January 2002
Volume 27
Number 1
Fluorescence In Situ
Hybridization for the
Detection of Urothelial
Ask Us
Abstracts of Interest
Test Updates:
• Clostridium difficile
Toxin Kit Change
• Human
Immunodeficiency Virus
Type 1 Genotyping
• Cystic Fibrosis Test
Method Change
• Growth Hormone
Method Change
• Monoclonal Protein
Urine Study Introduced
New Test
EBV PCR Quantitative
Stabile Building
150 Third Street SW
Rochester, Minnesota 55902
[email protected]
M a y o
R e f e r e n c e
S e r v i c e s
P u b l i c a t i o n
Fluorescence In Situ Hybridization for the
Detection of Urothelial Carcinoma
Each year, in the United States, there are
approximately 50,000 new cases of urothelial
carcinoma (cancer that arises from the lining of
the bladder, ureters, renal pelvis, and proximal
urethra). Most urothelial carcinomas (UC)
originate in the bladder.
Early detection of UC recurrence is key to longterm survival. Unfortunately, UC can be difficult
to detect. Early symptoms include blood in the
urine, or a frequent need to void that may be
accompanied by an inability to void. However,
these symptoms also are associated with bladder
stones, infections, or other benign conditions.
There are 2 main types of UC, papillary and
"flat." Approximately 80% of UC are papillary
tumors and approximately 20% are flat tumors.
Papillary UCs are visible by cystoscopy. They
tend to recur but not progress to invasive cancer.
Flat UC do not form an easily visible tumor
mass, are often clinically aggressive, and tend to
progress to invasive cancer.
When a UC is surgically removed, it is staged
and graded by the pathologist. Staging refers to
the extent of tumor spread, namely how deeply it
has invaded into the bladder wall (or ureters or
renal pelvis) and whether or not lymph node or
distant metastases have occurred. (See Table 1.)
Grading refers to how differentiated the tumor
cells appear when viewed under the microscope.
Pathologists’ assign a grade of 1, 2, or 3 to the
tumor, based on histologic criteria. Grade 3
tumors (poorly differentiated) have a tendency
to recur and progress from a lower to higher
stage. Most flat and invasive UC are grade 3
tumors, while most non-invasive papillary
tumors are grade 1 and 2. Higher stages and
grades are associated with a worse prognosis.
Stage and grade help determine the therapeutic
approach for individual patients.
Urine Cytology
Historically, the standard methods employed for
the detection and monitoring of UC have been
cystoscopy (where a urologist examines the lining
of the bladder with a cystoscope) and cytology
(where a pathologist examines cells that are
voided in the urine).
With regard to UC detection in urine specimens,
cytology has excellent specificity but suffers
from inadequate sensitivity. (See Table 2.) The
high specificity of urine cytology means that a
diagnosis of UC on urine cytology is almost
always correct (a low percentage of false
Table 1: Staging of Urothelial Carcinoma
non-invasive papillary tumor
carcinoma in situ
Extension into lamina propria
Extension into muscle layer of bladder, ureters, or renal
Extension through the muscle layer
Extension through muscle layer and into surrounding organs
Figure 1
Abnormal urine specimen. Trisomy 7, visible as 3 green
probe signals. CEP 3 (red); CEP 7 (green).
Table 2: Sensitivity and Specificity of Urine Cytology1
Sensitivity of Cytology by Grade
Grade 1
Grade 2
Grade 3
positives are seen). In contrast, its suboptimal sensitivity
for UC means that a negative result is not always
correct (eg, tumors are present, but not detected, and
thus the result is falsely negative).
Because patients with bladder cancer typically are
monitored for recurrence by urine cytology at periodic
intervals, false-negative test results may allow the
patient’s tumor 3 or more months to progress to a
higher (potentially incurable) stage.
Assays for Urothelial Carcinoma
chromosomes or loci. (Note: loci simply means location
on a chromosome and does not always imply that a
gene is present at that site.)
Centromeric probes are useful mainly for determining
how many copies of a chromosome are present in a cell.
Normal cells have 2 copies of each chromosome. (See
Figure 2.) Malignant cells frequently have more than 2
copies of the various chromosomes. Cells that have
additional copies of 2 or more different chromosomes
are very likely to be malignant. Locus-specific probes
provide information about whether genes are amplified
or deleted and this information may be of prognostic or
therapeutic importance (eg, Her-2/neu amplification in
breast cancer).
