Lab temperature control, clue cells, Kleihauer-Betke stain, fixing AFB smears, and sweat test utilization

June 1, 2002
Edited by Daniel M. Baer, M.D.

Lab temperature control

Q: Are there standards for laboratory environmental conditions? We have absolutely no control over air temperature in our lab. We freeze in the summer and swelter in the winter. This was not a problem in the days when we worked with the lab doors wide open, but now we must work with doors closed. We have complained, but with no effect. Id like some ammunition, if it exists. 

A: Both CAP and NCCLS mention lab environmental conditions in their documents, but neither has specific standards. 

The CAP inspection checklist question GEN.61300 asks: Are the room temperature and humidity adequately controlled in all seasons?

The preface to this question says: Ambient or room temperature and humidity must be controlled throughout the year to minimize evaporation of specimens and reagents, to provide proper growth conditions for room temperature incubation of cultures, and not to interfere with the performance of electronic instruments. Personnel comfort is important, but does not warrant a phase II deficiency if work is not compromised.

A temperature or humidity problem is a phase I deficiency.1

NCCLS discusses the need for temperature and humidity control in its Laboratory Design Guideline, 2 but does not set standards that should be met.

Some instrument manufacturers have temperature and humidity specifications in their instrument manuals.

Temperature and humidity control in laboratories is a fairly recent standard. Before the days of widespread air conditioning in buildings, labs with widely varying temperatures were common. This should be taken into consideration when reading older laboratory methods. In the British literature, room temperature incubation prior to the 1940s referred to 65 degrees F.

Daniel M. Baer, M.D.
Professor Emeritus
Department of Pathology
Oregon Health and Science University
Portland, OR


1. College of American Pathologists, Laboratory Accreditation Program Laboratory General Checklist, Question GEN.61300, 21 February 2000 edition.

2. NCCLS, Laboratory Design; Approved Guideline GP 18-A, 1998. Wayne, PA,
NCCLS. p 32.

Clue cells

Q: When performing a microscopic examination on urine sediment, I occasionally observe clue cells. Should clue cells be reported on the urinalysis report?

A: Epithelial cells that line all of the urinary and genital tracts are continuously sloughed off, and are occasionally seen in the urine sediment. The epithelial cells are of three types:
squamous, transitional (urothelial), and renal.1 Increased numbers of epithelial cells in the urine sediment is abnormal, but generally only the increased presence of renal tubular epithelial cells is clinically significant.2 Squamous epithelial cells
(SECs) line the female urethra, the distal portion of the male urethra, and the vagina. Large numbers of SECs in the urine sediment usually represent contamination from the foreskin in males or the vaginal contents in women. Occasionally this finding is the result of a rectovesical (rectum-bladder) or a rectovaginal fistula. 

Clue cell is a term for vaginal SECs that are coated with tiny coccobacilli such that the SEC border is obliterated or appears shaggy and may be dense enough to partially obscure the nucleus.3 The presence of clue cells in vaginal secretions is one of four findings used for the diagnosis of bacterial vaginosis (BV). Other laboratory signs include a vaginal pH greater than 4.5, a positive whiff test (fishy odor on addition of 10 percent KOH to vaginal secretions), and abnormal vaginal flora (absence of lactobacilli and increase of Gardnerella and anaerobes). Usually clue cells are visualized in vaginal wet mounts or stained smears. Although the presence of clue cells in urine sediment indicates a poorly collected specimen, it suggests that the woman may have BV and should be noted on the urinalysis report.

David Sewell, Ph.D., ABMM
Director of Microbiology
Veterans Affairs Medical Center
Portland, OR


1. Ringsrud KM and JJ Linne. Urinalysis and Body Fluids: A Color Text and Atlas. St. Louis, MO:
Mosby-Year Book, Inc. 1995.

2. Haber MH. Urine. In: McClatchey KD, ed. Clinical Laboratory Medicine. Baltimore, MD: Williams and Wilkins; 1994: 513-548.

3. Spiegel CA. Bacterial vaginosis: Changes in laboratory practice. Clin Microbiol News. 1999; 21: 33-37.

Kleihauer-Betke stain

Q: In our laboratory we offer the Kleihauer-Betke stain for the determination of fetal hemoglobin in red blood cells, but the procedure is difficult and time-consuming. Can you help us find an alternative? 

A: The Kleihauer-Betke stain has been used to differentiate normal adult hemoglobin-containing red cells from red cells containing fetal hemoglobin.1

If there is a fetal to maternal bleed during pregnancy which could trigger the production of maternal antibodies directed against the fetal cells resulting in hemolytic disease of the newborn, the early discovery can lead to the prevention of immunization by giving immune gamma globulin to the mother. 

In the past, the transfusion service tested the mothers blood for the presence of Rh D-positive red cells from the fetus. Now a positive result requires confirmation with a Kleihauer-Betke stain or flow cytometric quantitation of fetal hemoglobin. This is because the required dose of Rh immune globulin depends on the volume of the fetal-maternal bleed, with larger bleeds requiring larger doses of immune globulin.

Also there can be increased levels of fetal hemoglobin-containing red cells in thalassemia, sickle cell, and other anemias, and in such cases the determination of fetal hemoglobin-containing cells is a necessary diagnostic test. 

