Comparison Of Automated Platelet Counting

Methods In The Presence Of Interfering Substances

Steven Marionneaux1,2,3, Brenda Sarduy1, Nora Plante1, David Fagan1, Ann Marie Vega1, Denise Quinn1, Virgil Chan1. Don Wright4
1) St Vincent’s Comprehensive Cancer Center, New York, NY, United States; 2) New York Medical College, New York, NY, United
States; 3) University of Medicine & Dentistry of New Jersey, Newark, NJ, United States; 4) Abbott Diagnostics

INTRODUCTION FIGURE 3 FIGURE 4

Myelodysplastic syndrome (MDS) is a group of acquired clonal hematologic disorders characterized by ineffective
and disorderly hematopoiesis. The result is peripheral cytopenias and morphologic abnormalities in the
erythroid, myeloid, and/or megakaryocytic cell lines [Figures 1 & 2]. MDS related cellular abnormalities such as
microcytes/schistocytes and large/giant platelets have been shown to affect the accuracy of automated platelet
counting methods.2-5 The count can be falsely elevated in samples containing small red blood cells (RBC) of similar
size to platelets. Conversely, large/giant platelets may exceed the upper threshold of what is classified as platelets,
resulting in a falsely decreased count. In this study, we investigated the effect of these interferences on the accuracy
of four automated platelet count methods. The CD61 immunoplatelet count method served as the reference method.
CD61 has shown excellent agreement with the reference immunological method (CD41/CD61) in numerous studies.
Further, the CD61 immunological method is not affected by the presence of cellular abnormalities because platelets are
enumerated using a monoclonal antibody specific to platelets.6

FIGURE 1 FIGURE 5 FIGURE 6

FIGURE 2
FIGURE 7 FIGURE 8

MATERIALS AND METHODS

Analyzer results were carefully reviewed in order to detect potential platelet count interferences via inspection of
instrument scatterplots, histograms, indices, flags and quantitative results. Suspect samples were selected from
the routine workload and microcytes/schistocytes and large/giant platelets were confirmed microscopically by
experienced morphologists. Samples chosen for study included:
• Thirty (30) EDTA-anti coagulated samples with microcytes or schistocytes
• Twenty-nine (29) with large/giant platelets
• Samples were split and tested within eight hours of collection using
j Two optical based platelet count methods
s Bayer Advia 2120, Siemens, Tarrytown, NY
s Abbott CELL-DYN Sapphire, Abbott, Santa Clara, CA
CONCLUSIONS
• The Abbott optical and Coulter impedance methods significantly underestimated the platelet count in samples
containing large platelets. The Bayer optical count produced platelet counts that were falsely elevated when large
platelets were present.
• Our findings of falsely decreased platelet counts in the presence of large/giant platelets were consistent with
studies performed by Bowles et al. (2006)2 and Harrison et al. (2000)3
• In samples containing small RBC interference, the Abbott optical method showed the closest agreement with
CD61, while the other methods showed a statistically significant average positive bias of >10,000 platelets/uL

