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November 2002
Q. We use the Bio-Rad HPLC system, which like any other
available method does not have the ability to accurately quantitate
Hb A
2
in the presence of Hb S.
We know from the literature that Hb A
2
is helpful in diagnosing and interpreting sickle cell-beta thalassemias.
As a policy, we do not report Hb A
2
in sickle cell cases. Some physicians insist that a high A
2
level in sickle cell trait/disease is diagnostic and interpret it
as sickle cell-beta thalassemia. How important is the knowledge
of Hb A
2
for interpreting and
diagnosing sickle cell-beta thalassemias? How do you report variant
Hb fractions A
2
, F, S, and A in
sickle cell trait/ disease cases? What tests and parameters are
used to diagnose sickle cell-beta thalassemia? Is there a method
or technique for accurately determining Hb A
2
in the presence of HbS?
A. It is true that Hb A
2
is spuriously elevated in the presence of Hb S. For example, Hb
A
2
can be elevated to 3.5 to 4.5
percent in Hb S trait and to six to seven percent when homozygous
S is present. This is believed to be due to the presence of a Hb
S “adduct,” which co-elutes with Hb A
2
.
Many feel that this is likely glycosylated Hb S (Hb S1C). This topic
was discussed more fully in CAP Hemoglobinopathy Survey 2000 HG-A.
Review of previous CAP hemoglobinopathy Survey data shows that this
A
2
elevation is an issue, in particular
with high-performance liquid chromatography, or HPLC, methods, but
it can also occur to a lesser extent with many column methods. As
has been said in the hemoglobinopathy Survey critiques, measurement
of Hb A
2
by densitometry is not
an acceptable method for quantitating Hb A
2
.
The level of Hb A
2
, however,
is not critical in the diagnosis of Hb S/β-thalassemia. To
review, everyone has two copies of the beta globin gene, located
on each copy of chromosome 11. Thalassemia mutations that involve
the beta globin gene have been divided into β
+
(reduced production of Hb A from the affected beta globin gene)
and β
0
(no production of
Hb A from the affected beta globin gene). The accompanying table
summarizes the results that would be expected in a variety of disorders.
Thus, the combination of Hb S with a β
0
-thalassemia
mutation would be expected to produce electrophoretic results equivalent
to homozygous Hb S. Although microcytosis may suggest Hb S/β
0
-thalassemia,
concurrent iron deficiency also must be considered. The hallmark
of Hb S in combination with a β
+
mutation is percentages of Hb S greater than Hb A. The only other
condition that would produce a situation where Hb S is greater than
Hb A is recent transfusion in a homozygous S (or Hb S/β
0
-thalassemia)
patient. Values of Hb S in the range of 35 to 40 percent, regardless
of the elevation of Hb A
2
, therefore
are consistent with Hb S trait.
The Hb A
2
level thus is not
critical in diagnosing these disorders. In Hb S trait or homozygous
Hb S, we report the Hb A
2
level
obtained by HPLC, but we attach to our reports a comment that "Hb
A
2
can be elevated in the presence
of Hb S without implying concurrent β-thalassemia."
James D. Hoyer, MD
Department of Laboratory
Medicine and Pathology
Mayo Clinic
Rochester, Minn.
Advisor, CAP Hematology/Clinical
Microscopy Resource Committee Q. How many cassettes
should be processed by a histotechnologist during an eight-hour
shift?
A. No established comprehensive standard addresses
histology workload. Previously published CAP workload guidelines
for histopathology, based on data from the Laboratory Management
Index Program, say “each well-trained HT/ HTL can be expected to
produce approximately 3,000 slides per quarter, or 12,000 slides
per year. Included in these totals are 2,500 H&E slides and 500
common special stains, or 10,000 H&E slides and 2,000 common special
stains. If only rare special stains are requested, more set-up time
is required. Twelve thousand slides per year is equivalent to 50
slides per day. These are average numbers for a lab where much of
the work is not automated.”
In practice, a uniform standard across laboratories may be an
unrealistic goal because many factors influence the number of blocks
a histotechnician/technologist can cut in a given period. These
include:
- The experience level of the technician. A new employee or student
would be expected to cut at a slower rate.
- The case complexity. Biopsies, which require multiple levels
and careful trimming, require considerably more time than routine
cases (for example, uterus).
- The number of interruptions. Smaller laboratories, in which
the cutting technician may be answering the phone or receiving
special stain or recut requests, or both, will have lower productivity.
The most useful standard for employees in a given laboratory is set
by the supervisor or senior technologists, or both, based on past
productivity levels.
To address productivity in a more global sense, it is necessary
to assign work units to each of the varied tasks in histology, including
loading and maintaining processors, embedding, cutting, routine
staining, special stains, and immunohistochemistry. It is then possible
to benchmark units worked per hour. As with routine cutting, however,
the assignment of work unit values to a given task can only be done
realistically by the histology supervisor and pathologist at a given
site, taking into account economies of scale and levels of automation.
