College of American Pathologists
Printable Version

  Q & A





cap today

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

Member, CAP
Surgical Pathology Committee

With members of the
CAP/National Society
for Histology Committee:

Freida L. Carson, PhD, HT(ASCP)

Lena T. Spencer,

Vincent Della Speranza,

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 ┬Ám) 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.

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

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.

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