Section 4
PLATELET COUNTING
METHODS
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PLATELET COUNTING LEARNING GUIDE
DIAGNOSTICS
PLATELET COUNTING METHODS
MANUAL / MICROSCOPY
Indirect methods
Probably the first method for quantifying platelets in blood was the Fonio technique, developed by a Swiss
physician in the 1940’s. It is performed in a thin blood smear that is stained as for white blood cell differentiation.
Using a microscope with high magnification, the number of platelets is counted in relation to the red blood
cells. In a separate assay, the red blood cell concentration is quantitatively determined and finally, the platelet
concentration is calculated from the platelet/red cell blood ratio. The advantage of this method is that
platelets can be clearly distinguished from other small particles by their specific morphological characteristics.
Disadvantages are that the method is highly imprecise and time consuming. Some laboratories continue to use this
method as an approximate estimation for verifying automated platelets counts.
Direct methods
Several years after Fonio’s description, Feissly and Ludin suggested the use of phase-contrast microscopy for
platelet counting. This method was improved by Brecher and Cronkite and was later adopted as the international
reference method for platelet counting. A small amount of diluted, hemolyzed blood is brought into a calibrated
glass hemocytometer (counting chamber) with an engraved network of known dimensions, which enables
counting platelets in an exactly defined volume of fluid (Figure 6).
In skilled hands phase-contrast microscopy is accurate enough for clinical purposes, but the method has two
major disadvantages: high imprecision and potential interference by particles other than platelets. The high
imprecision is a direct consequence of the relative low number of platelets counted in the hemocytometer,
particularly in thrombocytopenia. Coefficients of variation of 20–40 % are not uncommon at all. Interference
may occur in conditions where small-sized cellular fragments of non-platelet origin circulate in blood. These may
be very difficult to distinguish from platelets. Leukocyte cytoplasmic fragments in some forms of leukemia are
a good example of this interference. In addition, microscopic platelet counting is time-consuming and requires
a high level of technical expertise, which is disappearing from many clinical laboratories after the widespread
introduction of automated platelet counting in hematology analyzers. In 2001, phase-contrast microscopy was
replaced as the international reference method and has become obsolete since.
3 mm
Cover Slip 0.1 mm depth
0.25 mm
1 mm
0.2 mm
1 mm
V Slash Moat
Ruled Area
W - WBC counting
P - Platelet counting
Figure 6. Phase contrast microscopy using hemocytometer
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IMPEDANCE
The principle of impedance counting, also known as the Coulter principle after its inventor Wallace Coulter, is the
passage of cells suspended in a known dilution through a small orifice. The electrolyte-containing diluent serves
as a conductor of a constant electrical current between two electrodes. Cells are poor electrical conductors and
as they pass through the orifice, they impede the passage of current, which is detected as an increase in electrical
resistance. Each cell will cause a resistance pulse, thus allowing cell counting. Furthermore, the magnitude of the
resistance peak is directly related to cell volume.38 Impedance counting is schematically illustrated in Figure 7.1.
Impedance platelet counting is typically performed in the presence of red blood cells (RBC), which are counted
simultaneously. Platelets are differentiated from RBCs based on histogram analysis of the accumulated resistance
events, meaning their size. Thresholds are used to find optimal separation between the two cell populations. Over
years of development, technical nuances have been introduced into impedance counting for improving accuracy.
One of the known limitations of impedance counting is the potential for a phenomenon called “recirculation”
that can cause falsely increased cell counts. This phenomenon (shown in Figure 7.2) occurs when cells that have
traversed the orifice become caught in eddy currents behind the orifice. These cells recirculate in and around the
detection zone and can be recounted, which obviously results in spuriously higher counts.
Direct current Direct current
Electrolyte/ Electrolyte/ Electrolyte/ Electrolyte/
diluent diluent diluent diluent
Pressure Pressure
Orifice Orifice
Figure 7.1. Principle of cell counting using impedance technology. Figure 7.2. Recirculation of cells trapped in the eddy current just
A vacuum draws the cells suspended in conductive diluent from left behind the orifice, giving rise to false recounts.
to right through the orifice. Passage of each cell is registered as a
peak in electrical resistance between the two electrodes.
