COAGULATION AND TRANSFUSION MEDICINE
Original Article
A Rapid Method to Isolate Platelets from
Human Blood by Density Gradient
Centrifugation
MAUREANE HOFFMAN, M.D., PH.D., 1 DOUGALD M. MONROE, PH.D., 2
AND HAROLD R. ROBERTS, M.D.2
Platelets can be damaged easily or activated during isolation,
making them unsuitable for functional studies. The most common
technique for isolating platelets involves centrifugation. Although
gentler methods have been devised to isolate platelets by density
gradient centrifugation or electrophoresis, these techniques either
result in a relatively dilute platelet preparation or are time-consuming. A simple, gentle technique for isolating concentrated
platelet preparations for experimental or clinical use is reported.
Freshly drawn whole blood was spun over a commercially available density gradient medium for 30 minutes. The mononuclear
cell layer (which also contains most of the platelets) was collected
and nucleated cells were pelleted by centrifugation. The recovery
of platelets was about 60%. Contamination with leukocytes was
less than 1%, and the platelet concentration was about 130% of
Platelets readily respond to physical or biochemical perturbation by activation. As the term is commonly used,
platelet "activation" refers to any or all facets of a complex
physiologic response, which includes shape change, release
of granule contents, expression of neoantigens, changes
in the functional status of platelet receptors, expression
of procoagulant activity, and aggregation. In vivo, activation changes the platelet from an relatively inert circulating particle to a key structural and biochemical feature of the hemostatic thrombus. Clinical platelet function
studies test the ability of a patient's platelets to respond
to activating stimuli, usually using aggregation as an endpoint. Therefore, it is critically important that platelets
blood concentration. Higher concentrations can be obtained if
more whole blood is layered onto the Mono-Poly Resolving Medium (MPRM; Flow Laboratories, McLean, VA). About 10%
of the platelets expressed the activation marker GMP-140 by
flow cytometric analysis. They could be activated by thrombin
so that 70% to 90% of the platelets expressed GMP-140. Thus,
this technique can rapidly and easily yield a functionally intact
platelet preparation. This preparation can be purified again if
needed. No specialized skills or equipment are needed. A significant advantage of the method is that platelets can be obtained
from thrombocytopenic patients in final concentrations that are
high enough to use for platelet function testing. (Key words:
Blood coagulation; Platelets; Techniques) Am J Clin Pathol 1992;
98:531-533
being used for functional or biochemical studies be isolated in a state that reflects, as closely as possible, their in
vivo (unactivated) state. At least biochemical parameters
of platelet function, such as von Willebrand factor content, are significantly affected by the method of platelet
isolation. Some currently used clinical tests can be performed on platelet-rich plasma.' This technique has the
advantages that platelets are not exposed to potentially
activating or damaging conditions, such as being pelleted
by centrifugation, and the preparation steps are quick,
easy, and require no specialized equipment. However,
platelet aggregation studies on platelet-rich plasma cannot
be interpreted when the platelet count is low. Also, specialized testing or research applications require that the
platelets be separated from potentially interfering plasma
components.
Therefore, a number of methods have been
From the Departments of Pathology and2Medicine, The Center for
developed
to
isolate blood platelets. The most common
Thrombosis and Hemostasis, University ofNorth Carolina, Chapel Hill,
methods involve centrifugation, which can activate or
North Carolina.
damage platelets, especially when they are pelleted several
Supported by the Division of Laboratory Medicine, University of North
times.
Gentler methods have been developed using density
Carolina Hospitals, Chapel Hill, North Carolina.
gradient
centrifugation2"4 or electrophoresis,5 but these
Received September 12, 1991; revised manuscript accepted for publication October 30, 1991.
methods tend to be quite time consuming or require speAddress reprint requests to Dr. Maureane Hoffman, Durham VA
cialized
equipment or expertise. Recently a density graMedical Center (113), Laboratory Services, Durham, North Carolina
dient centrifugation technique was reported that is faster
27705.
