College of American Pathologists

  Clinical Abstracts





September 2010

Michael Bissell, MD, PhD, MPH

Utility of genital Gram stains in the emergency department Utility of genital Gram stains in the emergency department

More than 19 million sexually transmitted diseases are diagnosed in the United States annually, and it is estimated that more than half of all Americans will acquire an STD at some point in their lifetime. Estimates from the 1992 to 1994 National Hospital Ambulatory Medical Care Survey indicate that women of reproductive age make 1.4 million visits to the emergency department per year for gynecologic reasons, with almost half relating to infectious diseases, including STDs, lower genital tract infection, and pelvic inflammatory disease. More than $8 billion annually is spent in the United States to diagnose and treat STDs and their complications. Testing and treatment of STDs often occurs in the emergency department, which serves as a medical safety net for underserved patient populations. Many patients at highest risk for STDs will present to the emergency department for care. When facing lengthy differentials for complaints such as pelvic or abdominal pain, emergency medicine physicians require rapid, inexpensive, and effective laboratory testing for accurate and timely diagnosis. The Gram stain is a rapid and economical test that historically has been used to diagnose the etiology of suspected undifferentiated genital infections. Although the genital Gram stain has not been studied extensively within the setting of an emergency department, research has been done to ascertain its utility in other settings. Several studies have shown conflicting results regarding diagnostic effectiveness. The authors conducted a study to gauge the usefulness of the genital Gram stain in an emergency department population versus the current gold standard, the DNA probe. A linked query of an urban, tertiary care, university-affiliated hospital laboratory database was conducted for all completed Chlamydia trachomatis and Neisseria gonorrhoeae DNA probes, Trichomonas vaginalis wet preps, and genital Gram stains performed on patients during emergency department visits between January and December 2004. Positive criteria for a Gram stain included greater than 10 white blood cells per high-power field, gram-negative intracellular/extracellular diplococci (suggesting N. gonorrhoeae), clue cells (suggesting T. vaginalis), or direct visualization of T. vaginalis organisms. DNA probes were used as the gold standard definition for N. gonorrhoeae and C. trachomatis infection. The authors found that of 1,511 initially eligible emergency department visits, 941 were analyzed (genital Gram stain and DNA probe results both present), with a prevalence for C. trachomatis or N. gonorrhoeae of 11.4 percent. A positive genital Gram stain was 75.7 percent sensitive and 43.3 percent specific in diagnosing C. trachomatis or N. gonorrhoeae infection, or both, and 80.4 percent sensitive and 32.2 percent specific when the positive cutoff was lowered to more than five white blood cells per high-power field. No Gram stains were positive for T. vaginalis (with 47 positive wet mounts), and clue cells were noted on 117 Gram stains (11.6 percent). The authors concluded that Gram stains in isolation lack sufficient diagnostic ability to detect C. trachomatis or N. gonorrhoeae infection in the emergency department setting.

Stefanski P, Hafner W, Riley SL, et al. Diagnostic utility of the genital Gram stain in ED patients. Am J Emerg Med. 2010;28:13–18.

Correspondence: Dr. John W. Hafner at

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Crossover study of two plateletpheresis systems Crossover study of two plateletpheresis systems