Figure 2
Normal Urine Specimen: CEP 3 (red); CEP 7 (green); CEP 17
(aqua); LSI 9p21 (gold)
The suboptimal sensitivity of urine cytology has
prompted medical scientists to develop new tests with
improved sensitivity for UC detection. Many of these
tests, such as BTA-stat and NMP-22, are based on the
detection of antigens that are seen at increased levels in
the urine of patients with UC. While some of these
assays significantly improve sensitivity for UC detection,
they all have poorer specificity than cytology (a higher
percentage of false positives). The lower specificity is
related directly to the fact that antigen levels also may be
increased as a result of inflammatory states.
Typical specificities for these new assays are
approximately 70% (approximately 30% of the positive
results are false-positives). Based on these specificities,
even with these newer tests, urologists often must
perform cystoscopy on these patients to determine if
UC truly is present.
Fluorescence In Situ Hybridization
Most solid tumors are characterized by chromosomal
abnormalities. Fluorescence in situ hybridization (FISH)
is a technique that utilizes fluorescently-labeled DNA
probes to detect chromosomal abnormalities. FISH has
applications in many areas including medical genetics,
obstetrics, hematology, and oncology. (See Figure 1.)
There are 2 general types of FISH probes, centromeric
and locus specific probes. A centromere is a constriction
point found in all chromosomes. Centromeric probes
(CEP) hybridize (attach) to the centromeres of the target
chromosomes, while locus-specific probes (LSI) are
directed to loci on specific chromosomes. Different
colors can be used to probe different chromosomes or
loci, allowing the laboratory to enumerate specific
FISH for Urothelial Carcinoma
Mayo's Cytogenetics Laboratory began development of
a FISH assay for the detection of UC in 1998. The
laboratory tested 10 different probes for individual and
combined sensitivity of urothelial cancer detection. Of
the 10 probes, 9 were CEP probes (CEP 3, 7, 8, 9, 11, 15,
17, 18, and X) and 1 was a 9p21 LSI probe.
The 9p21 LSI probe hybridizes to a region on the short
arm of chromosome 9 where the P16 tumor suppressor
gene resides. Genetic alterations that interfere with the
function of the P16 tumor suppressor gene (eg, deletion
of the gene) play an important role in the formation of
many UC. It was determined that a combination of CEP
3, CEP7, CEP17, and LSI 9p21 had the highest
sensitivity for UC detection. The CEP3, CEP7, CEP17,
and LSI 9p21 probes were labeled with red, green, aqua,
and yellow fluorophores (the fluorescent molecule that
is attached to the DNA probe), respectively and
combined into a single, multiprobe mixture. (See Figure
3.) This multitarget, multicolor probe set is now known
as UroVysionTM (Abbott Laboratories, Waukegan, IL).
UroVysion received FDA approval for monitoring UC
patients for tumor recurrence in July 2001.
Figure 3
Abnormal cell utilizing UroVysion probe set.
A subsequent study compared the sensitivity and
specificity of FISH to the FDA approved BTA-stat assay.3
This study demonstrated that the sensitivity of FISH and
BTA-stat were not significantly different statistically
(81% vs 78%, P=0.63), but that FISH was significantly
more specific than BTA-stat (96% vs 74%, P=0.0001).
The recently conducted FDA trial to evaluate UroVysion
found that FISH was more sensitive than cytology or
BTA-stat for UC detection, and more specific than BTAstat for UC detection.
Based on these and other studies, FISH has
demonstrated the best combination of high sensitivity
and high specificity of any test available for the
detection of UC. This improved analytic performance
(fewer false negative or false positive assays) should
improve the clinical management of patients who are
being monitored for UC recurrence.
Sensitivity and Specificity of FISH for Urothelial
Carcinoma Detection
Combining the 4 probes improved the sensitivity and
specificity of the assay compared to just 1 or 2 probes in
a mix. Fluorescence In Situ Hybridization (FISH) For
Urothelial Cancer, Urine (#81975) offers the high
specificity of urine cytology, but is significantly more
sensitive than cytology for the detection of UC.1,2
In a study performed at Mayo using the UroVysion
probe set, the overall sensitivity of FISH and cytology
were 81% and 59% (P=0.001), respectively in 75 patients
with biopsy-proven UC.1 When analyzed by grade
against cytology, FISH was significantly more sensitive
for grade 3 tumors, approached a statistically significant
difference for grade 2, and showed no statistical
difference for grade 1 tumors. (See Table 3.) The low
sensitivity of both FISH and cytology for grade 1 tumors
Table 3: Sensitivity and Specificity of FISH for UC1
Sensitivity of Cytology by Grade
Grade 1
Grade 2
Grade 3
is not surprising since grade 1 tumors tend to be diploid.
