College of American Pathologists
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February 2004

Clinical pathology abstracts editors: Michael Bissell, MD, PdD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus, and Ronald Domen, MD, professor or pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pa.

Intraoperative insulin measurements

Autonomous insulin production due to overconsumption of glucose causes organic hypoglycemia. The majority of patients with this condition have a single benign insulinoma, whereas multiple insulinomas are found in 13 to 24 percent of patients with hyperinsulinemia. The incidence of multiple tumors increases in patients with multiple endocrine neoplasia type I, with 10 to 40 percent of them being malignant. The ideal treatment for organic hypoglycemia is complete surgical excision of the hypersecreting tissue, which is often difficult to identify. Preoperative localization studies are of variable accuracy, but intraoperative ultrasound and pancreatic palpation localize 89 to 97 percent of insulinomas in patients. The four to 16 percent of tumors not recognized intraoperatively present a challenge. Another challenge is determining complete resection of the hypersecreting tissue. Most surgeons do so using the intraoperative increase of serum glucose levels after tumor excision, but this technique is often inaccurate. Intraoperative insulin levels have also been used prognostically. These studies have used immunoradiometric assays to confirm complete resection, but turnaround times of 30 to 60 minutes limit the intraoperative usefulness of the assays. The authors analyzed the use of a newly developed, eight-min. immunochemiluminescent assay for insulin that can provide useful information to the surgeon intraoperatively. Eight consecutive patients with organic hypoglycemia underwent pancreatic exploration. Using the immunochemiluminescent insulin assay, peripheral blood levels were obtained preoperatively, during resection, and at five-min. intervals after surgical excisions. The authors compared operative findings and outcome with intraoperative insulin/glucose ratios, glucose, and insulin levels. Using the return of insulin levels to normal range and insulin/glucose ratios of 0.4 or less 15 minutes after tumor resection as criteria to predict success, the outcomes of six patients were predicted correctly (five true positive and one true negative). One patient with nesidioblastosis had a false-negative result, and one could not be evaluated because of diazoxide medication. These criteria predicted postoperative absence of hypoglycemia with a specificity of 100 percent and accuracy of 89 percent.

Carneiro DM, Levi JU, Irvin GL. Rapid insulin assay for intraoperative confirmation of complete resection of insulinomas. Surgery. 2002;132:937-943.

Reprints: Dr. G.L. Irvin, III, Dept. of Surgery, P.O. Box 016310 (M-875), Miami, FL 33101-6310

Detecting breast cancer cells in bone marrow by flow cytometry

Quantitation of isolated tumor cells in bone marrow by immunocytochemical analysis has prognostic value, independent of axillary node involvement, for identifying breast cancer patients at high risk for poor outcomes. The authors used an ultrasensitive tumor-enriched flow-cytometric assay to determine the feasibility of detecting isolated tumor cells in the bone marrow of 19 patients with stage I/II breast cancer. Magnetic microbeads conjugated with an anti-cytokeratin 7/8 monoclonal antibody were used to remove epithelial cells and enrich tumor cells in bone marrow samples. A specific gate for MCF-7 breast cancer cells (gateMCF-7 cells) was used with a gate including all enriched bone marrow cells (gateenriched BM cells) in flow-cytometric analysis to enhance the specificity of the method. Ten patients (53 percent) were found to have cytokeratin-positive cells according to the enriched bone marrow cell gate, and six patients (32 percent) had CK+ cells according to the MCF-7 cell gate. The authors concluded that new strategies in nonmorphological ultrasensitive techniques might be useful to categorize patients with isolated tumor cells that have different tumor morphology and characteristics.

Lo C-Y, Luk JM, Tam SC. Applicability of intraoperative parathyroid hormone assay during thyroidectomy. Ann Surg. 2002;236:564–569.