Single-donor plateletpheresis products are preferred, especially for patients at high risk, such as hematology or oncology patients awaiting hemato-poietic progenitor cell transplantation or immunized patients. Apheresis systems based on different technical concepts and allowing single- or double-needle donations were developed by different manufacturers. For blood donation services, quality and donor comfort are the most important aspects of these products. Recently developed apheresis systems enable the simultaneous donation of platelet and plasma products and the production of double or even triple therapeutic platelet units from one donation. Total platelet count, residual white blood cell count, and absence of platelet aggregates are variables that contribute to product quality, while handling time for the entire procedure, loudness, and impact of the needle are variables that contribute to user friendliness and donor comfort. Because blood donation centers depend on the willingness of donors to give blood voluntarily and repeatedly, donor comfort is an essential criterion. The authors conducted a randomized crossover study to compare two manufacturers’ plateletpheresis cell separator systems based on product quality, number of platelet units per donation, and donor comfort. Forty-four females and 47 male donors were distributed to three body weight groups. Double platelet units with 6 x 1,011 platelets were collected from three Fenwal Amicus Crescendo (AC) and three CaridianBCT Trima Accel (TA) machines. Each donor made one donation on each randomly assigned system and answered a questionnaire about subjective donor comfort. The answers were scored from five (best) to one (worst). Based on 182 donations, with 91 donations each on the AC and TA separators, 179 runs resulted in double platelet units and three (2 x AC, 1 x TA) in single units. The white blood cell counts were below 1 x 106 in all but eight therapeutic units (8 x TA; mean, 1.98 x 106). The mean platelet yield (AC 6.00 x 1,011; TA 5.98 x 1,011), collection rate, and platelet extraction coefficient did not significantly differ between the two devices. Differences in donor comfort across all groups were only observed for the loudness of the instrument (4.63 AC versus 4.24 TA; P<0.001) and the subjective impression of the run time (4.24 AC versus 4.48 TA; P<0.05). Male donors weighing more than 88 kg preferred the TA instruments based on the criteria of impact of the needle, run time, overall experience (P<0.01 each), and willingness to donate on the same instrument again (P<0.05). Only minor differences were observed despite the fact that the AC separators are run with two needles and the TA with one needle.

Flesch BK, Adamzik I, Steppat D, et al. Paired crossover study of two plateletpheresis systems concerning platelet product quality and donor comfort. Transfusion. 2010;50:894–901.

Correspondence: Brigette Flesch at

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Detecting uropathologic strains of Escherichia coli Detecting uropathologic strains of Escherichia coli

Gram-negative rods are the major etiologic agents in urinary tract infections in humans, and Escherichia coli comprise most of these agents. In some cases, urinary tract infection (UTI) treatment is difficult because of persistent recurrences. Furthermore, UTIs often are asymptomatic at the beginning of the infection process. Particular phenotypic features of uropathogenic E. coli (UPEC) strains facilitate their persistence in urinary tracts and differentiate them from the other pathogenic and commensal E. coli strains. UPEC-specific virulence factors (VFs), which are mostly adhesins (P and S fimbriae), toxins (cytotoxic necrotizing factor type 1, α-hemolysin), bacteriocin (uropathogenic-specific protein), and siderophores (aerobactin and yersiniabactin), are important for colonizing the urinary tract. Type 1 fimbriae and afimbrial adhesin I are also beneficial in this type of infection. Furthermore, phylogenetic analyses have revealed that UPEC strains differ substantially from other E. coli strains. Pathogenic E. coli strains, including UPEC strains, belong primarily to groups B2 and D. In the case of E. coli, 16S rRNA gene-sequence analysis, phylogenetic studies, and VF profiles are valuable for detailed genetic identification. Polymerase chain reaction-based methods are efficient, inexpensive, and rapid. Two distinct prokaryotic repetitive elements have been used for gram-negative enterobacterial strain discrimination: repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. Because the ERIC-PCR band patterns were less complex than the REP-PCR band patterns, differences within the analyzed species were easier to distinguish with ERIC-PCR. The authors undertook the task of developing a novel genetic test, termed CGG-PCR, for differentiating and epidemiologically investigating UPEC strains and compared it to ERIC-PCR. For the comparison, the authors considered the following factors: cluster cutoff values, clustering capability with regard to virulence profiles, phylogenetic groups and quinolone susceptibility, reproducibility of band patterns, number of different profiles, and discriminatory indices. The authors’ assay was based on the presence in bacterial genomes of microsatellitestrinucleotide repeat sequences. Since the different trinucleotide repeat sequence elements vary in copy number and distribution in bacterial genomes, they may serve as valuable markers for phylogenetic and epidemiological studies. A (CGG)5 hybridization probe has been used successfully in conjunction with restriction fragment length polymorphism to type Mycobacterium tuberculosis. However, such hybridization techniques require isolating large amounts of genomic DNA and are time consuming and expensive. (GTG)5-PCR was also tested for its ability to track the origins of E. coli, Lactobacillus spp., and Enterococcus spp. isolated from various sources. The authors proposed an improved PCR methodology that employs an N6(CGG)4 primer with a high annealing temperature. Trinucleotide repeats are present on both DNA strands, allowing the authors to design a single PCR primer harboring the CGG motif that yields characteristic electrophoretic CGG-PCR band patterns. Considering the high reproducibility and specificity of the CGG-PCR profiles, this test has potential as an alternative to genotyping for E. coli strains and as an additional screening tool for rapidly and efficiently genotyping E. coli strains.

