Answering your questions

March 1, 2009
Mixing EDTA blood too long

Q If a CBC is collected in an EDTA tube, what is the
maximum amount of time it can be mixed on a mechanical rocker? Do any of
the cells break down if it is mixed too long?

A Blood samples collected in additive solutions for
subsequent hematologic evaluation must be thoroughly mixed prior to
analysis. Specimens are often placed on mechanical rockers to ensure
adequate homogeneity of the sample. Without sufficient mixing, cellular
elements are unevenly distributed, adversely effecting accuracy.1,2
Unfortunately, there are no guidelines specifically detailing how long a
specimen can be mixed on a mechanical rocker before cellular integrity
is compromised. There are, however, guidelines regarding specimen
stability in additive solutions, such as the additive EDTA (ethylenediaminetetraacetate),
without directly addressing the additional effects imposed by mechanical

The dimensions of the specimen-collection container
can influence the number of inversions required to attain an even
distribution of cellular contents necessary for accurate conventional
and automated complete blood count with differential (CBC/Diff)
analysis.1 A standard 10 to 14 x 75-mm tube with five mL of
blood and an air bubble, comprising at least 20% of the tube volume,
requires at least eight inversions.1,3 Additionally, in order
to maintain acceptable precision, tubes that have been standing must be
inverted at least 60 times, or placed on a mechanical rotator for two
minutes prior to analysis.2 According to the Clinical and
Laboratory Standards Institute (CLSI) guidelines, all tubes containing
additives except sodium citrate should be gently inverted at least five
to 10 times to mix the contents, unless otherwise specified by the
manufacturer, regardless of the device used for specimen collection.3
Tubes with sodium citrate, on the other hand, should be inverted three
to four times to thoroughly mix the contents.3

A logical question arises: Can a specimen be “overmixed”?
There are no guidelines explicitly directed at this inquiry; however,
studies have shown gentle handling of blood specimens, in general, is
imperative in preventing ex vivo mechanical trauma to red cells.4
Red-cell lysis can interfere with chemical and optical analyses, even
without a detectable visual change in the specimen.3 For
example, an increase in hemoglobin from a hemolyzed specimen will affect
the mean cell hemoglobin and mean cell hemoglobin concentration in
automated systems.3 For this reason, although there are no
specific guidelines outlining the length of time on a mechanical rocker,
minimal but adequate gentle mechanical rocking or inversion of a blood
sample is ideal.

Stability studies on blood samples collected in
various additive solutions (i.e., EDTA, potassium-ammonium oxalate, and
sodium heparin) have been performed.5,6 Brittin, et al,
demonstrated CBC parameters — including white blood-cell count, red
blood-cell count, hemoglobin, hematocrit (Hct), mean cell volume (MCV),
mean cell hemoglobin, and mean cell hemoglobin concentration (MCHC) —
are stable in EDTA for at least 24 hours in the refrigerator.5
For similar specimens stored between six to 24 hours at room
temperature, ensuing red cell swelling of approximately 1% to 3%
resulted in significantly different values for MCV, Hct, and MCHC when
compared to fresh specimens.5

In summary, adequately mixed, homogeneous blood
samples are required for accurate and precise CBC/Diff parameters when
measured by automated systems. There are published guidelines detailing
the minimum time a sample should be mixed2,3; however, there
are currently no guidelines directed at the maximum time a specimen may
continue to be mixed, whether by manual inversion or mechanical rocking.
As detailed here, samples collected in EDTA must be analyzed within 24
to 48 hours depending on the storage conditions of the specimen, which
indirectly suggests a maximum time spent mixing.

—Nicky Leeborg, MD

—Guang Fan, MD, PhD

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. Fairbanks VF, Fahey JL, Beutler E: Clinical
    Disorders of Iron Metabolism, 2nd ed. New York, NY: Grune &
    Stratton; 1971:178.
  3. Clinical and Laboratory Standards Institute
    (CLSI): Procedures for the Handling and Processing of Blood
    Specimens; Approved Guidelines – Third Edition
    . Wayne, PA. 2004.
    CLSI document H18-A3.
  4. Brittin GM, Brecher G, Johnson CA, et al.
    Stability of Blood in Commonly Used Anticoagulants. Use of
    Refrigerated Blood for Quality Control of the Coulter Counter Model
    S. Am J Clin Pathol. 1969;52:690-694.
  5. Lippi G, Blanckaert N, Bonini P, et al.
    Haemolysis: an overview of the leading cause of unsuitable specimens
    in clinical laboratories. Clin Chem Lab Med.
  6. Vogelaar SA, Posthuma D, Boomsma D, Kluft C:
    Blood Sample Stability at Room Temperature for Counting Red and
    White Blood Cells and Platelets. Vascular Pharmacology.
Calibration of digital thermometer

Q I am looking for procedures regarding the calibration
of digital thermometers. Currently, we are using a mercury NBS certified
thermometer to calibrate red-spirited filled thermometers but I want to
change to digital thermometers. I was unable to find clear guidelines
for calibration methods for digital thermometers, can you help?