Reprints: Chung Yau Lo, MS, FRCS (Edin), Division of Endocrine Surgery, Dept. of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam Rd., Hong Kong, China;

Utility of intraoperative PTH assays in thyroid surgery

The principal morbidities of thyroid surgery, which account for most medical litigation, are permanent hypoparathyroidism and recurrent laryngeal nerve damage. The nerve stimulator is increasingly used to monitor vocal cord function and prevent nerve injury during thyroidectomy. In contrast, surgeons have relatively little idea during surgery whether postoperative and permanent hypoparathyroidism is likely to occur. Efforts have been made to identify clinical risk factors for post-thyroidectomy hypocalcemia or long-term outcome. The indications for the operation, extent of thyroid resection, and anatomy of the parathyroid glands determine the ability to preserve parathyroid function. Clinically significant hypocalcemia following thyroid surgery is secondary to impairment of parathyroid function in most cases. Close monitoring of serum calcium levels is commonly used to identify postoperative hypoparathyroidism, but an ideal intraoperative assessment of parathyroid function is lacking. The intraoperative parathyroid hormone assay, or quick PTH assay, has been increasingly adopted to monitor the success of parathyroid surgery and to facilitate a focused approach during parathyroidectomy. It has been suggested that the quick PTH assay might have value in monitoring the outcome of thyroid surgery with respect to the development of postoperative hypocalcemia. The authors evaluated the applicability of the quick PTH assay for monitoring parathyroid function and identifying clinically significant hypocalcemia compared with postoperative serum calcium monitoring. The quick PTH assay was performed before and after thyroidectomy on 100 patients at risk of postoperative hypocalcemia and 20 control patients who underwent unilateral lobectomy. Postoperative serum calcium levels were closely monitored. Control patients had a normal but 38.9 ± 5.9 percent (mean ± SEM) decline in quick PTH after thyroidectomy. Eleven of 100 at-risk patients developed postoperative hypocalcemia. Hypocalcemic patients had significantly lower quick PTH values after thyroidectomy than did normocalcemic patients. Serum calcium was significantly lower in hypocalcemic patients the morning after the operation but not within six hours of the operation. A normal or less than 75 percent decline in quick PTH after thyroidectomy can identify normocalcemic patients during surgery, while serum calcium monitoring can take more than 24 hours. The authors concluded that the quick PTH assay can monitor parathyroid function during thyroidectomy and identify patients at risk of clinically significant hypocalcemia much earlier than serum calcium monitoring.

Lo C-Y, Luk JM, Tam SC. Applicability of intraoperative parathyroid hormone assay during thyroidectomy. Ann Surg. 2002;236:564–569.

Reprints: Chung Yau Lo, MS, FRCS (Edin), Division of Endocrine Surgery, Dept. of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Pokfulam Rd., Hong Kong, China;

Rapid detection of MRSA by multiplex PCR

Methicillin-resistant Staphylococcus aureus infections are associated with significant morbidity and mortality, especially in patients with bacteremia. It is, therefore, essential to rapidly detect methicillin resistance in staphylococci from bacteremic patients. Of the polymerase chain reaction-based assays for detecting MRSA, some require a pure culture of the organism, while others require long DNA extraction protocols. A rapid method for extracting DNA from blood cultures was described previously, but it requires several individual PCRs to detect all necessary products for identifying staphylococci. The authors described a rapid and simple multiplex PCR-based method for directly detecting MRSA from positive blood culture bottles, reducing the time necessary for identification from 24 to 48 hours to approximately three hours. A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 MRSA), 50 positive blood cultures that grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after five days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 hours. For the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2 percent and 100 percent, respectively. Thus rapid, direct detection of MRSA is possible.

Louie L, Goodfellow J, Mathieu P, et al. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol. 2002;40:2786–2790.

Reprints: L. Louie, Dept. of Microbiology, Sunnybrook and Women’s College Health Sciences Centre, B121-2075 Bayview Ave., Toronto, Ontario, M4N 3M5, Canada;

Is cystatin C better than creatinine?