Adamus-Bialek W, Wojtasik A, Majchrzak M, et al. (CGG)4-based PCR as a novel tool for discrimination of uropathogenic Escherichia coli strains: comparison with enterobacterial repetitive intergenic consensus-PCR. J Clin Microbiol. 2009;47:3937–3944.

Correspondence: Pawel Parniewski at

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Causes of death in hyper-IgE syndrome Causes of death in hyper-IgE syndrome

Hyper-IgE recurrent infection syndrome, or Job’s syndrome, is a rare primary immunodeficiency characterized by extremely increased serum IgE levels, eczema, recurrent infections, and a variety of connective tissue and skeletal abnormalities, including atypical facial appearance, failure to shed primary teeth, scoliosis, joint hyperextensibility, osteopenia with pathologic fractures, and craniosynostosis. Abnormalities of the humoral, cellular, and phagocytic compartments of the immune system are present, but they vary between patients and have not been fully characterized. Recurrent skin abscesses and pneumonias result most commonly from Staphylococcus aureus and other pyogenic bacteria. After acute pneumonias, characteristic pneumatocoeles that may become superinfected with gram-negative bacteria and fungal opportunists are formed. No autopsy series of individuals with hyper-IgE recurrent infection syndrome (HIES) and only limited review of pathologic specimens have been published. Therefore, the authors reviewed the available autopsies of patients with HIES followed at the National Institutes of Health, which is a referral center for HIES, to identify the causes of death and anatomic or pathologic correlates. They reviewed the medical records and autopsy slides for six patients with HIES who underwent autopsies at their institution. All six patients with HIES were women and ranged in age from 24 to 40 years. All had a history of cystic lung disease and had pneumonia at the time of death, with Pseudomonas aeruginosa and fungal organisms predominating. Pulmonary fungal vascular invasion with fatal hemorrhage was observed in three patients, and metastatic fungal disease to the brain caused by Aspergillus fumigatus and Scedosporium prolificans was observed in two patients. Four patients had evidence of renal tubular injury, which was likely from amphotericin B toxicity; three patients had glomerulosclerosis; and one patient had two kidney angiomyolipomas. The authors con-cluded that this series highlights the important role Pseudomonas and Aspergillus species play in patients with HIES who have cystic lung disease. Intensified antifungal and gram-negative bacterial prophylaxis should be evaluated as possible strategies to prevent these complications of infection in patients with cystic lung disease.

Freeman AF, Kleiner DE, Nadiminti H, et al. Causes of death in hyper-IgE syndrome. J Allergy Clin Immunol. 2007;119:1234–1240.

Correspondence: Dr. Steven M. Holland at

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Postmortem redistribution of fentanyl in blood Postmortem redistribution of fentanyl in blood

