Answering your questions on HEW certification vs. BS degrees, reporting spermatozoa in urine exams, Calcoflour stains, and hair analysis.

HEW certification versus BS

Q Under CLIA, what does a certificate from the Department of Health Education and Welfare (HEW) give you? Is it the same as a four-year Bachelor of Science degree, or an associates degree?

A Medical technologists who are HEW certified are recognized in the CLIA regulations in several ways. First of all, they are qualified as testing personnel for both moderate and high complexity tests. In addition, under section 493.1461(b)(3) of the Code of Federal Regulations, they are qualified to serve as general supervisors for high complexity tests., This section refers to the previous regulation published on March 14, 1990. In this regulation, for a HEW technologist to qualify, they would have been required to have 6 years of experience prior to February 28, 1992. However, since the last HEW exam was given five years prior to 1992, the CLIA regulations were amended to allow the persons who took the examination during the last year it was given (March 1, 1986 through December 31, 1987) to qualify for general supervisor with only five years of experience, as long as they obtained that experience by January 1, 1994. Therefore, persons holding HEW MT certificates are allowed to do essentially the same tasks as a person with an associate's degree, although they would be required to have more years of experience to be allowed to perform at the general supervisor level.

- Connie Laubenthal, MS, CLS(NCA)

Reporting of spermatozoa in microscopic urine exams

Q The hospitals in my area all have a general policy of not reporting spermatozoa seen in a urine microscopic exam. Recently there has been a debate about the appropriateness of this policy. Those in favor of not reporting the spermatozoa argue that their presence is not indicative of any medical condition and that historically, it has not been reported with results.

Also, those in favor of not reporting sperm, especially in a minor, argue that there is no chain of custody of the sample, therefore, you may be dealing with a sample that was accessioned incorrectly. On the other hand, those in favor of reporting the spermatozoa argue that the laboratory would be technically and legally remiss if it did not report it. Is it possible we could also be missing a case of child abuse? What is the opinion of the panel?

A The presence of sperm in urine can be the result of residual sperm in the urethra following ejaculation or from retrograde ejaculation. The latter is the expulsion of sperm into the bladder instead of through the urethra. It is an abnormal condition, almost always due to organic abnormalities such as prostate or bladder surgery or following a sympathectomy. If infertility is a problem, the finding of sperm in the urine could be of clinical significance. In most cases, it does not add useful information to the urinalysis. However, because of its occasional value, I believe that sperm should be reported with test results when present.

As for the possible legal issues, I believe that in the absence of sperm in vaginal fluid, it is unlikely that spermatozoa would be found in the urine of a female being examined for possible rape. In a male, spermatozoa in the urine would have little significance except to suggest that ejaculation, either through intercourse or masturbation had occurred. The presence of spermatozoa in either situation is not evidence of child abuse without other findings of history. Therefore the lab should not be concerned about possible legal issues or chain of custody.

- Daniel M. Baer

Calcofluor stains

Q I am able to see bright green fluorescent shaped crystals with the Calcofluor white stain droppers which I have been trying. Yeasts and molds are either nonfluorescent or extremely pale. On rare occasions, if I allow the slide to sit overnight, I can observe bright fluorescent yeast cells. The acridine orange fluorescent stain I use works fine on our fluorescent microscope. I am disappointed with the results I get with the Calcofluor white stain and wonder if you have any suggestions.

Wet potassium hydroxide preparations from the esophagus come down from the operating room in petri dishes. The slides are wet when the lab receives these specimens. Can the potassium hydroxide be added to these slides in the lab instead of in the OR, or would the dried sample on the slide be unacceptable for examination?

A Cellufluor, which was previously called Calcofluor White M2R, is a vital stain that is absorbed onto extracellular [Beta]-linked polysaccharides such as those composed of chitin and cellulose. The trans isomer absorbs ultraviolet light at 340-400 nm (peak 345-365 nm) and emits visible blue light at 400-440 nm. Cellufluor is very photosensitive and is susceptible to partial photo-polymerization; hence, solutions of Cellufluor should be protected from light in low actinic (red) glassware.