Bubendorf et al also have found that FISH using the
UroVysion probe set is just as specific as urine cytology,
but significantly more sensitive. 2
The FISH assay is performed by isolating cells from the
urine by centrifugation, fixing the cells, placing the cells
on a slide, doing the FISH hybridization and then
interpreting the slide with a fluorescence microscope. The
slide is scanned primarily, but not exclusively, for cells
that have abnormal cytologic features when viewed with
the DAPI nuclear counterstain. (See Figure 4.) These
features include nuclear enlargement, nuclear irregularity,
and mottled staining. The FISH signal pattern is assessed
primarily in cells with these features because these cells
have a much higher probability of having genetic
abnormalities indicative of malignancy.
Scanning significantly reduces the time required to
evaluate the slide, as some cells visualized with the DAPI
stain (eg, neutrophils) are clearly not neoplastic and can be
excluded from the evaluation. Scanning also increases the
sensitivity of the assay because the technologist scans the
entire slide and all cells on the slide, instead of evaluating
only a certain number (eg, 100) of consecutive cells.
The main type of abnormality that malignant urothelial
cells exhibit is "polysomy," where cells have gains of 2
or more chromosomes. A case is considered positive for
malignancy if 5 or more cells exhibit polysomy. The
cutoff of 5 or more cells with polysomy was based on
receiver-operator curves that determined this cutoff
gave the best combination of high sensitivity and
specificity. Some cases will have numerous abnormal
cells. Other cases will have few or no abnormal cells.
If a case is positive for cancer because it has 5 or more
abnormal cells, the percentage of cells that are abnormal
Figure 4
Polysomy usually results in an increased nuclear size. Cells can be
screened for abnormal morphology with DAPI stain, then assayed
with FISH to identify the specific abnormalities. Arrows indicate
abnormal cells.
copies of chromosome 9 having a deletion) with disomy
of other chromosomes (referred to as "homozygous 9p21
deletion" pattern). (See Figure 5.)
Figure 5
Homozygous 9p21 deletion. LSI 9p21 gold probes-note the
missing gold probe signals in tumor (arrowed) cells.
Specimen Requirement
is calculated by determining the percent of 100
consecutive urothelial cells that are abnormal by FISH.
Cells that are obviously neutrophils, squamous, or
other non-urothelial cells are excluded from these
counts. Some cases are high level positives with 30%,
45%, or even 95% of the cells being abnormal. Other
cases are low level positives with <10% of the cells
being abnormal. The percent abnormal cells roughly
reflects the patients tumor burden.
In our experience, the vast majority of UC’s are
detected because they shed polysomic cells. However,
there are other less common FISH abnormalities that
can be observed such as trisomy (ie, 3 copies) of one of
the chromosomes and disomy (ie, 2 copies) of the other
chromosomes, or homozygous 9p21 deletion (both
Fluorescence In Situ Hybridization (FISH) For
Urothelial Cancer, Urine (#81975) can be performed on
any type of urine specimen including voided urine, urine
obtained by catheterization, bladder washings, stomal
urine specimens, and ureteral washings. We recommend
collecting at least 30 mL of urine. However, it may not be
possible to obtain this volume for certain specimens, such
as ureteral washings. In those situations, the laboratory
will perform FISH despite the less than optimum
specimen volume.
An equal volume of 70%, 95%, or absolute ethanol
should be added (other preservatives, such as
PreservCyt, also are acceptable) to the urine submitted.
The refrigerated (not frozen) specimen should be
transported to Mayo Medical Laboratories (MML).
The laboratory routinely provides 2-day turnaround
for results.
Mayo currently is conducting studies to determine if
FISH may have a role in screening for UC. Meanwhile,
FISH offers a sensitive, specific, and non-invasive test
for monitoring patients with urothelial carcinomas.