Various approaches to resolve this artifact have been applied. Some instrument manufacturers use lateral flow
of reagents to sweep already counted cells away from the detection zone. Other manufacturers use a plate close
to the orifice, which ensures that any cell recirculation takes place away from the detection zone. These devices
are called after their inventor, von Behren’s plates (Figure 7.3). Another alternative is the use of hydrodynamic
focusing. This technique employs a sheath of fast moving fluid that guides and confines the cell suspension,
ensuring that during analysis the cells are continuously propelled forwards through and beyond the orifice and
therefore away from the impedance detection zone (Figure 7.4). One further advantage of using hydrodynamic
focusing is that it focuses the cells on the very center of the orifice Instruments that do not use hydrodynamic
focusing are prone to what is known as the “edge effect”. This phenomenon implies that cells flowing through
a simple bulk flow transducer may traverse the orifice at its center, but may also pass at the periphery of the
orifice. The consequence then is an irregular impedance profile, resulting in false estimates of cell size (Figure
7.5). Although electronic and algorithmic editing of the pulses can correct this phenomenon to some extent,
hydrodynamic focusing is the preferred means of resolving edge effects.
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Impedance analysis has some benefits. The method has historically been widely accepted and from an economic
perspective, impedance detectors can be cheaply manufactured. The disadvantage of impedance analysis is
that the discrimination between platelet and non-platelet events is purely based on size. Although normal sized
platelets and normal sized RBCs show little overlap, this may not be true in cases of pathology. Specific examples
of poor impedance separation are shown in Figures 7.6B and 7.6C. In some impedance analyzers attempts have
been made to optimize separation between platelets and RBCs, for example by using dynamic thresholding to find
valleys between the two cell populations. Alternatively, software algorithms have been developed that improve the
accuracy of platelet counts in comparison with the use of fixed thresholds.
Direct current
Direct current
Von
Behrens’s
plate
Impedance
Electrolyte/ Electrolyte/
diluent diluent
Diluent
sh eath
Sensing zone
Pressure
Impedance
Sample
injection
sh eath
Diluent
Impedance
Center of orifice
Orifice
Figure 7.3. A von Behren’s plate prevents Figure 7.4. Hydrodynamic focusing prevents Figure 7.5. The “edge” effect causes cells flowing
recirculation in the zone behind the orifice. recirculation as well as the edge effect. non-centrally through the orifice, resulting
in irregular impedance signals that no longer
represent the volume of the cells.
Volume (fL) Volume (fL) Volume (fL)
Figure 7.6A Figure 7.6B Figure 7.6C
Figure 7.6. Histograms of CELL-DYN Sapphire platelet impedance measurements. 7.6A: (left) normal platelet count and normal MCV; no overlap with RBC.
7.6B: (center) normal platelet count and low MCV; clear overlap between platelets and microcytic RBC. 7.6C: (right) very low platelet count; the separation
between platelets and non-platelets is difficult to define.
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OPTICAL
Optical platelet counting is based on the light scatter properties of blood cells and this technology is being used
by several manufacturers. Using flow cytometry, either two angles of light scatter are measured or single-angle
light scatter in combination with fluorescence that is generated by a dye binding to platelets is measured. The
advantage of a two-dimensional approach is that the resolution between platelets and non-platelet particles is not
based on size alone and therefore is more specific.40
In general, the low-angle scatter signal represents volume, whereas the higher-angle scatter is derived from the
cellular density. In the case of platelets, higher-angle scatter mainly represents granulation. In CELL-DYN Ruby,
0° and 10° light scatter are used, resulting in excellent separation between platelets and RBC (Figure 8.1). CELL-
DYN Sapphire utilizes 7° and 90° scatter and although the scatterplot looks somewhat different, it allows accurate
and specific delineation of the platelet cluster (Figure 8.2).
The precision of the CELL-DYN Sapphire is high, even at low platelet counts. This is because the instrument is
able to monitor the cell count as platelet events are being accumulated. If the analyzer detects a reduced platelet
count, then it automatically extends the platelet data acquisition time, thereby increasing the number of platelet
events to be counted. This eventually results in improved counting statistics.41 Also the fluorescent method can
reach very good precision.42 However, this method seems to suffer from systematic bias when compared with the
immunological international reference method.42,43
Despite two-dimensional optical counting being significantly less prone to interference by non-platelet particles
than impedance technology, there are still rare conditions where optical methods are sensitive to interference.
A newer version of the optical method for counting platelets is the Advanced MAPSS technology. This technology
utilizes 5 scatter signals (ALL, PSS and 3 IAS signals) to differentiate platelets from red blood cells based on size
and internal complexity of the cells. These multiple angles of scatter measurement enable better separation of
RBC from platelets, even in the presence of very small RBC or RBC fragments (Figure 8.3).
