Answering your questions

Aug. 1, 2009
Body-fluid pH testing

Q My question concerns the stability of pH in body
fluids. Historically, we only accept and perform body-fluid pH
measurement tests if the body fluids have been on ice and for less than
one hour. Is body fluid this unstable” I have found very little written
about this and have concerns that we may be unnecessarily rejecting
these samples. Can you also comment on the clinical use of this test”

A The importance of body-fluid pH likely varies by
fluid type. Pleural-fluid pH, collected from a thoracentesis procedure,
is probably the most important body fluid for which pH measurement is
indicated. Pleural-fluid pH may help to differentiate infectious from
malignant or other non-infectious sources of pleural effusion. In
addition, pleural-fluid pH less than 7.0 associated with pneumonia
indicates the need for immediate treatment and intervention, whereas a
pH greater than 7.4 may indicate that further drainage of the fluid may
not be required.1,2 There is some data available suggesting
that measurement of pleural-fluid pH should be performed only on iced
samples submitted in heparinized syringes, and that measurement should
occur on a blood-gas analyzer rather than by pH meter. Pleural-fluid pH
will change over time, and these changes could contribute to incorrect
diagnostic or therapeutic decisions. For this reason, your rejection
criteria for pleural fluids are entirely appropriate.

I am not aware of as much data about the use,
interpretation, and appropriate analytic criteria for other types of body
fluids. In the absence of any data or knowledge about how various other
body-fluid pH measurements might be used and how fast pH may change in other
body fluids, I would say that erring on the side of caution in rejecting
these other fluids is within reason.

—Brad Karon, MD, PhD


  1. Light RW, et al. Parapneumonic effusions. Am J Med.
  2. Varkey B. Pleural effusions caused by infection.
    Postgrad Med
    . 1980;80:213-216.
Specimen handling and storage

Q We have numerous satellite offices collecting lab
specimens. Non-lab personnel (medical assistants) who have only minimal lab
training usually staff these sites. We hear stories about specimens being
stored under various conditions prior to transport to the main laboratory
and are concerned with specimen integrity. We have had experience with some
sites storing their specimens on top of dorm-size refrigerators, which
typically put out a good amount of heat. Are there guidelines we can pass
along to standardize specimen handling and storage”

A Under CLIA requirements, it is the testing laboratory's
responsibility to ensure the integrity of the specimen through the entire
process, which includes from the time the specimen is drawn until the
results are reported. The guidelines for specimen handling and storage are
unique to each collection site and dictated by the testing procedure
provided by manufacturers or in-house performance specifications. Each test
will have specifications for how long the specimen is good at a certain

Under CLIA requirements, it is the testing laboratory's
responsibility to ensure the integrity of the specimen through the entire

In general, most routine chemistry tests should be
separated into serum or plasma within one hour to two hours of draw. After
separation, serum or plasma may be stable either refrigerated or at room
temperature for several hours, but this will vary by test and there are
notable exceptions, such as lactate and ammonia, which are not stable for
more than a few minutes. Coagulation tests in general should be separated
and frozen if delays of more than one hour to two hours will occur between
draw time and analysis. For hematology (CBC) testing, specimen stability
varies by instrument platform, but many are stable for up to 24 hours. An
in-service education and a tour of the testing facility can go a long way
toward educating non-lab personnel about the importance of specimen handling
and storage.

—C. Anne Pontius, MBA, CMPE, MT(ASCP)
CLMA President ;
Senior Medical Practice Consultant
State Volunteer Mutual Insurance Company
Brentwood, TN

Testing for NT-pro BNP

Q I am running a quick test for NT-proBNP with a cutoff of
450 pg/mL. Most of my patients are over 50 years of age. The test requires
two drops of serum. If the patient is positive, would it be acceptable to
run the test again with one drop of blood to give the physician some
guideline and assume that it is then positive for >900 pg/mL”

A Good laboratory practice and many regulatory agencies
require that all lab tests have a defined analytical measurement range (AMR)
and clinical reportable range (CRR). If a lab desires to report a result
beyond the AMR possible using undiluted blood, then the protocol for
dilution must be validated to ensure accurate results are reported after
dilution. In this case, you must validate that samples with known
concentrations of NT-pro BNP can be “diluted” by applying less sample, and
the results will be accurate by your method. My guess is that this will not
work, as most whole-blood immunoassays use little blood for actual testing
but may require more blood to provide sample flow through the various stages
of testing on the device.

Also keep in mind that depending upon where you are
located and what regulatory agencies oversee laboratory testing, alteration
of waived or moderate complexity (in the United States under the Food and
Drug Administration, or FDA) in this manner would turn this into a
high-complexity test. This would require a different level of validation for
the test and potentially change the personnel, quality-control, and
proficiency-testing requirements for the test. In summary, this is probably
not a good idea.

