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Evaluating platelet counting on a new automated analyser

Hitesh Tailor and Carol Briggs
2 June, 2014  
Assessment of the Sysmex XN haematology analyser in platelet counting is discussed
 
Hitesh Tailor
Carol Briggs
Department of Haematology,
University College Hospital,
London, UK
 
Platelets are essential to arrest bleeding, with the normal platelet count widely quoted as 150–400 x 109/l of whole blood.
Accurate and precise enumerations of platelets are essential in clinical haematology/oncology because they provide the data for diagnosis and guide treatment and maintenance of various haematological disorders. Platelets are essential for primary haemostasis and to prevent/arrest bleeding.
 
Analytical procedures
The four main analytical procedures for platelet counting are: 
  • microscopic manual counting using a Neubauer counting chamber(1) 
  • impedance analysis (available on most analysers)
  • optical light scatter/ fluorescence analysis using more up-to-date analysers(2)
  • immunoplatelet counting by flow cytometry.(3,4) 
The introduction of automated full blood cell counters using impedance technology resulted in a dramatic improvement in precision, with typical coefficients of variation (CVs) of less than 3%,(5,6) because much higher total numbers of platelets are counted. However, impedance platelet counting methods have significant limitations, despite their widespread use. One of the major problems is that cell size analysis cannot discriminate platelets from other similarly sized particles, such as small or fragmented red cells, immune complexes or large platelets.(7) These may be erroneously included in the platelet count, and in severely thrombocytopenic samples the number of interfering particles may even exceed the number of true platelets. Large or giant platelets may be excluded from the count on the basis of their size, because they cannot be resolved from red cells. More recently, multiple light scatter parameters and/or fluorescence, rather than impedance sizing alone have been introduced for platelet counting in automated haematology analysers. This has improved the ability of automated analysers to identify platelets. Despite these newer methods, there are still occasional cases in which absolute accuracy of the platelet count remains a challenge. 
 
Developments
The Sysmex XE-2100 (Sysmex, Kobe, Japan) was introduced in 19998 and is an automated haematology analyser using direct current sheath flow for impedance platelet (PLT) count, fluorescence flow cytometry for leukocyte differential, nucleated red blood cell (NRBC), reticulocytes and optical fluorescence platelet count (PLT-O) and with quantitation of reticulated platelets, expressed as the immature platelet fraction (IPF).(9,10) The IPF is identified by flow cytometry with the use of a nucleic acid-specific dye in the reticulocyte/optical platelet channel. The clinical usefulness of this parameter has been established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, particularly autoimmune thrombocytopenic purpura and thrombotic thrombocytopenic purpura, giving a high IPF whereas a low IPF in decreased bone marrow production. It as a predictor of platelet recovery following haematopoietic progenitor cell transplantation; the IPF rises one to two days before the platelet count and could allow for a decision to wait before giving a platelet transfusion as recovery is eminent.(11,12)
 
The decision to prophylactically transfuse platelets is dependent on the platelet count, careful regular clinical assessment and agreed local protocol: some say provided the patient is observed, platelet transfusions are not required whatever the count.(13,14) The ability to predict when platelet recovery will occur using the IPF should allow a more reasoned approach to platelet transfusions an increase in IPF demonstrates impending platelet recovery.
 
However apoptopic (dead cells with nuclear and chromosomal DNA fragmentation) white blood cells are sometimes stained by the reticulocyte stain on the XE-2100 giving falsely high results for platelet count and IPF. Apoptopic white cells are sometimes seen in patients undergoing chemotherapy where an accurate platelet count is required for decisions on treatment and /or platelet transfusions are needed.
 