The cause and manner of death established by forensic authorities in cases involving single or mixed drug ingestion must take into consideration postmortem redistribution. This is defined as the variation of drug concentration in blood samples taken from different sites—heart blood and femoral blood—and can be affected by the postmortem interval or time of death relative to specimen collection. As the interval between death and collection of blood becomes longer, drugs from tissues and organs that contain high drug concentrations redistribute owing to cadaver decomposition, leading to increased drug concentration in the blood. During the postmortem interval, drugs from the gastrointestinal tract, lungs, heart, or liver may travel via diffusion through blood vessels or via direct diffusion into other organs or vessels. Drugs that are highly concentrated in the liver, lungs, or myocardium redistribute quickly into heart blood, causing concentrations to increase. The most common postmortem samples used for forensic toxicologic analysis include femoral blood (per-ipheral), heart blood (central), urine, liver tissue, gastric contents, and vitreous humor. For most drugs, peripheral blood is regarded as the optimal sample for interpretation based on its greater distance from organs that may be influenced by postmortem redistribution (PMR) mechanisms. However, studies have demonstrated that alternative samples are more accurate in reflecting perimortem drug concentrations for particular drugs. For example, it has been shown that liver concentrations better represent true body burden of tricyclic antidepressants, which are highly concentrated in tissues that may break down after death and cause falsely increased concentrations of the drug in blood. In the same way, the unique PMR patterns of each type of drug must be taken into account in creating a complete picture of perimortem conditions. Fentanyl is a high-potency, rapid-onset synthetic opioid drug for treating chronic pain and is used as a surgical anesthetic. In the past decade, fentanyl misuse and abuse have steadily risen. Fentanyl toxicity causes decreased respiratory rate and depth, delirium, hypotension, bradycardia, and decreased body temperature, leading to death if untreated. The contribution of fentanyl to cause and manner of death in medical examiner cases is complicated by high amounts of the drug present in long-term users. This makes it more difficult for the medical examiner or coroner to determine whether increased fentanyl blood concentrations, beyond what was expected from a patient’s clinical history, contributed to the patient’s death. Considerable overlap exists between blood fentanyl concentrations in deaths attributed to fentanyl alone and those attributed to mixed drug overdose, as well as between concentrations found in fentanyl-related over-dose deaths compared with levels in hospitalized patients being treated for chronic pain. The possibility of a drug concentration falsely increasing in a blood sample as a postmortem interval lengthens makes defining therapeutic, toxic, and lethal ranges more difficult. The authors conducted a study to determine whether PMR of fentanyl occurs in femoral blood. They measured fentanyl concentrations in postmortem specimens collected in 20 medical examiner cases from femoral blood, heart blood, heart tissue, liver tissue, and skeletal muscle. In a subset of seven cases, femoral blood was obtained at two postmortem intervals—shortly after death and at autopsy. The mean collection times shortly after death and at autopsy were 4.0 and 21.6 hours, respectively. Fentanyl concentrations at those times ranged from undetectable to 14.6 µg/L (mean, 4.6 µg/L) and 2.0 to 52.5 µg/L (mean, 17.3 µg/L), respectively. Corresponding mean heart blood, liver tissue, and heart tissue fentanyl concentrations were 29.8 µg/L, 109.7 mg/kg, and 103.4 mg/kg, respectively. The fentanyl heart blood/shortly after death ratio (mean, 8.39) was higher compared with the corresponding heart blood/at autopsy ratio (mean, 3.48). These results suggest that postmortem redistribution of fentanyl can occur in femoral blood.

Olson KN, Luckenbill K, Middleton O, et al. Postmortem redistribution of fentanyl in blood. Am J Clin Pathol. 2010;133:447–453.

Correspondence: Fred S. Apple at

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444444444444444444 Tumor markers for bladder cancer surveillance

Cystoscopy remains the gold standard for detection and surveillance of bladder cancer. However, it can be uncomfortable for patients and costly for health care systems. Urinary cytology as a noninvasive alternative method is criticized as being too investigator dependent and having too low a sensitivity for detecting low-grade tumors. Therefore, this method is not considered safe enough for the followup of bladder cancer and is only recommended in combination with cystoscopy. Nevertheless, this method is still the reference standard for other noninvasive urine-based biomarkers for detecting new or recurrent bladder cancer. The search for better, highly sensitive and specific urine-based tumor markers is ongoing. Several markers have been approved by the FDA and various health care systems. The most widely used markers are ImmunoCyt (uCyt+), BTA Trak, BTA stat, NMP22, NMP22 BladderChek, and Uro-Vysion. All of them were, at least in some studies, superior to urinary cytology. However, none of these markers alone reached a sensitivity and specificity high enough to reduce safely the number of followup cystoscopies. Consequently, they are only recommended in combination with cystoscopy. One approach to improve noninvasive clinical detection of bladder tumor recurrence involved evaluating several urine-based tumor markers designed to increase the sensitivity of tumor detection. The authors conducted a study to investigate whether combinations of urine-based tumor markers, including urinary cytology, increase the sensitivity for detecting bladder cancer recurrence. Urinary cytology, NMP22, UroVysion (fluorescence in situ hybridization), and uCyt+ were evaluated in 221 patients during the followup of nonmuscle-invasive transitional cell carcinoma (NMI TCC) before cystoscopy (n=49) or with the suspicion of TCC recurrence before transurethral resection of the bladder (n=173). For all markers, alone as well as in all possible combinations (multimarker panels), the authors evaluated sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. Multimarker panels were considered positive if at least one marker was positive. The authors found no malignancy in 108 patients, whereas recurrent TCC was confirmed in 113 patients. Sensitivity and specificity were 84 percent and 62 percent for cytology, 68 percent and 49 percent for NMP22, 72 percent and 63 percent for FISH, and 73 percent and 62 percent for uCyt+, respectively. The negative predictive value was below 80 percent for all markers alone. Combinations of two and three markers increased the sensitivity as well as the negative predictive value to more than 90 percent and 80 percent by reducing specificity to an average of 44 percent and 35 percent, respectively. The most sensitive combinations were NMP22 and uCyt+ together with cytology and FISH, and uCyt+ together with NMP22 (sensitivity for both combinations, 98 percent). There was no further improvement when all four markers were combined. The authors concluded that combinations of tumor markers increased the sensitivity and negative predictive value for detecting recurrence of NMI TCC. A stepwise approach to tumor marker determination may be used to reduce the frequency of followup cystoscopies at a reasonable risk.

