Answering your questions

Dec. 1, 2008

Vaginal KOH preparations

Q Should vaginal KOH
(potassium hydroxide) preparations be heated? Should the KOH solution
contain DMSO (dimethyl sulfoxide)? What is the best KOH concentration to
use? We currently do not heat our KOH (10% solution) preps or use DMSO. We
have a physician who wants us to heat our slides and/or use DMSO to
completely clear our slides of cellular debris. Doing a literature search
seems just to have clouded the issue. I have found references for using 10%,
15%, and 20% KOH solutions. Some references suggest heating with Bunsen
burners (which we have not had in our labs in many years), and others say do
not use heat. Some places use KOH in a 40% DMSO solution, but most do not
mention DMSO use at all. Please let me know your recommendations on KOH
concentration, heating these preparations, and the use of DMSO. We are only
focusing on vaginal specimens — not skin KOH preparations where, perhaps,
more aggressive techniques are in order.

A Should vaginal KOH
preparations be heated? Heating the KOH prep is helpful to clear specimens
containing a large number of cells, such as skin and nails. It is not
necessary for vaginal preparations. Should the KOH solution contain DMSO?
DMSO is useful on specimens from skin, hair, or nails. Labs that use DMSO
with KOH find that they do not have to heat these specimens as the DMSO
gives more clearing of cells than KOH alone. It is not necessary for vaginal
preps. What is the best KOH concentration to use? Mycology references
recommend using KOH between 10% to 20%. The more cellular materials you have
in the specimen, the higher the concentration of KOH. Most clinical
laboratories use 10% and that would be sufficient for vaginal specimens.
Specimens containing more cells would be cleared easier with a 15% solution.
The procedure found on Doctorfungus.org1
references a 15% solution. Yeast can usually be visualized in a vaginal
smear using saline alone. It is also important to recognize that if the lab
is using KOH, it should also do a saline mount to check for
Trichomonas vaginalis
and clue cells. These would not be found in a
KOH prep.

—Lauren Roberts, MS,
Clinical Laboratory Science Program
Arizona State
Tempe, AZ


  1. The Laboratory. Lab Procedures. The Official Website of the Mycoses
    Study Group. Available at
    . Accessed November 20, 2008.

Further reading

Mahon CR, Lehman DC, Manuselis G. Textbook of
Diagnostic Microbiology
. 3rd ed. Philadelphia, PA: Saunders Elsevier;

Glucose collection guidelines

Q I am looking for
the most recent guidelines for specimen collection for glucose and how soon
the sample needs to be centrifuged after collection. Also, are there any
recent studies showing the effects of collecting in a lithium heparin vs.
sodium fluoride/potassium oxalate.

A According to the Clinical
and Laboratory Standards Institute guidelines on specimen handling,
specimens collected for glucose testing should be centrifuged and separated
from the cells within two hours of collection.1 To facilitate
separation from the cells, a gel tube with or without clot activator can be
used. Without centrifugation, glucose levels decrease significantly within
two hours at room temperatures. Warmer temperatures accelerate the decrease.
Since refrigeration of whole blood beyond two hours affects other analytes,
especially potassium, chilling a specimen for glucose and other analytes is
not recommended. When collecting into gel-separator tubes, it is important
that tubes are not recentrifuged if more than two hours have passed since
collection. Recentrifugation combines serum or plasma that has remained in
contact with the cells for a prolonged period with that which was timely
separated, resulting in a hybrid that will not likely yield an accurate

For the stabilization of glucose levels when
centrifugation is not possible, specimens should be collected into potassium
oxalate/sodium fluoride tubes (e.g., gray stopper). This additive provides
stability up to 24 hours if kept at room temperature — 48 hours when kept
refrigerated. In a study comparing glucose results in various tubes, no
changes in the concentration of glucose levels were observed when specimens
were collected into lithium-heparin or potassium-oxalate/sodium-fluoride
tubes.2 It stands to reason that glucose is more stable in
oxalate fluoride over time, but fresh samples should be equivalent.

—Dennis J. Ernst,
Center for Phlebotomy Education
Ramsey, IN


  1. Clinical and Laboratory Standards Institute.
    Procedures for the handling and processing of blood specimens. Approved
    Guideline — 2004. Wayne, PA: Clinical and Laboratory Standards Institute;
    CLSI Guideline H18-A3.
  2. Young D. Effects of Preanalytical Variables on
    Clinical Laboratory Tests
    . Washington, DC: AACC Press; 1997.

Peripheral blood smears for high platelet counts

Q What is the reason
to perform a peripheral blood smear on counts greater than 400 x 109/liter?
Our normal range is 140 to 400 x 109/liter. Would verification be
satisfied by retesting the platelet count electronically instead of

A The revised (04/06/2006)
College of American Pathologists criteria do not require verification of
platelet counts above the reference (normal) range; however, they do require
verification of results which fall outside the lower and upper reportable
(measurable, analytical measurement) range.

A reference range is the range of laboratory
test results expected for a given condition (usually health). The range
usually consists of the central 95% of the population of interest. The
reportable range
is the range that is defined by a minimum value and a
maximum value of calibration material.