The principle problems associated with the use of Cellufluor to stain fungi, regardless if they are yeasts or molds, include the 1) use of incorrect filters with epifluorescent illumination, 2) photo-polymerization of the stain when exposed to light for long periods of time, 3) precipitation of Cellufluor in the reagent bottles, and 4) fluorescence being masked by the presence of melanin in fungal cell walls. To avoid these problems, the correct filters need to be used. These filters are frequently not the ones used for other fluorescent stains such as acridine orange. Both the stock and working solutions in either water or potassium hydroxide must be protected from light. Precipitated Cellufluor should be removed by filtration of the stain solution. Even though a small amount of Cellufluor passes through the 0.45 [[micro]meter] filter, it is enough to stain fungal cells adequately. If the stain has been kept in the dark, the needed photostationary isomeric mixture of trans and cis molecules resulting in fluorescence develops upon exposure to long-wave ultraviolet light.

It should be the responsibility of the microbiologist to add the potassium hydroxide to clinical specimens. This ensures the proper concentration is used and all examination parameters are within accepted guidelines. Specimens that dry out prior to arriving in the microbiology laboratory can be placed in a leak-proof, capped tube containing a small amount of sterile saline or water. The potassium hydroxide can be added to the specimen in the laboratory. Dried specimens are unacceptable for mycology.

- Michael R. McGinnis

References

1. Moore, S.A. 1990. Cell size specific binding the fluorescent dye Calcofluor to budding yeast. Biochim Biophys Acta. 1035:206-213.

2. Polysciences, Inc. 1985. Data sheet 265.

Why hair analysis?

Q What is the rationale for performing hair analysis? How does it compare to urine analysis or blood analysis? What is usually measured in hair?

A Hair consists of a hair follicle and the hair shaft. The follicle contains both muscular and glandular components. The follicle is supplied by blood vessels, which bathe the follicle, and thus the follicle will absorb the various essential and potentially toxic elements. The transport is a one-way flow as the hair undergoes a hardening process as it approaches the skin. The hair shaft is that part of the skin which is above the skin surface. The hair stalk grows at approximately 1 cm each month. Thus, a 1-inch length of hair would represent about 5 months of growth or alternatively, a 5-month record of the elements that were incorporated into the hair follicle and then into the hair shaft. Human hair is made up of about 80% protein, 15% water, and some lipids and inorganic materials. The mineral content of the hair is 0.25% to 0.95% on a dry ash basis.

Hair analysis of trace metals is an excellent test for chronic exposure of toxic metals such as mercury. Hair element testing according to a study published by the Environmental Protection Agency in 1979 is "a meaningful and representative tissue for biological monitoring of most of the toxic metals."[1] Ideally, one could view hair analysis as a screening test, which, if positive, would lead to confirmatory testing in blood or urine. Elemental hair analysis includes the following potentially toxic metals: aluminum, arsenic, cadmium, lead, mercury and uranium.

- Bernard E Statland

Reference

1. U.S. EPA-600/4-049. August 1979.

Edited by Daniel M. Baer, MD, professor emeritus of Laboratory Medicine, Oregon Health Sciences University, Portland, Ore. Panelists (name following each answer indicates the responding panelist):

David Sewell, PhD, director of microbiology, Veterans Administration Medical Center, Portland, Ore. John A. Koepke, MD, professor emeritus of pathology, Duke University Medical Center, Durham, N.C. Byron A. Myhre, MD, PhD, professor of pathology and chief of clinical pathology, UCLA School of Medicine, Harbor General Hospital Campus, Torrance, Calif. Robert M. Nakamura, MD, senior consultant and chairman emeritus, department of pathology, Scripps Clinic and Research Foundation, La Jolla, Calif.