Horstmann M, Patschan O, Hennenlotter J, et al. Combinations of urine-based tumour markers in bladder cancer surveillance. Scand J Urol Nephrol. 2009;43:461–466.

Correspondence: M. Horstmann at marcus horst

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Role of PSA velocity thresholds in prostate cancer detection Role of PSA velocity thresholds in prostate cancer detection

Prostate-specific antigen-based prostate cancer screening may overdetect potentially insignificant prostate cancer. But clinical staging modalities are limited in their ability to accurately predict tumor biology, which has fueled an ongoing search for more specific markers of clinically significant prostate cancer. A promising marker is PSAV (rapidly increasing PSA). The authors’ research group previously noted a relationship between PSAV and Gleason grade in the radical prostatectomy specimen. Specifically, median preoperative PSAV was 0.84, 0.97, and 1.39 ng/mL per year in patients with a prostatectomy Gleason score of 6, 7, and 8 to 10, respectively (P=0.05). Other studies have shown a relationship between pretreatment PSAV and risk of prostate cancer-specific mortality. Together, these findings suggest that PSAV may be a surrogate marker for prostate cancer aggressiveness. Conversely, low PSAV (small PSA increase with time) may indicate a greater likelihood of indolent disease. The 2010 National Comprehensive Cancer Network Guidelines and other guidelines suggest a PSAV threshold of about 0.35 to 0.4 ng/mL per year in prostate cancer screening protocols. Because PSA kinetics were previously linked to prostate cancer-specific mortality, the authors conducted a study to determine whether prostate-specific antigen velocity is associated with clinically significant prostate cancer. For the study, 1,073 men with data available on prostate-specific antigen velocity and tumor volume underwent radical prostatectomy from 1992 to 2008. Insignificant cancer was defined by the Ohori criteria as organ confined, tumor volume of 0.5 cc or less, and no primary or secondary Gleason pattern four or five. The authors calculated the proportion of men with pathologically insignificant prostate cancer stratified by prostate-specific antigen velocity. They found that preoperative prostate-specific antigen velocity greater than 0.4 ng/mL per year was significantly associated with high-grade disease (P=0.008), positive surgical margins (P=0.003), and seminal vesicle invasion (P=0.007) at radical prostatectomy. Median tumor volume was also significantly higher in men with preoperative prostate-specific antigen velocity greater than 0.4 ng/mL per year (3.1 versus 2.4 cc; P=0.0001). Overall, 69 men (six percent) met the Ohori criteria for insignificant cancer. Patients with preoperative prostate-specific antigen velocity greater than 0.4 ng/mL per year were 50 percent less likely to have insignificant disease (10 percent versus five percent; P=0.003). The authors concluded that a prostate-specific antigen velocity threshold of 0.4 ng/mL per year was associated with the likelihood of insignificant prostate cancer. This suggests that prostate-specific antigen velocity may be a useful adjunct in prostate cancer screening to increase specificity for identifying patients with clinically significant disease.

Loeb S, Roehl KA, Helfand BT, et al. Can prostate specific antigen velocity thresholds decrease insignificant prostate cancer detection? J Urology. 2010;183:112–117.

Correspondence: William J. Catalona at

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