531
532
COAGULATION AND TRANSFUSION MEDICINE
Article
but still produces a relatively dilute platelet preparation.6
We developed a technique that allows the rapid isolation
of functionally intact purified platelets. It requires no specialized expertise or equipment or expensive reagents. Our
technique also allows platelets to be concentrated to the
point that platelet function studies can be performed on
samples from moderately thrombocytopenic patients.
MATERIALS AND METHODS
Mono-Poly Resolving Medium (MPRM) was obtained
from Flow Laboratories. It is a commercially available
mixture of Ficoll and Hypaque 85, with a density of 1.114.
The product was used as obtained from the manufacturer.
Blood was drawn from six normal volunteer donors
(three male, three female) through a 21-gauge or larger
butterfly into a syringe. All donors had platelet counts
between 200,000 and 300,000/juL. The blood was transferred immediately into a Vacutainer tube (Becton Dickinson, Mountain View, CA) containing citrate anticoagulant. The blood should not be allowed to be drawn into
the tube by vacuum, but rather run gently down the side
of the opened tube. In cases in which even small degrees
of platelet activation are to be avoided, prostaglandin E]
(PGEi) to afinalconcentration of 5 mg/mL can be added
to the Vacutainer tube. The undiluted blood (5 mL) was
layered on top of MPRM (4 mL) in a 15-mL sterile conical
bottom tissue culture tube. The sample was then centrifuged at ambient temperature (22 to 27 °C) for 30 minutes
at 350g. After centrifugation, the pattern of bands resembled that shown in Figure 1. The plasma was removed
from the tube and discarded. The uppermost leukocyte
layer (mononuclear cells and platelets) was removed in 1
mL. It was transferred to a polypropylene tube containing
1 mL Tyrode's buffer that had been warmed to 37 °C.
The buffer of choice for a given application (or plasma)
can be added instead of Tyrode's buffer at this time.
Warming the buffer is not essential, but we found that
platelets retained their functional integrity somewhat better if kept warm when possible. The cell suspension at
this step contained platelets and mononuclear leukocytes.
The nucleated cells were sedimented by centrifugation for
5 minutes at lOOg. The platelet-rich supernatant was removed and placed in a 37 °C warming block until use.
When the platelet preparation is held up to the light and
rocked gently, a swirling pattern should be visible and
aggregates should be absent. The platelet preparations
were counted on a Technicon H2 automated analyzer
(Miles Diagnostic, Tarrytown, NY).
At each step of the isolation process samples were fixed
in 1% paraformaldehyde for evaluation of activation.
Samples of the purified platelet preparation also were activated by incubation with 2 nmol/L thrombin (prepared
from human plasma, as previously described).7 Platelet
A.J.C.P. • I
Plasma
Mononuclear Cells & Platelets
Neutrophils
FIG. 1. Diagram of the appearance of the density gradient after centrifugation. The upper leukocyte band labeled "Mononuclear Cells and
Platelets" is collected.
activation was assessed by immunofluorescence staining
with an antibody directed against the platelet activation
antigen GMP-1408 provided by Dr. Rodger McEver of
the Oklahoma Medical Research Foundation. Platelet
samples were incubated overnight with 5 Mg/mL antiGMP-140 antibody, then with goat anti-mouse IgG-FITC
(Tago, Inc., Burlingame, CA) diluted 1:500 in Tyrode's
buffer with 1 mg/mL bovine serum albumin. The samples
were then analyzed on a FACScan flow cytometer (Becton
Dickinson). The detectors were set on the "log" setting,
and 10,000 events were counted per sample. The percentage of platelets expressing the activation marker was
then determined by comparison to a sample of platelets
incubated with only the secondary antibody.
RESULTS
After centrifugation, the whole blood separated into
bands, as shown in Figure 1. With samples from most
normal volunteers, the erythrocytes pelleted to the bottom
of the tube and were well separated from the leukocytes
and platelets. Above the erythrocyte pellet was a layer of
MPRM containing few, if any, cells. Above this layer were
two closely spaced bands of leukocytes. MPRM is marketed for the purpose of isolating neutrophils in a single
step. When heparinized blood was used, according to the
manufacturer's directions, the lower (neutrophil) band was
well separated from the upper (mononuclear cell) band.
