Optimizing platelet count in cats

Blood sampling from a patient who is scared can be very hard. To add a communication barrier to this is of course not ideal, but this is what veterinarians face every day. Besides the well-being of, and the difficulty to sample from the patient, the stress can also result in biological effects on blood cell populations. Such a stress-related change can, for example, be platelet aggregation.

This article summarizes tips and pointers on how to improve the platelet count in animals using automated veterinary hematology systems.

A common issue when analyzing platelets is the merging of the PLT peak with the RBC peak,
obscuring the valley in between these peaks. This issue can have several reasons, such as variability
in PLT size, activation and aggregation of platelets, and the presence of RBC fragments or
microcytic RBCs.
Platelet size and variability differs between animal species, for instance, cats have larger PLTs with
a greater variability in size (MPV 11–18.1 fL) as compared to dogs, pigs, and humans (MPV 7.6–8.3 fL)
(1). For cats, it is therefore a greater risk that the natural variation in size in between PLTs and RBCs
overlaps.

Aggregated platelets

Aggregation of PLTs is a major source of errors in the PLT count. The aggregation is a result of
the reactive nature of PLTs, which can be stimulated to aggregate by a variety of factors such
as adenosine diphosphate (ADP) and serotonin (excreted from the PLTs themselves), adrenalin,
vasopressin, collagen, and thrombin (1, 2). One theory for why PLTs from some animals, such as cats,
are more prone to aggregation than others is their larger size (therefore containing and releasing
more ADP and serotonin) and their sensitivity to stress-related substances such as adrenalin and
vasopressin (1).
If there is a high degree of PLT aggregation in the sample, this will lead to less single PLTs and
therefor a false low count, also called pseudo-thrombocytopenia.
Pseudo-thrombocytopenia can also be EDTA-induced. This is an in vitro phenomenon caused by
anti-PLT antibodies from the patient binding to EDTA-activated epitopes on the PLT (3).

PLT count

Aggregated platelets pose a problem both for automated and manual PLT count. For impedance technology, where the different cell types are discriminated by size, PLT aggregates can have the same size as RBCs and therefore causing the two peaks to merge.

If this happens, an instrument with a floating discriminator will not be able to find the minima between the peaks and the count is instead based on the beforehand set fallback discriminator of the hematology system, much like a fixed discriminator. It is therefore of great importance to understand the variability of PLTs and how these are distributed in the histogram so that the desired discriminator can be set accordingly (Fig 1). Furthermore, severe aggregation can also give effects on the WBCcount, as the PLT aggregates could potentially be mis-interpreted as lymphocytes (2).
Since automated count using optical technology do not only discriminate by size, the two populations will not merge in the same way. However, even these systems have a difficulty reading PLT aggregates as these have a different complexity than single PLTs and therefore a different scatter (1, 2). As the
scatter of PLT aggregates line up very close to the granulocytes (NEU, EOS, BASO), this may lead to a false elevation of these cells (4).
Since the aggregation results in an uneven distribution of PLTs, even manual count is affected. However, the manual smear should always be performed to confirm the presence of aggregates, especially if the PLT count from the automatic hematology system is unexpectedly low.

MPA-functionality

Compared to venous blood sampling, capillary blood sampling provides a less stressful experience for the animal and can therefore minimize stress-related effects on the blood cell populations – an important enabler for a reliable veterinary cell count. Capillary sampling also decreases the sample volume needed for analysis, which is especially important for small or dehydrated animals.
The micro-pipette adapter (MPA) featured on the Exigo H400 enables a complete blood count (CBC) from one drop of blood collected in a capillary sample tube – a functionality well suited for smaller animals (Fig 2).

Fig 1. The MPA capillary sample feature of the Exigo H400 is especially suitable for smaller animals such as cats.

Aperture optimization

In general, small cells demand a higher sensitivity of the analyzer to be counted correctly. To allow accurate count of the smaller blood cells, the Boule veterinary systems are equipped with a narrower capillary aperture for the RBC and PLT count compared with human cell counters. The narrower aperture (Fig 3) allows for smaller corpuscles such as the PLTs and certain RBCs to be detected with a higher sensitivity compared to a standard instrument.

Flexible discriminators

Boule systems employ a floating discriminator to separate the PLTs from the RBCs. The floating discriminator automatically finds the lowest point (valley) in between the two peaks and thereby gives an optimized separation. Should a valley not be found, the discriminator is set to a fallback mode which is a default setting in the systems. The Exigo H400 further offers the possibility to adjust the limits of the discriminator and the fallback.* This enables the laboratory to evaluate and set their own discriminator for PLT/RBC populations in different profiles, thereby making an informed decision on what to count as PLTs and what to count as RBCs.

To optimize the platelet count in animals, consider the following:

• MPA inlet for capillary blood sampling allows for a less stressful sampling method for the animal
as well as a minimized sample volume, suitable for smaller animals.
• Narrow cell count aperture to increase the sensitivity when counting smaller corpuscles
such as platelets.
• Floating and flexible discriminator settings, choose which cells to include.
• Flagging of suspected results.
Counting PLTs is inherently difficult due to the reactive nature of the cells. If the result is
unexpectedly low, or aggregation is suspected, always confirm the findings in a manual smear.

Download Optimizing platelet count in cats as pdf.

References

1. Elizabeth J. Norman et al. Prevalence of Low Automated Platelet Counts in Cats: Comparison with
Prevalence of Thrombocytopenia Based on Blood Smear Estimation. Vet Clin Pathol. 2001;30:137-140
2. Barbara Riond et al. Study on the kinetics and influence of feline platelet aggregation and Deaggregation.
BMC Veterinary Research (2015) 11:276
3. Véronique Baccini et al. Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking
Cellular Hematology Group (GFHC). J Clin Med. 2020 Mar; 9(3): 808.
4. Interpreting IDEXX ProCyte Dx* Hematology Analyzer Dot Plots. © 2011 IDEXX Laboratories, Inc.