A hematology laboratory must establish or verify the
upper and lower limits of all reportable parameters on the CBC instrument.
The results that fall outside these limits should be verified before

If the platelet counts fall below the reportable
lower limit, they must be re-analyzed by another method, such as manual
hemocytometry, semiquantitative blood-smear estimate, or flow cytometry. If
platelet counts are above the reportable range, they could be re-analyzed
using the minimal dilution necessary to bring the counts into the
instrument's analytical range.

When clinically appropriate, a laboratory may report
values as “less than” the lower limit or “greater than” the higher limit
(HEM.30250, Phase II).1

Blood-smear review to verify platelet count is
required in the following situations in which a hematology analyzer usually
shows a “flag” and abnormal histogram:

  • To prevent reporting of spurious thrombocytopenia when platelet
    clumps, giant platelets, or
    platelet satellitism are present (HEM.30300,
    Phase II)1;
  • When a significant number of microcytic erythrocytes, and/or small
    red-cell fragments are
    detected or suspected to verify the platelet
    count (HEM.30400, Phase II)1; and
  • Hemocytometry platelet counts should be correlated with a platelet
    estimate from a properly
    prepared blood film (HEM.33350, Phase II).1

In summary, the lab does not need to perform a
peripheral blood-smear review for a platelet count greater than reference
range but within the reportable range if there is no flag and no abnormal

—Sarah Henry, MD
Guang Fan, MD, PhD
Department of Pathology
Oregon Health and Science University
Portland, OR


  1. CAP Commission on Laboratory Accreditation,
    Hematology-Coagulation Checklist, HEM.30250, HEM.30300, HEM.30400,
    HEM.33350, revised 4/06/2006:


Henry JB, ed. Clinical Diagnosis and Management by
Laboratory Methods
. Philadelphia, PA: WB Saunders Company; 2001:139.

McKenzie SB, ed. Clinical Laboratory Hematology.
Upper Saddle River, NJ: Pearson Prentice Hall; 2004:938.

Drawing cardiac tests while transfusing

Q Is it all right to
obtain timed (Q6hr or Q8hr) cardiac tests, such as CPK (creatine
phosphokinase) and troponin, while the patient is receiving a blood

A Assays for cardiac
troponins are recognized as the most important lab tests for cardiac
diagnosis.1 Clinical consensus guidelines recognize measurements
of myocardial proteins in blood as an important tool in the evaluation of
myocardial necrosis. These guidelines cite the “nearly absolute myocardial
tissue specificity” and high sensitivity of cardiac troponins as a superior
assay compared to the best alternative — the MB (myocardial-specific)
fraction of creatine kinase (CK-MB).2 Specific guidelines
regarding monitoring markers of myocardial damage during transfusion are not
provided, however.

It is logical to assume that repeated transfusions
may dilute plasma levels of both cardiac troponins and CK-MB, giving falsely
decreased or false-negative results. Plasma dilution — specifically the loss
of plasma proteins due to hemorrhage and dilution secondary to colloid and
crystalloid transfusions — may cause the concentration of any plasma protein
to drop below the lower detection limit of a specific assay. In August 1986,
this phenomenon resulted in the unfortunate transplantation of HIV-infected
organs from a donor receiving a 56-unit transfusion for injuries suffered
during a motor-vehicle accident.3 Subsequently, transplantation
centers have been advised to reject organs from donors in whom plasma
dilution is sufficient to affect the outcome of communicable disease

In cases where patients are receiving large infusions
of blood products, additional clinical modalities, such as
electrocardiography and imaging (echocardiography and radionuclide
techniques), may be preferred for the acute detection of myocardial
necrosis. It should be noted, however, that cardiac troponins are slowly
released over the course of one to two weeks following myocardial
infarction, returning to baseline levels in the range of five to 10 days.1
Thus, cardiac-troponin levels should return to diagnostically elevated
levels in the setting of transfusion-related plasma dilution following
cessation of transfusion. It seems prudent and acceptable to check cardiac-troponin
levels while a patient is receiving a blood transfusion, with the caveat
that post-transfusional monitoring be continued in settings of excessive
transfusion requirements. We found no studies suggesting that transfusion
can falsely elevate troponin levels.

—Chris Rozelle, MD
Department of Pathology
Oregon Health and Sciences University
Portland, OR


  1. McPherson RA, Pincus MR, eds. Henry's Clinical
    Diagnosis and Management by Laboratory Methods
    , 21st ed. China: Saunders
    Elsevier; 2007.
  2. Joint European Society of Cardiology/American
    College of Cardiology Committee. Myocardial infarction redefined —A
    consensus document of The Joint European Society of Cardiology/American
    College of Cardiology Committee for the Redefinition of Myocardial
    European Heart Journal
    . 2000;21(18): 1502-1513.
  3. Centers for Disease Control and Prevention.
    Epidemiologic Notes and Reports Human Immunodeficiency Virus Infection
    Transmitted From an Organ Donor Screened for HIV Antibody: North Carolina.
    MMWR Morb Mortal Wkly Rep. May 22, 1987; 36(20):306-308.
  4. Ray G. Plasma Dilution. AATB Donor
    Eligibility Workshop. PowerPoint presentation. June 25, 2007.

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

MLO's “Tips
from the Clinical Experts” provides practical, up-to-date solutions to
readers' technical and clinical issues from a panel of experts in
various fields. Readers may send questions to Dan Baer by e-mail at
[email protected].