As you know, the test can be somewhat daunting, especially if it is requested infrequently. A blood film is reacted with a weak acidic solution which preferentially elutes adult hemoglobin from the red cells. The film is stained with hematoxylin and eosin and carefully examined under the microscope for the presence of so-called ghost red cells that have lost their content of hemoglobin. Especially low concentrations of such cells require counting larger numbers of red cells in order to ensure the precision necessary to determine the proper dose of immune globulin. Commercial kits (e.g., Sigma Diagnostics) are marketed and could help solve your problem. 

The NCCLS has recently published a comprehensive guideline for fetal red cell detection, and you will find a wealth of information on all of these methods in that document.2 

John A.
Koepke, M.D.

Professor Emeritus of Pathology
Duke University Medical Center
Durham, NC


1. Kleihauer, E, Braun, H & Betke, K. Demonstration of fetal hemoglobin in erythrocytes of a blood smear Klin Wochenschr 35: 637-638, 1957 

2. Fetal Red Cell Detection; Proposed Guideline H52-P. NCCLS, Wayne, Pennsylvania 2001. 

Fixing AFB smears

Q: What are the safety recommendations for fixing AFB smears prior to staining? We currently fix at 65 degrees C for two hours, but may switch to the one minute in absolute methanol method as described in the Clinical Microbiology Procedures Handbook.1 Is methanol fixation for one minute or heating at 65 degrees C adequate to kill M. tuberculosis on the smear?

A: The commonly recommended fixation method for AFB smears is heating at 65 degrees C for two hours; however, this method is not tuberculocidal.2,3 An acceptable alternative is to process a portion of the specimen by adding a volume of sodium hypochlorite equal to the volume of specimen, shaking briefly, and allowing the mixture to stand at room temperature for 15 minutes before centrifugation. This treatment effectively kills all mycobacteria; however, if you need to culture the specimen, another portion of the specimen must be processed.

The resistance of M. tuberculosis to disinfectants or fixation methods is greater than vegetative bacteria, but less than bacterial spores. Both ethyl and isopropyl alcohol are considered excellent tuberculocidal agents, but their activity is affected by the concentration of M. tuberculosis in the sample, the presence of organic material (e.g. sputum), the type of test performed to measure cidal activity of the disinfectant (suspension or carrier), and the time of exposure to the disinfectant. Best, et al. demonstrated that exposure to ethanol (70 percent) for one minute was effective against M. tuberculosis only in a suspension test in the absence of sputum.4 Methanol (80 percent) in a carrier test was effective after exposure for 30 minutes. Even the thickness of the smear affects the activity of the disinfectant; however, staining by either the Ziehl-Neelsen or fluorochrome method effectively inactivates M. tuberculosis on sputum smears.3

I was not able to find specific data relating to the viability of M. tuberculosis in a sputum smear after exposure to absolute methanol for one minute. In the absence of such data, the prudent action would be to assume that M. tuberculosis is viable until stained and handle the smear as you would after heat fixation. 

David Sewell, PhD, ABMM


1. Isenberg, H. I. Clinical Microbiology Procedures Handbook. American Society For Microbiology. Washington, D.C. 1992

2. Smithwick RW and CB Stratigos. Preparation of acid-fast microscopy smears for proficiency testing and quality control. J Clin Microbiol 8:110-111, 1978.

3. Goldfogel GA and DL Sewell. Preparation of sputum smears for acid-fast microscopy. J Clin Microbiol 14:460-461, 1981.

4. Best M, Sattar SA, Springthorpe VS and ME Kennedy. Efficacies of selected disinfectants against Mycobacterium tuberculosis. J Clin Microbiol 28:2234-2239, 1990. 

Sweat test utilization

Q: Respiratory allergies and asthma are common here in southwest Missouri, and some technologists feel that sweat testing is over-used. Any suggestions would be appreciated on how to encourage our physicians to show some restraint and order sweat testing only for chronic and not seasonal respiratory problems.

A: There are a variety of clinical presentations which suggest cystic fibrosis (CF) as a possible diagnosis. These are listed in Appendix A of NCCLSs guideline, Sweat testing: Sample collection and quantitative analysis.1 Because the majority of patients with cystic fibrosis (CF) present with acute or chronic respiratory symptoms, it is highly appropriate that a sweat test be ordered on a patient demonstrating symptoms of asthma or respiratory allergies. The most common clinical presentations are recurrent or chronic respiratory infections, failure to thrive, and chronic diarrhea, the three of which comprise the majority of illnesses in infants and young children who are diagnosed with cystic fibrosis. Unfortunately, these presentations are also frequent findings in normal infants, so a large number of sweat tests must be performed in order to rule out cystic fibrosis and to properly identify the few among the many who indeed have the disease. 

Most sweat tests performed will be negative for CF; for example, at our hospital, more than 90 percent of the sweat tests are negative. Our sweat testing workload increases during the late winter and early spring corresponding to an increase in respiratory infections. In conclusion, physicians should be encouraged, rather than discouraged, to order a sweat test in patients with symptoms suggestive of CF. 

Dr. Vicky A. LeGrys
School of Medicine
Division of Clinical Laboratory Science
University of North Carolina at Chapel Hill
Chapel Hill, NC 


1. NCCLS. Sweat testing: sample collection and quantitative analysis, approved quideline C34-A2. Wayne, PA. NCCLS 2000.

Daniel M. Baer is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLOs editorial advisory board.

© 2002 Nelson Publishing, Inc. All rights reserved.