—Brad S. Karon, MD, PhD

Hemolysis headaches

Q We have several off-site draw centers. Some techs
complain that specimens leaving their office

not identified as hemolyzed were
identified as such by the central lab. The specimens are spun in at the
off-site office, where centrifuges are inspected annually; we are pretty
sure the equipment is working appropriately. The off-site office staff
indicates the specimens are clotted for an appropriate time before spinning.
We thought the tube caps might be trapping some cells that then are “washed”
into the serum during transport. We have tried Greiner and BD tubes but have
not noticed a significant difference. The staff is frustrated to see clear
specimens going out only to see hemolyzed comments on returning reports. Why
we are experiencing this phenomenon” What we can do to prevent it”

A Investigating the cause of hemolysis is no small task.
Many patient and preanalytical variables can cause red blood cells to
rupture. Taking a systematic approach by ruling out the culprits one at a
time is likely to be the most effective strategy. You did not mention it,
but I assume the tubes in question have a gel barrier. Are the specimens
recentrifuged when they are received in the testing facility” Specimens
should only be centrifuged once. A second spin upon arrival, especially if
it is excessive, can hemolyze cells and cause rejection of tubes that looked
fine when they left the draw center. Additionally, if the specimen was
transported at temperature extremes, such as on ice, hemolysis can occur in
transit. When recentrifuged, the hemolyzed serum blends with the untainted
serum observed at the draw stations and leads to specimen rejection.

Are the specimens centrifuged at the draw centers in
fixed-angle or swivel-bucket centrifuges” If fixed-angle centrifuges are
being used, then the gel barrier, which will be thick on one side and
perilously thin on the other, could allow for red cells to pass into the
serum during transportation, especially if they are mishandled. Any
temperature extremes in transit may lead to hemolysis. Consider replacing
the centrifuges with the better swing-bucket models or buying double-gel
tubes to eliminate the thin gel interface that is characteristic of
fixed-angle centrifuges. Check your temperatures in transit, and avoid
letting tubes come in contact with ice.

Make sure those at the draw centers have the same sense of
what constitutes hemolysis as those who are rejecting the specimens.

Also consider that the problem may only be perceptual.
Make sure those employees at the draw centers have the same sense of what
constitutes hemolysis as those who are rejecting the specimens. Perhaps the
rejection criteria at the testing facility are more stringent than the
individuals at the draw centers realize. Showing them tubes with varying
degrees of hemolysis and pointing out those that are likely to be rejected
could help. The exercise might reveal that the draw-center staff was
underestimating the degree of hemolysis that warrants recollection. Those
who reject specimens upon arrival should all be following the same criteria.

—Dennis J. Ernst MT(ASCP)
Center for Phlebotomy Education
Ramsey, IN

Clot formation in blood-gas sample

Q A blood-gas specimen obtained by a physician on a newborn
was rejected because it had a clot that was expressed from the syringe just
before the attempt to put the sample on the blood-gas analyzer. The
physician wanted the specimen run anyway, stating that the clot was gone so
it would not hurt the machine. I called the manufacturer and inquired about
the validity of such a result. The manufacturer had no data and would not
state whether the results could be considered accurate. Please comment on
the appropriateness of following such a procedural step (i.e., squirt out
the clot and try to run the specimen).

A There are a number of potential problems that exist with
clot formation in blood-gas samples. First of all, adequate mixing of the
sample is important in measuring many of the analytes including hemoglobin,
pO2, pCO2, and electrolytes. Clot formation prevents
the sample from mixing thoroughly because of elements trapped in the clot.
For example, any red blood cells trapped in a clot may falsely decrease
hemoglobin measurements. In addition, ions such as ionized calcium will be
used in the clot formation and could be falsely low in a measured sample.
Most importantly, a sample that is not thoroughly mixed will produce
spurious pO2 results.1

Another potential problem is introduced when the clot is
expelled from the syringe. Any exposure to air can affect a number of
parameters including pH, pCO2, and pO2. Changes in pCO2
also affect the measurement of ionized calcium.1 Exposure could
occur at the time of expelling the clot by direct exposure to air or by the
introduction of air bubbles into the sample. Clinical practice guidelines on
arterial blood-gas analysis recommend voiding any results from samples that
contain a blood clot because of the potential for spurious results.2

—Liz Jaben, MD
Pathology resident
Mayo Clinic
Rochester, MN
—Brad S. Karon MD, PhD


  1. CLSI. Blood Gas and pH Analysis and Related
    Measurements; Approved Guideline – Second Edition.
    CLSI document
    C46-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
  2. Shrake K, Blonshine S, Brown R, et al. AARC clinical
    practice guideline, sampling for arterial blood gas analysis. Respir
    . 1992;37:891-897.