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Accuracy of the Coulter Impedance Platelet Count Method in Samples Containing Large/Giant Platelets or Schistocytes/Microcytes

Steven Marionneaux , Elaine Keohane , Brenda Sarduy , Nora Plante , Ann Marie Vega , Denise Quinn , David Fagan 1 St. Vincents Comprehensive Cancer Ctr., New York, NY, United States 2 University of Medicine and Dentistry of New Jersey, Newark, NJ, United States
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PURPOSE
Evaluate the effect of large/giant platelets and schistocytes/ microcytes on the accuracy of the Coulter impedance platelet count. Determine if the Coulter platelet fitting algorithm and platelet R code are useful in providing an accurate platelet count and alerting the operator when platelet interferences are present.

MATERIALS AND METHODS


Sample Analyzed by Coulter LH 750: Results Reviewed for Potential Platelet Count Interference Suspect Large/Giant Platelets
OR

DATA ANALYSIS
Data were grouped by type of interference and R code vs No R code Linear regression and paired t-Test used to compare impedance and phase platelet counts. Paired t-test determined statistical significance of the differences between methods. Data was also plotted on Bland Altman difference plots (Bland & Altman, 1986). Percent success rate of the platelet fitting algorithm was calculated

Large/Giant Platelet Group (n=24): Bland Altman Difference Plot


Difference between the reference and impedance method (y) against the mean of the two methods (x). Plus and minus two standard deviations of the differences and the confidence intervals are indicated.

DISCUSSION
Large/Giant Platelet Group
Our finding of lower impedance platelet counts compared to the reference method in samples with large/giant platelets was consistent with the findings of Bowles et al. (2006) and Harrison et al. (2000) A platelet R code to indicate further evaluation and verification of the impedance count was triggered in only 50% (12/24) of the samples with large/giant platelets. However, the impedance platelet counts were significantly lower than the reference method in both the R and non-R groups. According to the Coulter reference manual (Beckman Coulter, Inc., May 2007), the operator may report results with high confidence in platelet counts not flagged with R. In the samples without an R code, the platelet count may not be further investigated, resulting in the reporting of an erroneously low platelet count This study suggests that laboratories using the Coulter LH750 on patients with large/giant platelets should use alternate methods, in addition to the R code, to detect large/giant platelet interference. One such method used in this study was a careful examination of the platelet histogram.

The impedance method underestimated the platelet count in all 24 samples tested.

Suspect Microcytes/Schistocytes
AND

INTRODUCTION
The impedance method is associated with excellent accuracy and precision in the majority of samples tested (Briggs, Harrison, & Machin, 2007). However, because the method is based on cell size, the platelet count can be falsely elevated in samples containing very small red blood cells of similar size to platelets (Pinkowski, 1999). Conversely, the count can be falsely decreased in samples with platelets that are larger than the upper threshold limit of what the analyzer classifies as platelets (Bowles, Bloxman, Perry, & Baglin, 2006; Harrison, Horton, Grant, Briggs, & MacHin, 2000). The St. Vincents Comprehensive Cancer Center recently opened a myelodysplastic syndrome (MDS) research and treatment center. As a result, the number of patients with questionable impedance platelet counts has increased due to the red blood cell and platelet abnormalities associated with this condition. These suspect platelet counts are recognized by abnormal histograms and/or the lack of a fitted platelet curve. In the latter case, the absence or presence of an R code signifies a high or low confidence in the accuracy of the count. Our policy is to perform the phase microscopy method on all suspect samples. This study was done to assess the effect of large/giant platelets and schistocytes/microcytes on the accuracy of the Coulter impedance platelet count and to determine if the platelet fitting algorithm and R code are effective in recognizing inaccurate counts.