However, when citrate anticoagulant was used, as for the
isolation of platelets, the bands were more closely spaced.
nber 1992
HOFFMAN, MONROE, AND ROBERTS
Rapid Method to Isolate Platelets from Human Blood
533
TABLE 1. RECOVERY OF PLATELETS AND LEUKOCYTES FROM DENSITY GRADIENT*
Leukocytes
(X106)
Fraction
Platelets
(XIO*)
Percentage
Recovery
Total
PMN
Lymphocytes
Whole blood
Platelet preparation
1344
710
53
24.4
1.7
11.9
0.2
10.3
1.5
* Figures are the total number of cells from a single preparation of S mL whole blood. In six
preparations, the total platelet recovery was always between 50% and 60%. and the leukocyte
contamination was always less than 0.5%.
PMN = polymorphonuclear cells.
The platelets localized in the mononuclear cell band. In
some cases the two leukocyte bands were so closely spaced
that some of the neutrophil band was collected with the
monocyte band. This was generally not a problem because
the nucleated cells were sedimented in the next centrifugation step. The platelet preparation obtained by this
procedure generally contained about 50% of the total
platelets from the whole blood sample. There was always
less than 0.5% contamination of the platelet preparation
by leukocytes, most of these being lymphocytes (Table 1).
To test the functional integrity of platelets isolated on
MPRM, we examined the activation state of the isolated
platelets. When whole blood was fixed immediately in
paraformaldehyde, 1.7 ± 0.3% of the platelets were GMP140 positive (n = 6). The value presumably reflects the
number of "activated" platelets circulating in vivo and
those activated during blood collection. After isolation on
MPRM, 3.9 ± 2.5% of the platelets were GMP-140 positive. After incubation for 15 minutes with 2 nmol/L
thrombin, 77.8 ± 9.4% of the platelets were GMP-140
positive. The platelet preparation obtained by centrifugation over MPRM can be gel filtered by standard techniques if it is desirable to remove all traces of plasma
proteins and MPRM from the platelet preparation for
more specialized studies.
tion can be used for functional testing of samples from
moderately thrombocytopenic patients. It is, of course,
possible to concentrate platelet preparations by sedimenting and resuspending the platelets. However, pelleting the platelets can result in activation or damage. We
satisfactorily isolated platelets from patients with platelet
counts between 50,000 and 10,000/ML (data not shown).
An additional advantage of platelet separation on a density
gradient is that the platelets are removed from most
plasma components. Thus, any plasma substance that
might interfere with or augment platelet aggregation tends
to be removed. The preparation obtained by our method
can also be subjected easily to gel filtration as a further
purification step. Gelfiltrationdilutes the starting sample
by a factor of two. Therefore, it is again desirable to start
with a relatively concentrated preparation.
The isolation of platelets on MPRM is not necessary
for routine platelet aggregation studies in the clinical laboratory. However, the technique could be very useful for
specialized or research applications, especially conducting
platelet function testing on thrombocytopenic patients.
DISCUSSION
Many clinical and research applications require the
isolation of blood platelets by techniques that do not
damage the cells or alter their functional status. Several
methods have been reported to achieve this goal. We now
report a density gradient separation method that has the
advantages of being simple, gentle on the platelets, and it
produces a concentrated platelet preparation. We evaluated the activation state of the isolated platelets by a very
sensitive technique—detection of GMP-140 expression
by flow cytometric analysis.9 Even using this sensitive
measure of activation, the platelets obtained by our technique were only minimally activated during isolation.
Furthermore, the isolated platelets were functionally intact
because they were readily activated by thrombin. Obtaining a concentrated platelet preparation is a significant advantage of the isolation technique. The platelet prepara-
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