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Techtalk August2010
Techtalk August2010
Techtalk August2010
Volume 8, No. 3
August 2010
specimen from clotting. It functions by binding the calcium in the blood, which is an essential component of
the coagulation cascade.1 The specimen can be analyzed as whole blood or centrifuged to separate plasma.
EDTA is the most commonly used additive to perform hematology testing. The whole blood sample is used
to perform a complete blood count (CBC) on various types of automated instruments.
References:
1. C
LSI Guideline, Tubes and Additives for Venous Blood Specimens Collection; Approved
Standard Fifth Edition, H01-A5, 2003.
2. Rodak, B. Diagnostic Hematology, 1996, WB Saunders Co.
3. www.healthatoz.com/healthatoz/Atoz/ency/platelet count.jsp.
4. BD Vacutainer Evacuated Blood Collection System Product Insert, 11/2007.
5. Kjeldsberg, C., Hershgold, E., Spurious Thrombocytopenia. JAMA, Feb. 11, 1972, Vol. 227 No.
6. BD LabNotes, Vol.14, No.1, 2004.
7. CLSI Guideline, Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture;
Approved Standard Sixth Edition, H03-A6, 2007.
8. CLSI Guideline, Procedures and Devices for the Collection of Diagnostic Capillary Blood
Specimens; Approved Standard Sixth Edition, H04-A6.
9. CLSI Guideline, Procedures for the Handling and Processing of Blood Specimens for Common
Laboratory Tests; Approved Standard Fourth Edition, H18-A4, 2009.
10. Hagerman, R. Ethylenediamainetetraacetic acid (EDTA) Dependant Pseudothromocytopenia:
A Case Report of an Incidental but Important Finding. www.priory.com/med/casepresentations.
11. Hsieh, A.T., Chao, T.Y., Chem, Y.C. Pseudothrombocytopenia Associated with Infectious
Mononucleosis. Archives of Pathology and Lab Med.
Vol. 127 No. 1, e17-e18.
12. Qureshi, B. EDTA-Dependent Pseudothrombocytopenia. LabMed. Aug. 1998, Vol. 29 No.8,
471-473.
13. Gulati, G., Asselta, A., Chen, C. Using a Vortex to Disaggregate Platelet Clumps. Lab Med.
Oct, 1997, Vol. 28 No. 10, 665-667.
14. Sakurai S., Shiojima I., Tanigawa T., et al: Aminoglycosides prevent and dissociate the
aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia.
Br J Haematology. 1997; 99:817-23.
Platelets play an important role in hemostasis
by forming a platelet plug at the site of vessel trauma
and serving as the phospolipid foundation for fibrin
deposition. The glycoprotein receptors on the platelet
surface and sub-surface allow the reception of external
messages from vascular and circulatory sources. Signals
are transmitted across the platelet membrane to second
messengers controlled by calcium mobilization or
calcium sequestration.2
BD Diagnostics
Preanalytical Systems
1 Becton Drive
Franklin Lakes, NJ 07417
www.bd.com/vacutainer
continued on reverse side
The most common cause of platelet clumping in an EDTAanticoagulated specimen is improper mixing of the tube. It is
recommended that the EDTA tube be inverted eight to 10 times
immediately after the specimen is collected.4 Fewer inversions may
result in incomplete mixing of the additive in the blood and therefore,
platelet clumping.
Improper collection of the blood sample may cause thrombin
release and a falsely low platelet count due to platelet aggregation.5
This may be due to an excessively traumatic venipuncture or
inadequate anticoagulation.
A BD Microtainer Tube with EDTA should be inverted 10
times after collection, and mixed 10 times prior to testing. When
performing the skin puncture, it is best to avoid scraping the skin with
the collection tube, as this may result in platelet clumping and/or
clotting of the specimen.6
All blood collection tubes should be filled to their stated draw volume. Overfilling an EDTA
tube can result in an improper blood to additive ratio. Insufficient EDTA in the sample may contribute
to platelet clumping and/or clotting of the blood.9
Another cause of platelet clumping is EDTA-induced pseudothrombocytopenia. This
phenomenon is believed to be caused by EDTA-dependant platelet agglutinins or antibodies present
in the plasma. The clumping occurs due to an alteration of the platelet surface glycoproteins when
they are incubated with a calcium chelator such as EDTA. These modified platelet antigens then
react to anti-platelet autoantibodies (immunoglobulins of both IgG and IgM types to form the large
agglutinates.)10
EDTA-induced pseudothrombocytopenia can be exhibited in normal, healthy individuals
and those with diseases such as human immunodeficiency virus (HIV), rubella, cytomegalovirus
(CMV), autoimmune disorders, thrombotic disorders and infectious mononucleosis.10 In infectious
mononucleosis, the patient may have increased levels of cold agglutinins, which may contribute to
platelet agglutination.11
= 1 Inversion
BD Diagnostics
Preanalytical Systems
When a platelet, white cell and/or red cell count is flagged on an electronic counter, there are
several steps that can to be taken in order to get an accurate platelet count:12
2. Re-collect the patients blood into a sodium citrate tube. Platelets will generally not exhibit
clumping in sodium citrate. When running the citrate tube on the instrument, multiply the
platelet count by 1.1 to get the correct value.
3. If the patient has infectious mononucleosis, or a cold agglutinin is suspected as the reason
for platelet clumping, collect the blood into an EDTA tube that has been pre-warmed to
37C in a water bath.
4. The sample can be vortexed, which will cause the platelet clumps to break apart and they
can then be counted more accurately. However, be aware that too vigorous mixing can, by
itself, cause platelet activation.13
Venipuncture:7
The BD Vacutainer Blood Collection System
Closure Color
Mix by
Inverting
Collection Tube
or
or
or
8 to 10 times
Citrate Tube*
3 to 4 times
BD Vacutainer SST
Gel Separator Tube
5 times
Serum Tube
5 times (plastic)
none (glass)
BD Vacutainer Rapid
Serum Tube (RST)
5 to 6 times
BD Vacutainer PST
Gel Separator Tube
With Heparin
8 to 10 times
Heparin Tube
8 to 10 times
EDTA Tube
8 to 10 times
EDTA tubes
Order of Draw
8 to 10 times
BD Vacutainer PPT
Gel Separator Tube with K 2EDTA
Order
of Draw
8 to 10 times
Additive
Mix by
Inverting
8x
363706
Microcollection:8
365965
EDTA tubes
365985
Heparin tubes
Serum tubes
K2EDTA
Lithium
Heparin
10x
Lithium
Heparin and
Gel for Plasma
Separation
10x
NaFl/Na2EDTA
10x
365987
365992
365967
Clot Activator
and Gel
for Serum
Separation
5x
365978
No Additive
365963
0x
Once laboratory personnel understand the reasons and possible causes for platelet clumping in
an EDTA-anticoagulated blood specimen, it can be much easier to deal with clumped platelets when
they occur, and in many circumstances, prevent them from occurring in the first place.