TechTalk

Volume 8, No. 3

August 2010

Author: Leslie Magee

BD Global Technical Services receives
many questions about BD products.
To address these questions, we have developed
a periodic news bulletin called Tech Talk.

Q. Why do platelets sometimes clump in EDTA Tubes?

A. EDTA (ethylenediaminetetraacetic acid) is an anticoagulant added to blood collection tubes to keep a

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.

Please call BD Global Technical Services for clinical support material.

BD Global Technical Services: 1.800.631.0174
BD Customer Service: 1.888.237.2762
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2010 BD
08/10
VS8877

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

Platelet clumping may be suspected when the

platelet count result is below the lower limit of the
reference interval, particularly when the patient has no
clinical basis for a low platelet count. Platelet clumps
commonly elicit a platelet count flag on modern
automated analyzers. For some analyzers, the white cell
and red cell counts, or red cell parameters, may also be
flagged. Confirmation of clumping is best achieved with
microscopic examination of a Romanowsky-stained smear.

Electronic counting instruments, where particles are sorted based on a specific size range, will
sometimes produce artificially low platelet counts. This can occur for several reasons. Clumps of platelets
will not be counted as part of the platelet count because clumps exceed the upper size exclusion limit for
platelets. Depending on the instrument type and cell counting technology being used, the clumps may be
counted as white cells, potentially leading to falsely elevated white cell counts (generally with flags, as
described above) or as red blood cells, leading to errors in red cell size determination (MCV) and red cell
indices derived from MCV (ie, MCHC). These erroneous red cell results will also often be flagged, just as
the white cell count would be. A falsely low platelet count may also occur if a platelet and another blood
cell pass through the counter at the same time. The instrument will not count the larger cell because of the
size exclusion limits, but the platelet may accidentally be missed as part of the count. Susceptibility to such
errors varies with the instrument type and technology. Lastly, a falsely elevated platelet count can result
from red cell fragments in the blood that can occur due to ruptured red blood cells.3

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

The BD Vacutainer Blood Collection System

Possible Causes of Platelet Clumping

It is important to follow the recommended order of draw for

venipuncture and skin puncture collections. The purpose of the order
of draw is to avoid possible cross contamination of additives from one
tube to the subsequent tube being drawn. The correct order of draw is
as follows:

Mix Tubes by Inverting

the Recommended
Number of Times

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

Managing Platelet Clumping in the Lab

BD Technical Services 1.800.631.0174

BD Customer Service 1.888.237.2762
www.bd.com/vacutainer
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. 2010 BD 03/10 VS5734-5

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

1. Examine a stained blood smear microscopically to verify the platelet clumping.

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

5. Aminoglycosides (e.g. kanamycin) have been reported to be effective in dissociating platelet

clumps in cases of EDTA-induced pseudothrombocytopenia.14

Venipuncture:7
The BD Vacutainer Blood Collection System

Order of Draw for

Multiple Tube Collections
CLSI recommended Order of Draw (H3-A5, Vol 23, No 32, 8.10.2)

Closure Color

Mix by
Inverting

Collection Tube

BD Vacutainer Blood Collection Tubes (glass or plastic)

or

or

or

Blood Cultures - SPS

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

Blood culture tubes or bottles

Sodium citrate tubes

 Serum tubes with or without clot

activator, with or without gel

Heparin tubes with or without gel

EDTA tubes

Sodium fluoride tubes

BD Microtainer Tubes
with BD Microgard Closure

Order of Draw

8 to 10 times
BD Vacutainer PPT
Gel Separator Tube with K 2EDTA

Fluoride (glucose) Tube

Order
of Draw

8 to 10 times

*When using a winged blood collection set for venipuncture and a

coagulation (citrate) tube is the first specimen tube to be drawn, a
discard tube should be drawn first. The discard tube must be used to fill
the blood collection set tubings dead space with blood but the discard
tube does not need to be completely filled. This important step will
ensure maintenance of the proper blood-to-additive ratio of the blood
specimen. The discard tube should be a nonadditive or coagulation tube.

NOTE: Always follow your facilitys protocol for order of draw

Additive

Mix by
Inverting

8x
363706

Microcollection:8

365965

EDTA tubes

365985

Heparin tubes

Sodium fluoride 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.