Optical PLT
250
6535
200
150
0°
IAS3
100
50
90°
0
0
0 50 100 150 200 250 0 6535
7° IAS2
RBC 10°
Figure 8.1. Optical platelet scatterplot of Figure 8.2. Optical platelet scatterplot of Figure 8.3. Intermediate angles of light scatter
CELL-DYN Ruby, displaying 0° against 10° CELL-DYN Sapphire showing 90° against plot. This scatterplot shows the separation of
light scatter. The platelets (yellow dots) are 7° light scatter. The two lines are dynamic the PLT and the RBC populations based on two
well separated from non-platelet particles thresholds for separating platelets (yellow angles of intermediate light scatter (IAS2 vs.
(black dots) and from the red blood cells dots) from non-platelet particles (black IAS3). RBC (red) smaller in size are positioned
(red dots). dots), which can be located both above near the platelet cluster (yellow); however,
and below the platelet cluster. differences in the internal complexity of RBC
and PLT enables differentiation between them.
This suggests that the platelet count is not
affected by the presence of microcytic RBC.
In patients with normocytic RBC, there is a
wider separation between the RBC and PLT
populations due to differences in both size and
internal complexity.
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IMMUNOLOGICAL
The most reliable technology for measuring platelets is based on monoclonal antibodies to platelet-specific
surface antigens. The international reference method employs dual-color immunofluorescence flow cytometry
using a mixture of two different antibodies, CD41 and CD61.44 Platelets are identified by their reaction with these
antibodies and the platelet/erythrocyte ratio is determined by selective gating. Subsequently, erythrocytes are
counted in a separate hematology analyzer, which eventually allows calculation of the platelet concentration.
This ICSH reference method is a two-platform technique that cannot be fully automated.44 This fact and the
requirement for an experienced flow cytometrist render it difficult to perform the reference method on a routine
basis in the hematology laboratory.
The CELL-DYN Sapphire offers a variant of the ICSH reference method. It is a fully automated, single-color flow
cytometric technique using CD61 monoclonal antibodies that can easily be run in a routine setting by staff that is
not experienced in flow cytometry. It is essentially identical to the method that was developed for the CELL-DYN
4000 back in the 1990’s.45,46
In short, the analyzer dispenses a small aliquot of blood into a tube that contains lyophilized FITC-labeled CD61
monoclonal antibodies. When the reaction mixture incubates, the CD61 monoclonal antibodies bind to specific
epitopes on the platelet surface membrane. Subsequently, the mixture is further diluted and then passed through
the optical flow cell of the analyzer. Two angles of light scatter are measured (7° and 90°), along with the FL1
fluorescence signal that comes from cell-bound FITC, representing CD61. Through knowledge of the dilution
used, the flow rate and the duration of analysis, the instrument is able to directly calculate the concentration
of platelets, the CD61 positive events. CD61 negative, non-platelet events are automatically excluded from the
analysis (Figure 9.1).
CD61 CD61 II
neg pos
FL1 CD61
FL1 - CD61 7°
Optical PLT
CD61 I
90°
90°
7° 7°
Figure 9.1. The CELL-DYN Sapphire CD61 immunoplatelet method. Upper left: histogram of FL1 fluorescence for defining CD61-positive events as platelets. Upper
right: FL1 against 7° scatterplot showing platelets (large green cluster) as well as platelet-erythrocyte coincidence events (small cluster on the right). Lower left: 90°
against 7° scatterplot in which all CD61-positive events are colored green and non-platelet events colored black. In this plot the dynamic thresholds are identical with
those in the Optical PLT scatterplot (lower right).
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Several studies have demonstrated very close correlation between the CD61 immuno-platelet method and the
ICSH reference method. 47,48 Moreover, it has been shown that the precision of the Sapphire CD61 method is
excellent, possibly even better than the CD41/CD61 ICSH reference method.47 In severe thrombocytopenia with
platelet counts ranging between 5 x 109/L and 10 x 109/L, the coefficients of variation of the CD61 assay were
found to be 1.6–2.3 % only, whereas the corresponding data of the ICSH reference method were 3.8–5.6 %.41 Some
authors recommend the CD61 immunoplatelet assay as the method of choice for samples with low platelet count
and from neonates.49
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