Platelet distribution curves: interpretation, potentials and limitations

Sysmex Xtra Online | June 2011

Automated determinations of platelet counts have replaced chamber counts in laboratories for years already and have greatly simplified and clearly improved cell count determination. Counting chambers have either long been collecting dust in some drawers or are only used to check seemingly doubtful values. One of the most important reasons for automated counting is aside from time savings the clearly lower statistical error due to the greater number of cells examined. However, despite the automated cell count, releasing the platelet value will require a more precise review. On most haematology analysers, the platelet count is done by impedance measurement. To determine cell concentration, cells are passed one after the other through a capillary opening (Fig. 1). When a cell passes the measuring orifice, the electrical voltage above the transducer is changed and results in an electrical signal which is proportionate to the cells volume. These impulses result in a volume distribution curve for the corresponding cell population. The sum of impulses within a certain size distribution will result in the corresponding cell number. This measuring principle is further improved through hydrodynamic focusing. This centred stream principle will jacket the stream of particles by a sheath flow so that the particles are passed centrally and one after the other through the measuring capillary (Fig. 2). This almost completely excludes any interference factors such as double passages (coincidences), recirculation,

impedance

direct voltage source (appr. 100 Volt) constant direct current external electrode capillary orice blood cells

vacuum

internal electrode

blood suspension sample cup

Fig. 1 The capacitance measurement principle

Fig. 2 Sheath flow principle with hydrodynamic focussing

Sysmex Xtra Online | June 2011 | 6 pages Platelet distribution curves: interpretation, potentials and limitations

etc. and cells will thus be counted with utmost precision. This also applies for counts with extremely low and extremely high PLT concentrations. Despite all the technologies ensuring a safe result, the assessment must take into account that a cell count is primarily a particle determination which takes only the cell volume into account. Red blood cells (RBC) and platelets will be separated from each other due to their cell volume.
40 fL

PL 100%

12 fL

PU

20% 10 fL 20 fL 30 fL

Aside from numerical platelet values, the analyser also mostly supplies a volume distribution curve which is very important for determining the platelet morphology and thus provides still further information on the platelet value. The volume distribution curve (histogram) presents the sum of all impulses of the respective cell population of a specific size (Fig. 3).
Fig. 3 Display of a platelet histogram

Platelet and red blood cells populations are separated by means of flexible discriminators. Due to the flexible discriminators PL (lower discriminator) and PU (upper discriminator) (Fig. 3), the populations will be optimally separated from each other. Platelets of a physiological size of 812 fL are measured in a range between 230 fL with the flexible discriminator PU setting the optimum separation from the red blood cells. They have a size of 80100 fL and are recorded from 25 fL to 250 fL. Due to this procedure of the automated haematology instrument, technical measuring errors may occur in the analysis of certain samples. Platelets having a greater volume than 30 fL, or red blood cells smaller than 25 fL can falsify the measuring result. Also, other very small particles such as cytoplasm fragments may be erroneously counted as platelets. The most frequent interference factors are giant platelets and fragmentocytes. But microerythrocytes and platelet aggregates are also responsible for wrong counting results. In such cases, mere volume separation is not sufficient for unambiguous cell count analysis, and it is necessary to verify the cell count. Because of a cell volume > 30 fL, giant platelets are taken for red blood cells. Under the microscope, this is evident by a massive anisocytosis of platelets, with platelets which partly reach the size of a red blood cell. Even larger platelet aggregates can be erroneously counted among the red blood cells, while smaller, looser aggregates are torn apart due to the sheath flow method. Fragmentocytes and microerythrocytes, in turn, are counted among platelets. In all these cases, however, an abnormal volume distribution curve and thus possibly a false measurement can be detected. The volume distribution curve should always begin at the baseline and end at the baseline. If deviations from the baseline are detected, the flexible discriminator will search for the optimum position to separate the two populations from each other. In these cases, the analyser generates an alarm to make

Sysmex Xtra Online | June 2011 | 6 pages Platelet distribution curves: interpretation, potentials and limitations

the user aware that the value should be checked either by a more precise evaluation of the histogram or by the actual verification of the platelet value by means of another method. If a false analysis of platelets is suspected, the counting value can be reviewed by the method of the conventional counting chamber. However, it must be taken into account that the counting chamber analysis is subject to many manual errors and requires a certain experience and routine. A second possibility is the semiquantitative estimation of platelets by means of the Fonio method. This method allows an orientation evaluation of the cell count and an accuracy check of the automatic count. With a 1,000-fold magnification (eyepiece 10, lens 100), one platelet per display is equivalent to twenty-thousand platelets/L in circulating blood. In case of a discrepancy between the automated analysis and the estimated value, the chamber count should be used. Immunological marking of platelets with specific, monoclonal antibodies is a good possibility to check the value but unfortunately, it is also very expensive and not always suitable for routines. Another alternative to check the platelet value is a staining of platelets by means of an RNA fluorescence dye. The platelet RNA will be stained and based on the fluorescence activity and the volume of cells the exact platelet value, including giant platelets, will be determined by automation. Most Sysmex instruments integrate RNA staining of platelets as a method and, in case of interferences, switches over to the fluorescence platelet value by means of an automatic algorithm.