Glomerular filtration rate is the volume of plasma that can be completely cleared of a substance by the kidneys per unit of time. Gold standard measurements of GFR involve estimating the clearance of exogenous substances such as inulin, iohexol, 51Cr-EDTA, 99mTc-labeled diethylenetriamine pentaacetic acid (DTPA), or 125I-labeled iothalamate. These techniques, however, are time consuming, labor intensive, expensive, and require administration of substances that make them incompatible with routine monitoring. Endogenous substances such as blood urea nitrogen and serum creatinine (SCr), therefore, are commonly used to estimate GFR. Although widely used, these markers are not ideal and do not perform optimally in certain clinical settings. The authors systematically reviewed the potential utility of cystatin C, especially in patient populations in which CysC may have an advantage over routinely used endogenous markers of GFR. They extensively reviewed publications, primarily from the last five years, that address the development of methods to measure CysC, reference intervals, and the diagnostic accuracy of CysC to assess GFR. Between June 2000 and September 2001, they searched Medline using “cystatin C” as a text word. The authors included in their review those articles that examined more than 75 individuals (except for renal transplant studies) or used accepted gold standards for assessing GFR, or both. Seventeen studies that provide reference interval data for several populations were reviewed. Twenty-four of the studies draw conclusions about the utility of CysC versus SCr or creatinine clearance, or both, with 20 providing data on the sensitivity and specificity of CysC for detecting impaired GFR. Studies of reference intervals for CysC overwhelmingly demonstrated that CysC values in blood are independent of age and gender. Of the 24 studies that examined clinical utility, 15 concluded that CysC is superior to SCr, and nine concluded that CysC is equivalent but provides no advantage. Summary receiver operating characteristic plot analysis of 20 studies that provide sensitivity and specificity data strongly suggested that CysC will be superior to SCr for detecting impaired GFR. The authors concluded that CysC performs at least as well as SCr in the population at large and that it is likely to be superior to SCr in specific patient populations.

Laterza OF, Price CP, Scott MG. Cystatin C: an improved estimator of glomerular filtration rate? Clin Chem. 2002;48:699–707.

Reprints: Mitchell Scott, Dept. of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110; mscott@ labmed.

European proficiency testing standards for lead and aluminum

The passage of legislation in many countries pertaining to heavy metal testing has increased awareness of the importance of using properly validated methods. It is accepted that truly independent assessments of laboratory performance are provided by properly constructed and managed surveillance programs, external quality assessment schemes, and proficiency testing. These not only measure participant performance but also provide a stimulus for improving accuracy. Schemes relating to occupational or environmental laboratory medicine, or both, are organized from at least nine countries of the European Union. Most use similar protocols to monitor performance, but each has its own technique to define a satisfactory standard of performance, even though clear guidelines for statistical evaluation of performance have been developed. Divergence occurs when organizers use techniques that are complementary to those of other programs within the same country to provide harmonization within that nation. A consequence of these variations in evaluating performance is that different schemes have the potential to give conflicting conclusions, even from the same raw data. Even within the same country, multiple criteria may exist for judging laboratory performance. The criterion set for blood lead in the United States by the Occupational Safety and Health Administration for occupational monitoring purposes is ± 60 µg/L or ± 15 percent, whichever is greater. Clinical laboratories, however, must comply with the performance criteria for blood lead set under CLIA ’88—that is, ± 40 µg/L or ± 10 percent, whichever is greater. The authors analyzed real results for blood lead and serum aluminum assays reported by participants in external quality assessment schemes in Italy and the United Kingdom. The results were evaluated according to individual scheme scoring criteria and then used to produce z scores using scheme-based between-laboratory standard deviations as the estimate of variability to determine whether simple performance-derived quality specifications produced better agreement among schemes. The schemes gave conflicting assessments of participants’ performance—participants judged to be successful by one scheme could be defined as performing inadequately by another. An approach proposed by Kenny, et al (Scand J Clin Lab Invest. 1999;59:585), which uses clinical inputs to set targets for analytical imprecision, bias, and total error allowable, was used to further define quality specifications. The authors suggested that the CLIA ’88 recommendations for blood lead (± 40 µg/L or ± 10 percent of the target concentration, whichever is greater) could be used as a quality specification, although they recommended a revision to ± 30 µg/L or ± 10 percent. For serum aluminum they suggested a suitable quality specification of ± 5 µg/L or ± 20 percent of the target concentration, whichever is greater. These specifications may be used to compare laboratory performance across schemes.

Taylor A, Angerer J, Claeys F, et al. Comparison of procedures for evaluating laboratory performance in external quality assessment schemes for lead in blood and aluminum in serum demonstrates the need for common quality specifications. Clin Chem. 2002;48:2000–2007.

