HEMATOLOGY
Gene L Gulati, PhD, SH(ASCP)DLM
Using a Vortex To Disaggregate
Platelet Clumps
Clumping of platelets in EDTA-anticoagulated
blood is perhaps the most common cause of false
decreases in platelet counts generated by auto-
mated analyzers. To obtain accurate platelet
counts in such cases, laboratorians commonly
obtain and analyze additional blood specimens,
which are either anticoagulated with a different
anticoagulant, such as sodium citrate, or diluted
directly into a diluent containing ammonium
oxalate.1'"3 Thus, the patient is subjected, often to
his or her dislike, to a second venous or capillary
puncture. We present our experience with an
alternative approach that does not require collec-
tion of a second specimen yet offers reliable
platelet counts, at least in some cases, from EDTA-
anticoagulated blood specimens that reveal
platelet clumps on Wright-Giemsa-stained
smears. Our approach is to mix the EDTA-antico-
agulated blood by vortex and rerun the specimen
through the automated analyzer to obtain the
platelet count.
Materials and Methods
We used 188 EDTA-anticoagulated blood speci-
mens that revealed platelet clumps on Wright-
Giemsa-stained smears. These specimens, which
came from 163 patients, were collected in
lavender-topped tubes (3 mL Hemogard or 5 mL
Vacutainer [Becton Dickinson, Rutherford, NJ]),
and were subjected to a vortex and repeat CBCs.
Although the specific diagnoses of many of the
patients providing the blood specimens were
unknown, a small number of patients had known
diagnoses, including liver disease, renal disease,
urinary tract infection, diabetes, and sickle cell
disease. Nearly 90% of the blood specimens in
the study had one or more abnormal (elevated or
decreased) cell counts (white cell, red cell, or
platelet). An automated analyzer (Coulter-STKS,
Coulter, Miami) was used to perform the CBCs.
The analyzer was calibrated using S-Cal (from
Coulter), and daily quality control was performed
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Amy Asselta, MS, MT(ASCP)SH
Conrad Chen, H(ASCP)
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ABSTRACT Clumping ofplatelets in blood anticoagulated
with EDTA is a main cause of reports of falsely low platelet
counts by automated analyzers. To obtain reliable platelet
counts in such cases, laboratorians often collect and analyze
a citrated or nonanticoagulated blood specimen. The patient
thus is subjected to a second venous or capillary puncture,
and the availability of results is delayed. Our data on an
alternative approach suggest that reliable automated platelet
counts can be obtained from many blood specimens that
reveal microscopic platelet clumps after mixing them by
vortex and rerunning the samples through the analyzer.
Mixing the blood by vortex at the maximum possible speed
for I to 2 minutes disaggregated platelet clumps completely in
44% of specimens and partially in another 49% of specimens.
using three levels of 5C controls (from Coulter), From the
Hematology
according to manufacturer's instructions.4 The
Laboratory, Thomas
samples were subjected to a vortex on a vortex Jefferson University
mixer (Pulse Vortexer, Glas-Col, Terre Haute, Hospital,
Ind). Although either an aliquot or the entire Philadelphia. Ms
specimen can be subjected to vortex, we chose Asselta is currently
at Coulter
the latter to avoid the extra step of portioning,
Corporation, Miami.
consumption of another tube and transfer
pipette, and the possibility of inadequately mix-
Reprint requests to
ing the specimen in the process of sampling by
Dr Gulati, 307
aliquot. We found that 1 to 2 minutes' vortex at Pavilion Bldg,
or near the highest setting (8-10 on a scale of Thomas Jefferson
1-10) on the vortex mixer was adequate to disag- University Hospital,
gregate platelet clumps. Soon after vortex (within 125 S 11th St,
1-2 minutes), we reran each specimen through Philadelphia, PA
19107.
the automated analyzer for a CBC, made and
stained a smear with Wright-Giemsa stain, and
examined the smear microscopically for platelet
clumps. We compared postvortex results on the
188 specimens with the corresponding prevortex
(initial) results (Fig). The parameters evaluated
were WBC count, platelet count, and the smear
review finding as to the presence or absence of
platelet clumps (Table). The WBC count was
OCTOBER 1997 VOLUME 23, NUMBER 10 LABORATORY MEDICINE 66
 postvortex platelet counts and WBC counts
in these samples were as unreliable as the corre-
sponding prevortex cell counts.

