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  Q and A




January 2009

Fredrick L. Kiechle, MD, PhD

Question Q. How important is it to provide a physician with the immature reticulocyte fraction and the mean reticulocyte volume?

A. The CAP Hematology/ Clinical Microscopy Resource Committee feels that when any new parameter becomes potentially available, the laboratory needs to consult with its clinicians to see if they want it reported and understand the effect on laboratory practice such reporting will entail. Some reports, for instance, suggest that immature reticulocyte fraction is of interest in managing bone marrow transplant patients, so measuring and reporting it could be desirable in such a population. With any parameter, the use and limitations must be clearly understood and the result provided must be well validated.

Robert Novak, MD
Department of Pathology
Children’s Hospital Medical Center of Akron
Akron, Ohio

Immediate past chair, CAP Hematology/
Clinical Microscopy Resource Committee

Question Q. Our histopathology lab performs direct immunofluorescent stains for renal and skin biopsies. Are positive and negative controls required for these stains? If so, what would be an acceptable schedule for doing the controls, and where can our lab obtain them?

A. I have had a great deal of experience performing direct immunofluorescence, or DIF, on renal and skin biopsies and firmly believe positive and negative controls are necessary. Any institution capable of this type of offering (DIF testing, interpretation, report) will also have available tonsil/adenoid specimens that can be sampled, frozen, tested, and validated as positive control material. It is not difficult to do. These tissues can even be pre-cut onto slides and stored frozen, but it is not necessary to do so. The frozen tissue control blocks can be stored frozen in OCT compound that is wrapped in aluminum foil and sealed in airtight bags. These tissue control specimens can be continually renewed. They are common in the surgical pathology laboratory.

An acceptable schedule for controls is as follows: one set of controls for each run—IgG, IgM, IgA, C1q, C3, C4, fibrinogen, and so on. Multiple cases can be performed in a single run with one set of controls. Some institutions place the control and patient sections on the same slide.

Validation and titration studies should be performed on each fluorescein-isothiocyanate (FITC) conjugated (or other) antibody used for testing. The tissue controls described above are ideal for these studies. They should be treated just like the patient samples. That is, if patient samples are placed into Michelís fixative, the control tissues should be as well.

Positive controls simply tell you the test is working. The negative control, done by substituting non-immune Ig serum at a similar dilution (for example, 1/200) on the patient tissue, is the true indicator all is well with the assay (even though this negative control serum is not FITC-conjugated).

Sometimes it is difficult to determine whether the sensitivity of the assay is appropriate. This can be gauged only by observing the staining from day to day, as with IHC. Using positive cases like renal biopsies with systemic lupus erythematosus, membranous glomerulonephritis, or IgA disease is a good way. A skin biopsy with pemphigus is also useful.

In renal biopsy pathology, the light microscopic findings often correlate very well with the DIF findings (that is, mesangial disease observed by light microscope versus mesangial IgA/C3-positive DIF; lumpy-bumpy basement membrane deposits observed in light-microscopy examination versus definitive pattern of granular IgG observed in the peripheral capillary loops). This gives the user some degree of confidence in DIF stain findings. If recorded, these cases can be used to gauge sensitivity and specificity with primary antibodies. It is a matter of record keeping.

Robert L. Lott, HTL(ASCP)
Manager, Anatomic Pathology
Trinity Medical Center/LabFirst
Birmingham, Ala.

Question Q. Our laboratory is closed on weekends and holidays. Is it acceptable to use a “hi/lo” digital thermometer to cover the period when no one is in the lab? The storage devices are not on a monitoring system.

A. It is acceptable to use hi/lo digital thermometers or ìmin/maxî thermometers to monitor temperatures when no staff members are in the laboratory. Small laboratories that perform limited services often use such devices.

This is addressed in laboratory general checklist item GEN.41042, which reads:

Are refrigerator/freezer temperatures checked and recorded daily?
Note: This checklist question applies to refrigerators/freezers containing reagents or patient/client specimens.“Daily” means every day (7 days per week, 52 weeks per year). The laboratory must define the acceptable temperature ranges for these units. If temperature(s) are found to be outside of the acceptable range, the laboratory must document appropriate corrective action, which may include evaluation of contents for adverse effects.

It is imperative that the laboratory validate this temperature-monitoring system before implementing it and verify thermometer accuracy periodically as part of its equipment maintenance program. If the monitoring system indicates that a temperature fell outside the acceptable range, then the laboratory must undertake pre-established corrective measures to ascertain whether the contents have been adversely affected and to remedy any problems.

Linda D’Agostino
Senior Technical Specialist,
Division of Laboratory Accreditation
College of American Pathologists
Northfield, Ill.

Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.