Reprints: Andrew Taylor, Centre for Clinical Science and Measurement, School of Biomedical and Life Sciences, University of Surrey, Guilford, Surrey GU2 7XH, United Kingdom;

VRE in dogs and humans

Enterococci can acquire and disseminate antimicrobial resistance determinants, including those to aminoglycosides and glycopeptides. These organisms cause serious illness in immunocompromised patients. High-level resistance to gentamicin is usually due to the presence of the bifunctional Aac6'-Aph2" aminoglycoside-modifying enzyme, which is highly potent and confers resistance to all the clinically useful aminoglycosides, with the exception of streptomycin. The aac6'-aph2" genes have, in most cases, been localized to plasmids as part of transposon Tn5281. Association of aac6'-aph2" with a transposon further aids in the rapid dissemination of the resistance marker. The appearance of high-level gentamicin resistance in clinical medicine in 1979 had a substantial negative effect on the treatment of severe enterococcal infections. The only available antibiotic left by the mid-1990s to successfully treat enterococcal infections was vancomycin. Over the past decade, however, there has been a rapid surge in the prevalence of vancomycin-resistance among enterococci, predominantly of the vanA and vanB genotypes, and glycopeptide-resistant enterococci have emerged worldwide. The vanA resistance locus, which is most prevalent, consists of a cluster of seven genes present on Tn1546, a 10.8-kb transposon that has been identified in enterococci isolated in the community and from sewage, animal feces, and raw meat, suggesting that each of these may act as a reservoir of resistant enterococci and the vanA gene. Epidemiological studies in Europe have suggested that vancomycin-resistant enterococci are transmitted horizontally from animals to humans. In view of the possible involvement of companion animals in the spread of antibiotic-resistant enterococci to humans, the authors characterized gentamicin and vancomycin resistance among 35 enterococcal isolates recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital from 1996 to 1998. One Enterococcus faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC>32 µg/mL) and gentamicin (MIC>2,048 µg/mL). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from vancomycin-resistant E. faecium. Sequence analysis revealed a particular form of Tn1546 that has only been described in human clinical VRE isolates unique to the United States. This is the first report of a VRE isolated from a companion animal in the United States.

Simjee S, White DG, McDermott PF, et al. Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci. J Clin Microbiol. 2002;40:4659–4665.

Reprints: S. Simjee, U.S. Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Rd., Laurel, MD 20708;

A rapid test for hepatitis B mutations

The hepatitis B virus is a partially double-stranded DNA molecule virus replicating by reverse transcription via an RNA intermediate. Mutations to the HBV genome are common, and the majority of chronic HBV infections are associated with the emergence of mutations. Some of these mutations result in diverse viral populations and different clinical outcomes. Those in the surface, core, precore, and basal core promoter regions appear to have the greatest clinical relevance. Mutations have also been described after antiviral therapy and vaccination. Detecting these mutants is essential to understanding HBV’s natural history and response to antiviral therapy and immunoprophylaxis. Detection of HBV mutants is performed primarily by restriction fragment-length polymorphism analysis, gap ligase chain-reaction assay, or direct sequencing. These methods, however, are not ideal for routine clinical use. A new high-speed thermal cycler recently was used to determine HBV-DNA levels and to detect the presence of YMDD mutants. This technology allows for real-time monitoring of sensitive and specific amplification of the melting behavior of hybridization oligonucleotides. The authors reported the sensitivity and specificity of real-time polymerase chain reaction with fluorescent hybridization probes for HBV mutant detection. They examined three common, clinically relevant HBV mutations: an S-escape point variant at nt 587, a precore stop codon point variant at nt 1896, and double variants at nt 1762/1764 in the basal core promoter region. Two probes were designed for each mutation: anchor probes were 3’ labeled with fluorescein and sensor probes, 5’ labeled with LC-Red 640, and 3’ phosphorylated. The authors then determined temperatures for each probe melted from amplification products in a melting program. They analyzed sera from 12 patients, each containing identified HBV mutants (six S-escape, one precore, one BCP, and four mixed precore and BCP), and five control sera from patients with wild-type virus. Direct sequencing was used to confirm genomic sequences of mutant and wild-type viruses. Real-time PCR with fluorescent hybridization probes identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the three mutants were distinguished from wild-type (>4°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL, and as few as five percent of mutant among wild-type virus were detected. The authors concluded that real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants.

Zhang M, Gong Y, Osiowy C, et al. Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis. Hepatology. 2002;36:723–728.

Reprints: Dr. Gerald Minuk, Liver Diseases Unit, John Buhler Research Center, University of Manitoba, 715 McDermot Ave., Winnipeg, Manitoba, R3E 3P4, Canada;