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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; cylo@hkucc.hku.hk
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; cylo@hkucc.hku.hk
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;
lisa.louie@swchsc.on.ca
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. wustl.edu
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; A.Taylor@surrey.ac.uk
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; ssimjee@cvm.fda.gov
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;
gminuk@cc.umanitoba.ca
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