j Two electrical impedance methods

j With mean platelet counts of approximately 40,000/uL, falsely elevated platelet counts of >10,000/uL
are likely to be significant and may affect decisions regarding platelet transfusions
s Beckman-Coulter LH750, Beckman-Coulter, Hialeah, FL
• Falsely elevated platelet counts due to circulating microcytes/schistocytes were also previously reported by
s Abbott CELL-DYN Sapphire (data not shown) Kunicka et al. (2000)4 and Pinkowski et al. (1999)5
j CD61 immunoplatelet method served as the gold standard. • This study suggests that laboratories should be aware of limitations in platelet counting methods when large/
• Platelet counts from each method were compared with CD61 platelet counts using the paired t-test giant platelets and/or microcytes/schistocytes are present
(p<0.05) for determination of statistical significance j Circulating cellular abnormalities are not limited to MDS patients, in fact, they are not uncommon
• Differences in individual samples were visualized using Bland Altman plots. findings in most hospital/lab settings:
s Large/giant platelets
• Associated with active marrow thrombopoiesis and represents newly regenerated platelets
RESULTS v Post Chemotherapy
v Consumptive platelet process
• Among the fifty-nine patient samples tested, most had a diagnosis of MDS. Due to limitations in sample
o DIC
volume, 22 samples containing large/giant platelet and 23 with microcytes/schistocytes were tested on the
o TTP
Advia. Platelet counts ranged from 3000 – 447,000/uL, although mean platelet counts in each comparison
o HUS
were in the thrombocytopenic range. Five samples produced no result on the Sapphire impedance method
• Immune thrombocytopenia
as the platelet count was below the linear limit of 20,000/uL. One sample with large platelets produced an
• Chronic myeloproliferative disease
erroneous result on the Sapphire optical method and was excluded.
• Bernard-Soulier & May-Hegglin anomalies
• The CD61 immunoplatelet count was compared with the phase microscopy method across all 59 samples
s Microcytes, schistocytes or both
and showed good agreement (y = 0.9196x - 0.6001 (r = 0.9594)). These results were consistent
• Iron deficiency
with a previous report that compared these methods in samples that did not contain significant platelet
• Thalassemia
count interferences.1
• Microangiopathic hemolytic anemias
• In Tables 1 and 2, the paired t-test results are shown for each method vs. the CD61 reference method in the • Severe burns
large/giant platelet and microcyte/schistocyte groups, respectively. Individual differences between CD61 • Alternate methods for counting platelets should be utilized when appropriate. However, choices in alternate
platelet counts versus differences between methods are depicted in Bland Altman difference plots (Figures platelet counting methods are limited:
3-8)7. In samples containing large/giant platelets, the Sapphire optical and Coulter impedance methods j Manual phase microscopy count
significantly underestimated platelet counts, and the Advia significantly overestimated platelet counts. s Tedious, time consuming and poorly reproducible
Circulating microcytes/schistocytes resulted in a statistically significant positive bias in all methods (although j Abbott CD61 method
the Abbott optical difference of 3,400 is not likely to be medically significant). s Based on antigenic markers (GPIIIa) found only on the surface of platelets.
s The antiCD61 method for counting platelets and has not been shown to be affected by
TABLE 1
circulating interferences
Platelets CD61 Test s Therefore the CD61 immunoplatelet count is a reasonable alternative to the manual phase method
Large/Giant Confidence Std. error
(x1000/uL) (x1000/uL) Method - t P
Platelet Analysis Interval of Diff. for enumerating platelets in these problematic samples
Mean (SD) Mean (SD) CD61
s In our lab we have successfully implemented the CD61 immunoplatelet count as an
Sapphire Optical (n=28) 70 (51.1) 86 (61.1) -16.0 -24.9 to -7.1' 4.4 3.68 0.001 orderable, billable, reflex test
s CD61 has eliminated 4-6 manual phase platelet counts per day and has improved accuracy
Bayer Optical (n=22) 99.6 (58.6) 90.9 (55.0) 8.7 4.1 to 13.3 2.2 3.93 0.0008 in patients with circulating interferences

Coulter Impedance (n=29) 72.9 (62.6) 88.1 (61.0) -15.5 -23.5 to -7.4' 3.8 3.94 0.0004

REFERENCES
1. Gill, JE, et al, A rapid and accurate closed-tube immunoassay for platelets on an automated hematology analyzer, (2000)
TABLE 2 American Journal of Clinical Pathology 114, 47-56.
Platelets CD61 Test 2. Bowles, K. M., Bloxman, D. M., Perry, D. J., & Baglin, T. P. (2006). Discrepancy between impedance and immunofluorescence
Microcyte/Schistocyte Confidence Std. error platelet counting has implication for clinical decision making in patients with idiopathic thrombocytopenic purpura. British
(x1000/uL) (x1000/uL) Method - t P
Analysis Interval of Diff.
Mean (SD) Mean (SD) CD61 Journal of Haematology, 134, 320-322.
3. Harrison, P., Ault, K. A., Chapman, S., Charie, L., & Davis, B. (2001). An interlaboratory study of a candidate reference method
Sapphire Optical (n=30) 46.23 42.85 3.4 0.85 to 5.90 1.2 2.274 0.0105 for platelet counting. American Journal of Clinical Pathology, 115, 448-459.
4. Kunicka, J. E., Fischer, G., Murphy, J., & Zelmanovic, D. (2000). Improved platelet counting using two-dimensional laser light
scatter. American Journal of Clinical Pathology, 114, 283-289.
7.59 to
Bayer Optical (n=23) 51.57 41.28 10.28
12.98
1.298 7.92 0.0001 5. Pinkowski, R. (1999). Difference between impedance and optical platelet count methods in patients with microcytosis of red
blood cells. Laboratory Hematology, 5, 22-27.
8.84 to
6. Ault K.A., Mitchell J., Knowles C., VanHove L. (1997). Implementation of the immunological platelet count on a hematology
Coulter Impedance (n=30) 57.4 42.85 14.55 2.79 5.21 0.0001
20.26 analyzer – the Abbott CELL-DYN 4000. Laboratory Hematology, 3: 125-128.
7. Bland, J. M., & Altman, D. G. (1986). Statistical methods for assessing agreement between two methods of clinical agreement.