Richard W. Brown, MD
Medical Director,
Core Histology Laboratory
Memorial Hermann Healthcare System
Houston
Member, CAP
Surgical Pathology Committee
With members of the
CAP/National Society
for Histology Committee:
Freida L. Carson, PhD, HT(ASCP)
Lena T. Spencer,
MA, HT(ASCP)HTL, QIHC
Vincent Della Speranza,
MMS, HT(ASCP)HTL
Sue E. Lewis, HTL(ASCP)
Q. Do prosthetic particles polarize when viewed
with a polarizing microscope? I am trying to develop a procedure
for staff to deal with physician requests to determine if there
are particles from prosthetic devices in synovial fluid.
A. The presence of particles in synovial fluid that
appear to be derived from the articular surface of prosthetic hips
and knees has been studied, often in an effort to predict prosthesis
wear and nonseptic prosthesis failure. A correlation has been sought
between the morphology and number of these particles and prosthesis
problems using many techniques, including light microscopy with
polarizing filters, light microscopy with oil red O staining, scanning
electron microscopy, and various particle-counting methods. The
latter two usually are performed after digestion of the synovial
fluid with a strong base (NaOH or KOH) to remove organic materials
before analysis. Because the question involves analyzing synovial
fluids in a clinical laboratory, the discussion will be limited
to issues with light microscopic methods.
You must first consider the type of prosthesis you are dealing
with to anticipate what type of particles you might encounter. In
knee prostheses, the articular surface is usually a high-molecular-weight
polyethylene, so polyethylene particles would be sought. In hip
prostheses, metal-on-metal prostheses have been associated with
the presence of metal fragments, whereas hip replacements with a
polyethylene articular surface yield both metal and polyethylene
particles. Metal particles cannot be polarized or stained and need
an approach such as spectroscopy to be identified reliably. Thus,
in the clinical laboratory, polyethylene particles are the most
likely to be identified.
Polyethylene particles show birefringence with polarized light
and stain with oil red Ο, though neither technique is obviously
specific for polyethylene.
1
This
nonspecificity of light microscopy is demonstrated in one study
of particles in the synovial fluid of patients receiving hip prostheses,
where about 50 percent of preoperative specimens were reported by
the laboratory to have polyethylene particles, when the status of
the patient was unknown to the laboratory.
2
Furthermore, it has been demonstrated that polyethylene particles
are present in synovial fluid in most patients with knee prostheses,
if carefully sought, and that it is the number and size of the particles,
not merely their presence, that correlate with prosthesis problems.
3,4
Some have suggested it is the small globular particles (diameter,
<5 µm) that are most significant, as these can be phagocytized by
monocytes, resulting in the release of inflammatory cytokines, which
lead to osteolysis and prosthesis failure. These particles are not
as easily detected by light microscopic techniques as larger elongated
forms, which are readily detected but may not be as clinically significant
as the smaller particles.
In view of these considerations, merely finding apparent prosthesis
fragments in synovial fluid may not be of particular value to your
clinicians, and you may want to discuss with them alternative approaches
to optimize the utility of the fluid analysis to their patients.
References
1. Peterson C, Benjamin J, Szivek J, et al. Polyethylene
particle morphology in synovial fluid of failed knee arthroplasty.
Clin Orthop. 1999;359:167-175.
2. Dorr L, Hilton K, Wan Z, et al. Modern metal
on metal articulation for total hip replacements. Clin Orthop.
1996;333: 108-117.
3. Bosco J, Benjamin J, Wallace D. Quantitative
and qualitative analysis of polyethylene wear particles in synovial
fluid of patients with total arthroplasty. Clin Orthop.
1994;309:11-19.
4. Calonius O, Saikko V. Analysis of polyethylene
particles produced in different wear conditions in vitro. Clin
Orthop. 2002;399:219-230.
Robert Novak, MD
Department of Pathology
Children’s Hospital
Medical Center of Akron
Akron, Ohio
Vice Chair, Hematology/Clinical
Microscopy Resource Committee Q. Prostate-specific
antigen has become man’s best friend as a screening tool. However,
many manufacturers say their test should not be used for screening
but rather for charting disease course or treatment. Further, CAP
Surveys (ligand) data show wide variance of PSA values between methods,
yet most rely on the same 0-4.0 ng/mL reference range. (Some have
begun reporting age-related ranges.) Are we, or should we be, heading
toward an International Normalized Ratio in PSA testing? What is
the status of the use of age-related normal ranges for PSA in serum?
Is there justification for continuing to perform prostatic acid
phosphatase in the clinical laboratory?
A. These questions pertain to the measurement of
prostate-specific antigen for prostate cancer screening. PSA is
a serine protease of molecular weight 34 kD that is produced by
the epithelial cells lining the acini and ducts of the prostate
gland. Proliferation of these cells, due to a benign or malignant
process, causes the concentration of PSA in blood to increase. Men
with early prostate cancer have, on average, higher serum PSA levels
than men without cancer, but large overlap between the two populations
makes reliable discrimination impossible. Choosing a cutoff involves
the usual tradeoff between sensitivity and specificity. At the commonly
used fixed cutoff of 4.0 ng/mL, sensitivity and specificity are
each perhaps in the 70 to 75 percent range.