Clostridium difficile Toxin Kit Change
1. Halling KC, King W, Sokolova IA, et al: A comparison of
cytology and fluorescence in situ hybridization for the
detection of urothelial carcinoma. J Urol, 2000;164(5):1768-75
2. Bubendorf L, Grilli B, Sauter G, et al: Multiprobe FISH for
enhanced detection of bladder cancer in voided urine
specimens and bladder washings. Am J Clin Pathol
3. Halling KC, King W, Sokolova IA, et al: A comparison of
BTA-Stat, hemoglobin dipstick, telomerase, and UroVysio™
assays for the detection of urothelial carcinoma in urine. J
Urol (accepted for publication)
Mayo has converted Clostridium difficile Toxin, Feces
#8131 from the Clearview® kit for detecting C difficile
toxin in feces, to a Primer® kit. The Primer kit detects
both toxin A and toxin B (the Clearview kit detected
only toxin A). It has been shown that some diseasecausing strains of C difficile produce only toxin B.
Therefore, immunoassays which detect only toxin A
will provide false-negative results for those strains.
Monoclonal Protein Urine Study
Mayo has implemented a new ordering strategy for
detection of monoclonal proteins in urine. Monoclonal
Protein Study, Urine #8823, (MPSU) provides total
protein, immunofixation, and protein electrophoresis
testing. This test is useful for the detection and
immunotyping of abnormal protein in urine. The
specimen requirement is listed below.
The introduction of MPSU coincides with a method
change in the urine total protein test and deletion of
Immunoelectrophoresis, Urine #8179, from the test
offerings. The urine total protein test method will
change from a Coomassie Blue stain to a Pyrogallol Red
stain. Pyrogallol Red stain can result in a higher value
for some monoclonal proteins. Consequently, for
patients who have previously had a positive test with
Coomassie Blue stain, MML will assess the patient's
next specimen with both stains to provide an individual
comparison. This service will be offered until June 2002.
Specimen Requirement
30 mL urine from a 24-hour collection. Although a
24-hour specimen is recommended, a random
specimen will be tested if sent. Send three 10-mL
specimens refrigerated in plastic 13-mL urine tubes.
Preferred: plain
Acceptable: toluene or thymol
In addition, Electrophoresis, Protein, Urine #82441
preservative options have also changed:
Preferred: plain
Acceptable: toluene or thymol
Human Immunodeficiency Virus Type 1 Genotyping
Information Requirement
Human Immunodeficiency Virus Type 1 (HIV-1)
Genotyping, Plasma #82340 should be performed only
on patient specimens that have a viral load ≥1000
copies/mL, as the test does not detect below this limit.
MML now requires a recent viral load result (performed
within 14 days of genotype draw) be included with the
test request. If this information is not provided, the
specimen will be held until the information is made
available to MML.
If the patient does not have a current viral load result,
Human Immunodeficiency Virus Type 1 (HIV-1) RNA
by Polymerase Chain Reaction (PCR), Quantitative,
Plasma #80928 can be added to the test request and will
be performed at an additional charge. If the resultant
viral load is ≥1000 copies/mL, genotyping will be
performed. If the viral load is <1000 copies/mL, the
genotyping test will be canceled.
Growth Hormone Moves to Automated Method
Growth Hormone, Serum #8688 was converted to an
FDA-approved automated chemiluminescent
immunoenzymatic assay. This method is more sensitive
and provides better precision. The new assay is
calibrated against pituitary hGH 80/505* and yields
results that are lower than the previous method. As a
result, the reference values have been adjusted. The
preferred specimen transport temperature has changed
from frozen to refrigerated (frozen specimens will still
be assayed).
New Reference Values
≤1.5 ng/mL
Females: ≤4.0 ng/mL
Previous Reference Values
≤5 ng/mL
Females: ≤10 ng/mL
*World Health Organization 1st International Standard for
growth hormone, derived from human pituitaries (not
recombinant), given the code number 80/505 by the National
Institute of Biological Standards and Control.
Cystic Fibrosis Test Method Change
The American College of Obstetrics and Gynecology and the American College of Medical Genetics released
recommendations on mutation testing for cystic fibrosis. In keeping with those recommendations, Mayo has converted
Cystic Fibrosis, Diagnosis and Carrier Detection #9497 to a polymerase chain reaction-based assay using line probe
(reverse blot) technology in combination with confirmation sensitive gel electrophoresis for all 25 mutations specified
for population-based carrier screening. Additionally, this test also detects 2143delT, 3905insT, 394delTT, E60X, Q552X,
and S1251N. Intron 8 poly T testing is performed as a reflex test when appropriate. There are related changes to the
reference value and CPT codes. Additionally, the test now will be performed twice a week, on Monday and Wednesday.