A Accurate temperature measurement is recognized as an
essential component to many clinical laboratory analyses. Guidelines
have been published that describe how to properly calibrate other
(secondary) temperature sensors using standard reference material (SRM)
thermometers. In particular, CLSI Document 12-A2 (1) describes this
procedure. These guidelines state that “sensors that might be calibrated
include other precision liquid-in-glass thermometers, small industrial
platinum-resistance thermometers, thermometer probes using thermistors,
electronic fever thermometers, thermal-dilution catheters, and others.”
The proper procedure in calibration includes proper immersion of the SRM
thermometer and the secondary sensor to be calibrated in a liquid bath
whose volume is at least 100 times that of the total volume of the
devices placed in it.

—Steven C. Kazmierczak, PhD, DABCC

Department of Pathology

Oregon Health and Science University

Portland, OR


  1. Clinical and Laboratory Standards Institute.
    Temperature Calibration of Water Baths, Instruments, and Temperature
    Sensors — Second Edition. Approved Standard — 1990. Wayne, PA:
    Clinical and Laboratory Standards Institute; CLSI Document 12-A2.
Ictotest black color

Q When performing Ictotest for bilirubin confirmation,
we observed a shade of gray (light) or black color — not blue or purple.
The technologists have doubts about color interpretation. Is it a
positive reaction? Is there any correlation of bilirubin in serum with
the bilirubin in urine? Is there any serum bilirubin threshold level for
excretion of bilirubin in urine as there is for glucose?

A This raises an important issue regarding urinalysis
interpretation of bilirubin confirmation with the Ictotest tablet
method. Bilirubin is detected first by the dipstick and then confirmed
with the Ictotest. In my experience, I have never observed a gray or
black color and, therefore, would not report this test finding as
positive. I would check to ensure that 1) the test kit is not outdated,
has been properly stored, and has not been contaminated; 2) there is not
a technical personnel issue of color blindness; and 3) not only check
the manufacturer's insert but also contact the manufacturer for
information on this observation.

In response to the second part of your question,
there is a correlation between bilirubin in the serum and bilirubin in
the urine. Using glucose as an example, as referenced in your question,
the threshold is based on renal tubular absorption. This threshold is
lowered if there is injury or damage to the renal tubular cells, causing
a spillage of glucose into the urine. In the case of bilirubin, the
threshold is based on the ability to be conjugated by protein (amino
acids); therefore, when there is a decrease in plasma proteins for a
variety of reasons, there will be spillage of bilirubin into the urine.
The detection of bilirubinuria indicates pathologic conditions such as
hepatitis, cirrhosis and biliary obstruction. Review of standard
textbooks in urinalysis is recommended for further discussion.

—G. Berry Schumann, MD (deceased)

Former medical director, Schumann Diagnostics

Neutrophlic hypersegmentation index

Q We received an order for a neutrophlic
hypersegmentation index. Could you please explain how to calculate and
arrive at a result for this test? What is the reference range for this
test? I saw mention of this test on the Internet but no explanation and
find none in books.

A Normal neutrophil granulocytes are characterized by a
number of nuclear lobes (segments) in the range of two to five. Normally
there are two lobes in 10% to 30% of segmented neutrophils; three lobes
in 40% to 50%; four lobes in 10% to 20%; and five lobes in fewer than
5%.1 A so-called neutrophil right shift (that is, increase in
the number of lobes), occurs in leukemias, chronic nephritis, diseases
of the liver, cancer, sepsis, and vitamin B12, and folate deficiency.

Neutrophil hypersegmentation has clinically been
widely used as indication of B12 and folate deficiency. For this reason,
attempts have been made to quantify the neutrophil right shift. There
are at least three methods to accomplish this:

One method is 'lobe average'. It is
simply calculated by determining the sum of lobes present in a certain
number of neutrophils and dividing it by the cell number. Normal values
are between 2.5 and 3.3 in different studies.2 Right shift
would be signified by an increase in average lobe count.

Another index is the percentage of neutrophils
with five or more lobes
, which is considered an indication of
right shift at more than 3%. The third is the segmentation index
(also called hypersegmentation index). It is
calculated as:

Number of neutrophils with 5 lobes or more x 100

 Number of neutrophils with 4 lobes

Values greater than 16.9 are characteristic of
hypersegmentation. The number of neutrophils counted was 200 and 500,
respectively, in different parts of the study mentioned.3 For
practical purposes, 200 cells appears to be a sufficient number.

A thorough quantification of neutrophil
hypersegmentation in vitamin B12 deficiency showed that the different
calculation methods had different sensitivities.3 The
segmentation index was superior by identifying hypersegmentation in 64%
of 25 patients with serum B12 levels below 150 pg/mL, whereas the lobe
average identified 16% and the “number of cells with five or more lobes”
identified 44%. In summary, the segmentation index is the most sensitive
method of determining a right shift in the neutrophils in vitamin B12

—Winfried Reichelt, MD, PhD

—Guang Fan, MD, PhD

Oregon Health and Science University

Portland, OR


  1. McPherson RA, Pincus MR.
    Henry's Clinical Diagnosis and Management.
    21st ed. Saunders, Elsevier; 2007;474.
  2. Bain BJ.Blood Cells. A
    Practical Guide.

    3rd ed. Wiley-Blackwell; 2006;91-92.
  3. Edwin E. The segmentation of polymorphonuclear neutrophils. Acta
    Med Scand
    . 1967;182:401-410.

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