Parameters facilitating platelet histogram interpretation: Aside from an evaluation of the histogram, there are also different additional parameters such as P-LCR, MPV, PDW and PCT, providing a multitude of information on platelet morphology.

Sysmex Xtra Online | June 2011 | 6 pages Platelet distribution curves: interpretation, potentials and limitations

PL 100%

12 fL

PU

P-LCR 20% 10 fL Fig. 4 Display of the P-LCR 20 fL 30 fL 40 fL

P-LCR (platelet large cell ratio) The P-LCR indicates the percentage of large platelets with a volume > 12 fL. Aside from the two flexible discriminators which delimit the volume distribution curve, there is additionally a fixed discriminator at 12 fL (Fig. 4). The share of platelets >12 fL in the total platelet number is presented in %. The standard range is 1535 %. An increase of the parameter may be an indication for platelet aggregates, microerythrocytes and giant platelets. PDW (platelet distribution width) The PDW indicates the platelet distribution width measured at 20 % relative height of the total height of the curve (Fig. 5). An increased PDW is an indication for the anisocytosis of platelets. Standard PDW ranges from 9 to 14 fL.

PL

12 fL

PU

100% 20%

PDW

10 fL

20 fL

30 fL

40 fL

reference range 914 fL Fig. 5 Display of the PDW

MPV (fL) =

Pct (%) PLT (x 103/L)

Calculation

MPV (mean platelet volume) This parameter provides a statement on the MPV between the lower discriminator PL and the upper discriminator PU. The standard range is between 812 fL.

Pct (platelet crit) The platelet crit is equivalent to the sum of platelet impulses which are individually detected by means of the impedance measurement principle and thus it is the equivalent to the haematocrit of the red blood cells. If there is an abnormal height of the volume distribution curve at the lower or upper discriminator so that one or several of the indicated parameters can no longer be determined, the parameters will be marked by - - -; the platelet value will be marked and the analyser generates an alarm. In this case, the histogram curve must be checked for correctness of the result. For a more precise evaluation of a platelet histogram, the curves can be divided into three different categories. We call these curves A, B and C curves.

Sysmex Xtra Online | June 2011 | 6 pages Platelet distribution curves: interpretation, potentials and limitations

PL 100%

PU

20% 10 fL Fig. 6 Display of the A curve 20 fL 30 fL 40 fL

A curve: The platelet histogram starts at the baseline but does not end there since there is an interference with microerythrocytes. Due to the extremely small erythrocytes, there will be no clear volume separation between the two cell populations. Since, however, the overlapping of both cell populations is approximately the same on both sides of the discriminator, this platelet value can be used if the MCV shows a standard value. In this case, a chamber count would be statistically much more imprecise. B curve: Here again, the platelet histogram does not end on the baseline. Deviation from the baseline is major and there is no separation between the two populations. There is a total overlapping of both populations. A possible cause might here be fragmentocytes. Fragmentocytes slip into the platelet population and do not allow any clear separation between red blood cells and platelets. In any event, this platelet value is questionable and requires verification. C curve: This case also requires verification of the platelet value. The flexible upper discriminator always looks for the optimum place to separate the populations. However, due to giant platelets or micro-clots, no clear separation can be found; instead, there is major overlapping. The platelet value should be verified, for example, by means of the Fonio method.

PL 100%

PU

20% 10 fL Fig. 7 Display of the B curve 20 fL 30 fL 40 fL

PL 100%

PU

20% 10 fL 20 fL 30 fL 40 fL

Fig. 8 Display of the C curve

Sysmex Xtra Online | June 2011 | 6 pages Platelet distribution curves: interpretation, potentials and limitations

Abnormal platelet distribution on the lower discriminator PL is rather rare. This will be triggered by particles with a volume < 2 fL. Cause for it could be an increased blank value, bacteria, fungi or cell debris. In this case, the instruments blank value should be checked and, if necessary, the reagent should be replaced.
Potential platelet interference factors in impedance measurements Giant platelets PLT
n Evaluate n

histogram If necessary, check by Fonio method or chamber count n Optical (Fluorescence platelets), if available (e.g. XT-4000i, XE-5000)
n

Fragmentocytes

PLT

Fragmentocytes and platelets have no clear volume separation n Evaluate histograms n If necessary, check by Fonio method or chamber count n Optical (Fluorescence platelets), if available (e.g. XT-4000i, XE-5000) Microerythrocytes and platelets have no clear volume separation n Evaluate histograms n If necessary, check by Fonio method or chamber count n Optical (Fluorescence platelets), if available (e.g. XT-4000i, XE-5000) n Evaluate histograms n With implausible thrombocytopenia, clarify an EDTA-induced n Pseudothrombocytopenia through a second sample in citrate
n

Microerythrocytes

PLT

Platelet aggregates

PLT

Table 1

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