New Reference Values
Racial or Ethnic
Ashkenazi Jewish
Mixed European
Northern European
Hispanic American
African American
Asian American
Mutation Detection
Rate - Using ACOG 25
mutation panel
Data not available
Carrier risk - prior to
Cystic Fibrosis Carrier
Carrier risk - with a
negative test result for
ACOG panel
Data not available
Table adapted from the ACMG position statement of CF screening (Genetics in Medicine 2001; 3:149-154)
Previous Reference Values Omitted
New CPT Codes
Previous CPT Codes
Abstracts of Interest
Clostridium difficile–Associated Diarrhea and Colitis
Said Fadi Yassin, MD; Tonia M. Young-Fadok, MD, MS; Nizar N. Zein, MD; And Darrell S. Pardi, MD
Clostridium difficile is a spore-forming toxigenic bacterium that causes diarrhea and colitis, typically after the use
of broad-spectrum antibiotics. The clinical presentation ranges from self-limited diarrhea to fulminant colitis and
toxic megacolon. The incidence of this disease is increasing, resulting in major medical and economic
consequences. Although most cases respond quickly to medical treatment, C difficile colitis may be serious,
especially if diagnosis and treatment are delayed. Recurrent disease represents a particularly challenging
problem. Prevention is best accomplished by limiting the use of broad-spectrum antibiotics and following good
hygienic techniques and universal precautions to limit the transmission of bacteria. A high index of suspicion
results in early diagnosis and treatment and potentially reduces the incidence of complications.
Mayo Clinic Proceedings 2001;76:725-730
Meeting Calendar
Interactive Satellite Program . . .
February 12, 2002
Advances in the Management of Rheumatoid Arthritis
Presenters: Harvinder S Luthra, MD; Alexander P Ruggieri, MD
Moderator: Steven R Ytterberg, MD
March 12, 2002
Diabetes Prevention and Management
Presenter: Mehmood A Khan, MD
Moderator: Robert M Kisabeth, MD
Upcoming Education Conferences . . .
March 12-13, 2002
Practical Spirometry
Course Director: Paul D Scanlon, MD
Rochester, Minnesota
May 1-3, 2002
Integration Through Community Laboratory Insourcing:
From Mission Statement to Successful Implementation
Hilton in the Walt Disney World®
Lake Buena Vista, Florida
For additional information regarding the above programs, please contact the
Mayo Reference Services Education Office at 1-800-533-1710.
( US
Does the new Lyme vaccine cause a positive Lyme serology test?
Yes. The screening enzyme-linked fluorescent immunoassay (ELFA) may show a positive ELFA result in subjects who
have received the Lyme vaccine, but who have not been exposed to Lyme disease.
For this reason Lyme Disease Antibody, Confirmation, Serum #9535 includes a Western Blot test. The Western blot test is required
to distinguish a vaccine response (1 band [OpsA] only) from true exposure (5 or more bands).
What is the benefit of ordering your amplified direct TB test over your routine culture method for TB?
Conventional culture methods typically take 2-8 weeks to detect Mycobacterium tuberculosis, although results may be
available as early as 1 week in uncommon cases. MML offers rapid detection of M tuberculosis by Mycobacterium
Tuberculosis, Amplified Direct Test, #81383. The M tuberculosis Direct (MTD) test provides detection of M tuberculosis complex
ribosomal ribonucleic acid (rRNA) in less than 1 hour. While the laboratory charges for the direct test are higher than the
laboratory charges for traditional culture, early detection and prompt treatment is critical to reduce the spread of the disease.
Rapid diagnosis also allows more appropriate use of isolation rooms and reduced hospital expenses.
Editorial Board:
Communiqué Staff:
Jane C Dale, MD
Managing Editor: Denise Masoner
Tammy Fletcher
Medical Editor: Jane C Dale, MD
Suzanne Leaf-Brock
Denise Masoner
Anita Workman
Contributors: David J Brandhagen, MD; Virgil F Fairbanks, MD; Kevin C Halling, MD, PhD;
Darrell S Pardi, MD; Stephen N Thibodeau, PhD
The Communiqué is published by Mayo Reference Services to provide laboratorians with
information on new diagnostic tests, changes in procedures or normal values, and continuing
medical education programs and workshops.
A complimentary subscription of the Communiqué is provided to Mayo Medical Laboratories’
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