1
Evaluating prostate cancer screening is also controversial because
it can be argued that some cancers are so slow-growing they are
better left undetected. PSA may be man’s best friend, but some might
say that prostate cancer screening is a dog.
Despite its imperfections, PSA is the best biochemical test for
prostate cancer. Prostatic acid phosphatase, or PAP, appears to
offer no additional benefit for screening, diagnosing, staging,
or monitoring. PAP may come into play with those rare patients who
have a prostate cancer that does not secrete PSA, and forensic labs
measure it to test for the presence of semen, but most clinical
labs are encouraged to abandon the test.
2
Several years ago a large, multicenter evaluation of prostate
cancer screening compared the first commercial test for PSA (the
Hybritech assay, now marketed by Beckman Coulter Inc.) to digital
rectal examination in men over age 50.
3
It was found that PSA was more effective than DRE but that each
technique picked up some cancers that the other missed; thus screening
was most effective when the modalities were combined. Based on this
evidence, the Food and Drug Administration allowed the manufacturer
to claim that "the test is effective in the detection of prostate
cancer when used in combination with digital rectal examination
(DRE) in men aged 50 years or older." Some other manufacturers of
PSA tests may be permitted to claim only that their test is useful
for monitoring prostate cancer and may even caution against using
their test for screening. That does not mean these other brands
are ineffective for screening, only that effectiveness has not been
proved to the FDA.
But as the questioner points out, all brands of PSA test are not
equivalent. Differences observed in proficiency tests can be misleading
because the tests are conducted with artificial materials that differ
in important ways from authentic patient specimens. Nevertheless,
commercial assays are known to have differed in absolute calibration
and sensitivity to different molecular forms of PSA, in particular
free PSA versus complexes with antiproteases such as alpha-1-antichymotrypsin.
The latter differences cannot be eliminated by mathematical adjustment
analogous to the INR, which adjusts prothrombin time measurements
for the varying sensitivities of thromboplastin reagents. The trend,
however, has been toward harmonizing commercial PSA assays.
4
In summary, the sensitivity and specificity of PSA testing for
prostate cancer screening are (a) fairly low; (b) dependent to some
extent on the brand of assay; and, of course, (c) dependent on the
cutoff chosen. Because (a) is inescapable, discussion of (b) and
(c) is somewhat mitigated. Many laboratories continue to use the
fixed cutoff of 4.0 ng/mL that was established in earlier studies.
3
It has the advantages of simplicity and wide acceptance. With a
fixed cutoff, however, specificity of testing will decline with
age because as a man ages, the prostate tends to enlarge. Age-adjusted
cutoffs—for example, as proposed by Oesterling, et al
5
—achieve
more uniform specificity across the male lifespan. Some, however,
have criticized the use of higher cutoffs in older men because,
naturally, they lower the sensitivity.
6
Other approaches to improving the PSA test include use of PSA
velocity, PSA density, and measurement of free or complexed PSA.
Reported benefits are controversial, but it is safe to state that
in no case is the detection of cancer as efficient as one would
desire.1 More discussion of these and other issues related to PSA
testing can be found in a best practice policy issued by the American
Urological Association, available at www.cancernetwork.com/journals/oncology/o0002e.htm.
As of Jan. 1, 2000, Medicare began to reimburse PSA tests to screen
for prostate cancer. Coverage applies to one test per 12-month period
in men over 50 years of age.
References
1. Bunting PS. Screening for prostate cancer with
prostate-specific antigen: beware the biases. Clin Chim Acta.
2002; 315: 71-97.
2. Bunting PS. Is there still a role for prostatic
acid phosphatase? CSCC position statement. Clin Biochem.
1999;32: 591-594.
3. Catalona WJ, Richie JP, Ahmann FR, et al. Comparison
of digital rectal examination and serum prostate specific antigen
in the early detection of prostate cancer: results of a multicenter
clinical trial of 6,630 men. JUrol. 1994; 151: 1283-1290.
4. Chan DW, Sokoll LJ. WHO First International
Standards for prostate-specific antigen: the beginning of the end
for assay discrepancies? Clin Chem. 2000; 46:1291-1292.
5. Oesterling JE, Jacobsen SJ, Chute CG, et al.
Serum prostate-specific antigen in a community-based population
of healthy men. JAMA. 1993; 270: 860-864.
6. Slovacek KJ, Riggs MW, Spiekerman AM, et al.
Use of age-specific normal ranges for serum prostate-specific antigen.
Arch Pathol Lab Med. 1998; 122: 330-332.
Jay L. Bock, MD, PhD
University Hospital SUNY
Stony Brook University
Stony Brook, NY
Member, CAP Therapeutic
Drug Monitoring/Endocrinology
Resource Committee
George Klee, MD, PhD
Mayo Clinic
Rochester, Minn.
Vice Chair, CAP Therapeutic Drug Monitoring/Endocrinology
Resource Committee
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