The Sysmex XE-5000 was launched in 2007. This analyser performs all blood cell enumeration in the same way as the XE-2100 but with added parameters for red cell indices useful for the diagnosis of functional iron deficiency and monitoring erythropetin treatment.(15)
 
The new XN Modular analyser was introduced in 2011. The red cell parameters and impedance platelets are measured in the same way as previous XE series instruments, but several new channels have been introduced. The new features on the instrument are: white cell and NRBC channel, all samples include the NRBC count; white cell precursor channel; false positive flags for blasts, abnormal lymphocytes and atypical lymphocytes are reduced significantly without a statistical increase of false negatives;  low white cell count mode; an extended count is more precise and provides an accurate differential.(16)
 
Impedance platelet counting results are similar to the XE-2100. This is not surprising because it is the same technology.  For the purpose of this article, the most important is the dedicated fluorescent platelet channel (PLT-F). Optical platelets can still be analysed in the reticulocytes channel if desired, however the PLT-F dedicated channel gives more precise and accurate results on low platelet counts and samples with interfering substances, however flags suggesting an optical platelet count is needed, unreliable impedance count, on the XE-2100 were seen on 27% of abnormal samples but only 9% of samples on the  required confirmation using the PLT-F with the XN. This is a reduction of repeat sampling and improvement of workflow as reflex testing is automatic for the PLT-F on the XN but  on the XE-2100 the sample has to be manually selected and re-tested.(16)
PLT-F and IPF are stained by a proprietary fluorescent dye based on oxadine. Included are a side fluorescence light that gives information on DNA/RNA content of the cell, a side scattered light that gives information on intracellular structure of the cell, and a forward scattered light, which gives information on cell size.
 
The PLT-F channel can be selected for testing on any sample or only used as a reflex test if there are abnormal red cells, or platelet size histograms or, if the platelet count is below a pre-set limit, determined by the user, then an extended platelet count is performed (five times longer) to increase accuracy and precision.
 
The PLT-F method was superior to the XE-2100 optical method when compared with the flow cytometric reference method on samples with a platelet count of 30 x 109/l or less, (XE-2100 R2 0.500 and XN 0.875).(14) These results are very important because this is the most common threshold for platelet transfusion and accuracy is needed to ensure patients receive, or do not receive, transfusions where indicated.(13)
 
The precision of platelet counts on the XN is also impressive because of the extended platelet counts. PLT-F precision showed a CV of only 4% on platelet counts of between 9 x 109/l and 39 x 109/l. This gives far superior precision than previously documented on the impedance platelet counts from the Sysmex XE-2100 with an average CV of 13.6% on counts in this range.(17)
 
Conclusions
Overall the platelet counts on abnormal samples, fragmented red cells, large platelets and low counts on the XN system is excellent when compared with the reference flow cytometric method.
 
Acknowledgements
This work was supported by an unrestricted educational grant to University College London Hospitals.
 
References
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  3. Harrison P et al. Immunoplatelet counting: a proposed new reference procedure. Br J Haematol 2000;108: 228–35.
  4. International Council for Standardisation in Hematology Expert Panel on Cytometry: International society of Laboratory Hematology Task Force on platelet Counting. (2001). Platelet counting by the RBC/platelet ratio method. A reference method. Am J Clin Path 2001;115(3):460–4.
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  9. Briggs C et al. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004;126:90–9.
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  12. Briggs C et al. Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation. Transfus Med 2006;16(2):101–9.
  13. Wandt H et al. Safety and cost effectiveness of a 10 x 109/L trigger for prophylactic platelet transfusions compared to with the traditional 20 x 109/L trigger: a prospective comparative trial in 105 patients with acute myeloid leukemia. Blood 1998;91:3601–6.
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  15. Thomas DW et al. Guideline for the laboratory diagnosis of functional iron deficiency. Br J Haematol 2013; 161:639–48.
  16. Briggs C et al. Performance evaluation of the Sysmex Haematology XN modular system. J Clin Pathol 2012;65(11):1024–30.
  17. De la Salle BJ et al. The accuracy of platelet counting in thrombocytopenic blood samples distributed by the UK National External Quality Assessment Scheme for General Haematology. Am J Clin Pathol 2012;137:65–74.