread variability in the performance of the sepsis screen, the one most frequently implicated is lack of uniform inclusion criteria, especially with regard to severity of underlying illness. However, there is a paucity of published data to substantiate this assertion. Most studies do not define objective severity criteria, such as Score for Neonatal Acute Physiology (SNAP) scores, for their patient populations. Early onset sepsis (EOS) accounts for 67 percent of all episodes among hospitalized neonates. The authors hypothesized that the severity of underlying sickness may alter the interpretation of the sepsis screen for diagnosing EOS, and they conducted a prospective cross-sectional study to evaluate this hypothesis. They enrolled in their study consecutive neonates with clinically suspected EOS and performed blood culture and sepsis screen (CRP, absolute neutrophil count, immature-to-total ratio, and microerythrocyte sedimentation rate). Exclusion criteria were prior antibiotic exposure, nonavailable reports, and contaminated cultures. Score for Neonatal Acute Physiology Perinatal Extension (SNAPPE-II) was used to categorize neonates into mild to moderate (score, 40 or less) and severe (score, 40 or more) illness. Sepsis was defined as positive blood culture, and positive screen as two or more parameters positive. Of 125 subjects, 86 had mild to moderate illness and 39 had severe illness. Twenty-eight (22 percent) subjects had sepsis. Sensitivity, specificity, negative predictive value, positive predictive value, likelihood ratio of positive test, and likelihood ratio of negative test of the sepsis screen and screen parameters were similar between the two groups. The sensitivity of the screen was 37.5 percent and 25 percent for mild to moderate illness and severe illness, respectively. Only immature- to-total ratio values correlated with SNAPPE-II scores in patients with sepsis (Spearman’s rho, 0.4; P=0.036). The authors concluded that the severity of underlying illness does not alter the performance of the sepsis screen in diagnosing culture-positive EOS.

Mahale R, Dutta S, Ahluwalia J, et al. Baseline illness severity does not alter accuracy of neonatal sepsis screen. Am J Perinatol. 2010;27:327–332.

Correspondence: Dr. Sourabh Dutta at

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CA 125 immune complexes in ovarian cancer patients with low CA 125 CA 125 immune complexes in ovarian cancer patients with low CA 125

Clinical CA 125 concentrations are increased to more than 35 kU/L in about 80 percent of women with epithelial ovarian cancer. They are correlated directly with disease stage and inversely with survival. The kinetics of this marker during chemotherapy predict disease status and survival, and concentrations after therapy are used to monitor disease recurrence. Use of CA 125 has been suggested for distinguishing benign from malignant pelvic masses and, in combination with ultrasound, as a potential screening tool for ovarian cancer. Therefore, the ovarian cancer case with low CA 125 concentrations is problematic. About 20 percent of women with ovarian cancer have low CA 125 concentrations, and this important tumor marker cannot be used to monitor their disease. The measured concentration for mucin 1 (MUC1), or the tumor marker CA 15-3, can be lowered in breast and ovarian cancer patients when circulating immune complexes containing antibodies bound to the free antigen are present. Because CA 125 and MUC1 are related members of the mucin family, the authors sought to determine whether circulating immune complexes might also exist for CA 125 and interfere with its clinical assay. They developed an antigen capture-based assay to determine the presence of circulating immune complexes for CA 125. They put mouse antibodies to CA 125 onto nanoparticle slides, incubated them with patient serum, and added Cy5-tagged goat antihuman IgG antibodies. Fluorescence intensities were read and normalized to the intensities for glutathione S-transferase A1 as a control. The authors found that assay results for 23 ovarian cancer cases with high CA 125 concentrations, 43 cases with low CA 125 concentrations, and 19 controls (mean CA 125 concentrations, 2,706, 23, and 11 kU/L, respectively) revealed mean fluorescence intensities for CA 125 circulating immune complexes of 2.30, 2.72, and 1.99 intensity units, respectively. A generalized linear model adjusted for batch and age showed higher CA 125 circulating immune complex fluorescence intensities in low CA 125 cases than in high CA 125 cases (P=0.03) and controls (P=0.0005). Four ovarian cancer patients who had recurrent disease and always had low CA 125 values had a mean CA 125 circulating immune complex value of 3.06 intensity units (95 percent confidence interval, 2.34–4.01 intensity units). The authors concluded that these preliminary results suggest the existence of circulating immune complexes involving CA 125, which may help explain some ovarian cancer cases with low CA 125 concentrations.

Cramer DW, O’Rourke DJ, Vitonis AF, et al. CA 125 immune complexes in ovarian cancer patients with low CA 125 concentrations. Clin Chem. 2010;56:1889–1892.

Correspondence: Daniel W. Cramer at dcra

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