Discussion
Using a vortex disaggregated platelet clumps in 93%
of the EDTA-anticoagulated blood specimens that
originally revealed microscopic platelet clumps. It
did so completely in 44% of specimens and partially
A representative
blood smear (Wright-
included because platelet clumps are known to in an additional 49%. All specimens that revealed

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Giemsa, original falsely elevate the automated WBC counts.5-7
magnification X500)
showing a platelet Results
aggregate (clump),
We noted complete disaggregation of all platelet
prevortex (A), and
clumps with a subsequent increase in platelet
disaggregated platelets,
postvortex (B). counts in 82 (43.6%) of the 188 specimens (see
Table). The postvortex increase in platelet counts
averaged 72.6% with a range of 5.2% to 300%.
The increase in the platelet count was often
accompanied by a decrease in the WBC count,
which averaged 11.9%. The maximum decrease
in the WBC count noted was 44.3%.
In an additional 93 specimens (49.5%),
using a vortex increased the platelet count by an
average of 111% (range 8.6%-611.1%) but
disaggregated only some of the platelet clumps.
The accompanying decrease in the postvortex
WBC counts averaged 11.2%. The maximum
decrease in the WBC count was 42.9%.
The remaining 13 specimens (6.9%) showed
either no change in the degree of platelet clumping
or increased platelet clumping. Consequently, the
complete disaggregation of platelet clumps after the
vortex yielded reliable platelet counts, as judged by
the platelet estimates from the smears, when rerun
through the automated analyzer. The postvortex
platelet counts from this group of specimens can be
reported without any reservation, thereby avoiding a
second venous or capillary puncture of the patient.
Although not accurate, the postvortex automat-
ed platelet counts obtained on the specimens that
revealed a lesser degree of platelet clumping (ie,
those with a decreased number of clumps, smaller
clumps, or both) on postvortex smears also may be
reported with appropriate comments about clump-
ing status, particularly when the counts are within
the normal range or increased. To be considered
reportable, the postvortex automated platelet
counts should be significantly (more than 10%)
greater than the prevortex counts, and the postvor-
tex smears should reveal either no platelet clumps
or a significantly lesser degree of platelet clumping.
Our search for possible causes of the incom-
plete disaggregation of platelet clumps in some

PREVORTEX AND POSTVORTEX PLATELET COUNTS, WHITE BLOOD CELL COUNTS, AND
BLOOD SMEAR FINDINGS

Specimen Group No. With Platelet Platelet Count (109/L) WBC Count (109/L)
(N = 188) Clumps on Smears Mean Range Mean Range
Complete Disaggregation*
(n = 82)
Prevortex 82 153 15-470 10.7 1.8-46.7
Postvortex 0 252 34-651 9.5 1.7-47.0
Partial Disaggregation^
(n = 93)
Prevortex 93 118 7-422 12.1 3.1-36.7
Postvortex 93 189 18-669 10.5 3.0-34.3
No Disaggregation*
(n= 13)
Prevortex 13 101 21-259 9.3 2.3-17.1
Postvortex 13 79 16-216 9.1 2.3-17.1
* Specimens revealing complete disaggregation of all platelet clumps after the vortex procedure
t Specimens revealing disaggregation of some platelet clumps after the vortex procedure.
$ Specimens revealing no change or increased platelet clumping after the vortex procedure.

LABORATORY MEDICINE V O L U M E 28, NUMBER 10 OCTOBER 1997

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 specimens and the failure to disaggregate platelet
clumps in others by vortex did not yield any
unequivocal results. With the exception of small
platelet clumps, which generally were disaggre-
gated by vortex, neither the sample volume
(0.5-4 mL) of the specimen subjected to vortex
nor the size of platelet clumps as revealed by the
blood smear seemed to influence the end result.
Increasing the vortex time beyond 2 minutes did
not improve the chance of disaggregating platelet
clumps. Setting the vortex mixer at less than the
indicated speed or for less than half a minute at
the indicated speed, however, seemed to decrease
the chances of disaggregating the platelet clumps.
Because we performed all of the postvortex speci-
men analyses (repeat CBC and smear review)
immediately after vortex (within 2 minutes), we
can draw no conclusions about the possibility
that platelet clumps, once disaggregated by vor-
tex, may reclump after some time. We did note,
however, that the platelet clumps, after being dis-
aggregated by vortex, remained disaggregated in
the only two specimens we reanalyzed (by CBC
and smear review) 1 hour after vortex.
As expected, the lower postvortex WBC
counts more closely matched the WBC estimates
from the blood smears than the corresponding
prevortex WBC counts did. These observations
corroborate previously published reports of
pseudoleukocytosis associated with platelet
clumps.5~7 Although we did not investigate the
effect of vortex on the differential leukocyte
count, none is expected, based on our
observation that the vortex did not alter white
cell morphology in any way in any specimens that
underwent vortex. In fact, the morphology of all
cellular elements of blood remained unperturbed
after vortex, with the exception of degranulation
(usually partial) of some platelets noted in a few
cases. Although we also did not systematically
study the effect of vortex on red cells, a cursory
review of the CBC results indicated no significant
change in any of the red cell parameters after
mixing by vortex. Similarly, no significant change
was apparent in any of the CBC parameters after
subjecting a group of specimens that had no
platelet clumps to vortex. These observations
alleviated our concern that using a vortex might
cause damage to some of the cellular elements.
Conclusion
Reliable automated platelet counts can be obtained
from EDTA-anticoagulated blood specimens that
reveal platelet clumps on smears. Such specimens
should be mixed by vortex for 1 to 2 minutes at or
near the highest speed setting. Subjecting the
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patient to a second venous or capillary puncture to
obtain an accurate platelet count should be
reserved for cases in which the vortex is either
unsuccessful or not feasible. This procedure is not
recommended for blood specimens that reveal fib-
rin strands along with platelet clumps on the
smears; the integrity of such specimens is ques-
tionable for use in obtaining reliable results.®
Acknowledgment
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The authors thank the hematology laboratory staff of the
Thomas Jefferson University Hospital for their technical help
in completing this project.
References
1. Mant MJ, Doery JCG, Gauldie J, Sims H.
Pseudothrombocytopenia due to platelet aggregation and
degranulation in blood collected in EDTA. Scand J. Haematol.
1975;15:161-170.
2. Berkman N, Michaeli Y, Or R, Eldor A. EDTA-dependent
pseudothrombocytopenia: a clinical study of 18 patients and
review of the literature. Am! Hematol. 1991;36:195-201.
3. Cornbleet PJ, Astarita R, Wolf PL. White blood cell and
platelet disorders. In: Howanitz JH, Howanitz PJ, Cornbleet
PJ, et al, eds. Laboratory Medicine: Test Selection and
Interpretation. New York, NY: Churchill Livingstone;
1991:592-594.
4. Coulter Corporation. Coulter-STKS Manual. Miami, Fla:
Coulter Corporation; 1992.
5. Solanki DL, Blackburn BC. Spurious leukocytosis and
thrombocytopenia: a dual phenomenon caused by clumping
of platelets in vitro. JAMA. 1983;250:2514-2515.
6. Savage RA. Pseudoleukocytosis due to EDTA-induced
platelet clumping. Am J Clin Pathol. 1984;81:317-322.
7. Lombarts AJPF, DeKieviet W. Recognition and preven-
tion of pseudothrombocytopenia and concomitant
pseudoleukocytosis. Am ] Clin Pathol 1988;89:634-639. c
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For more information about Laboratory Medicine, call Laura Pelehach
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VOLUME 28, NUMBER 10 LABORATORY MEDICINE 667