SAMPLES
Smear Review & PLT Estimate to Confirm Presence of Interference
50 consecutive samples with suspect histograms were selected for smear review 40 met criteria for study: 24 with large/giant platelets and 16 with schistocytes/microcytes. 10 did not meet criteria due to lack of observed interference (1) or presence of both interferences on the smear (7), or disagreement between the Diagnosis Number duplicate phase counts (2) MDS 41 Subjects were 32 to 83 years Multiple Myeloma 3 with 27 males and 23 females; Liver Cancer 2 Most had a diagnosis of MDS (See table) Aplastic Anemia 1
Colon Cancer Rectal Cancer Thalassemia 1 1 1

Difference between methods

Average of MP and Phase

Excluded if Both Types of Interferences Present

Sample Selected for Study if Contains Large/Giant Platelet OR Schistocytes/ Microcytes

Sample Excluded From Study if No Interference Present

In the Bland Altman plot above, most of the individual differences between the impedance and reference methods clustered near the mean difference (-27,000/uL). There were 2 outliers, from liver patients, in which the impedance method underestimated the platelet count by >2SD of the mean difference.

Tech 1 Phase Platelet Count

Tech 2 Phase Platelet Count

The table below depicts how frequently the clinical severity of thrombocytopenia in individual patients was downgraded by using results of the phase microscopy reference method instead of the impedance method. Clinical severity of thrombocytopenia was downgraded in 16/24 (67%) patients and in half of the patients in the non-R Group
Clinical Severity Group Change Severe to Moderate Moderate to Mild Moderate to Very Mild Mild to Very Mild Very Mild to Normal Total Patients that Changed Groups Platelet R Group Platelet No R Group Number of patients (%) Number of patients (%) N=12 N=12 1 (8) 0 4 (33) 3 (25) 1 (8) 1 (8) 10/12 (83) 4 (33) 0 2 (17) 0 6/12 (50)

References
Bowles, K.M., Bloxham, D.M., Perry, D.J., & Baglin, T.P. (2006). Discrepancy between impedance and immunofluorescence platelet counting has implications for clinical decision making in patients with idiopathic thrombocytopenia purpura. British Journal of Haematology, 134, 320-322. Bland, J.M., & Altman, D.G. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet, i, 307-310. Beckman Coulter, Inc. (May 2007). Coulter LH 750 Reference Manual. Hialeah, FL. Briggs, C., Harrison, P., & Machin, S.J. (2007). Continuing developments in the automated platelet count. International Journal of Laboratory Hematology, 29, 77-91. Harrison, P., Horton, A., Grant, D., Briggs, C., & MacHin S. (2000). Immunoplatelet counting: a proposed new reference procedure. British Journal of Haematology, 108, 228-35. Pinkowski, R. (1999). Difference between impedance and optical platelet count methods in patients with microcytosis of red blood cells. Laboratory Menatology, 5, 22-27.

Agreement of Platelet Counts within 15,000/uL for PLT < 100,000/uL or 15% for PLT > 100,000/uL
No ree Ag ent m A gr eem ent

RESULTS
Large/Giant Platelet Group (n=24)
Most samples were thrombocytopenic. Counts ranges from 17,000/uL to 166,300/uL. Impedance method was lower than phase method in all samples tested with a mean difference of 27,000/uL. Twelve had impedance counts with R code and 12 did not Differences between the impedance and phase counts were greater in the R group (34,900/uL) compared to the non-R group (19,100/uL) All t-test comparisons (All Patients, R Group, and No-R Group) showed statistically significant differences between the Coulter LH impedance platelet count and reference method (see below)
Impedance Mean (x1000/uL (CI for mean) All Patients 51.2 N = 24 (38.2 64.2) R Group 55.8 N = 12 (30.8 80.7) No R Group 46.6 N = 12 (34.0 59.2) Reference Mean (x1000/uL) (CI for mean) 78.2 (62.4 94.0) 90.7 (61.9 119.5) 65.7 (51.2 80.3) Mean Difference (x1000/uL) (CI for mean) -27.0 (-34.0 - -20.1) -34.9 (-47.5 - -22.3) -19.1 (-22.9 - -15.4) SD of t-value P-value Difference 16.5 19.9 5.9 -8.1 -6.1 -11.2 <.001 <.001 <.001

Sample excluded from study

Average Phase Count = Reference Method

Thrombocytopenia classification (x1000/uL) as described by Bowles et al. (2006): Severe: <20; Moderate: 20 49; mild: 50 69; very mild: 70 - 149

A: Microcytes/Schistocytes

B: Large/Giant Platelets

Schistocyte/Microcyte Group (n=16)


The average impedance platelet count was higher than the reference method (+5,400/uL), but was not statistically significant
Mean Impedance Reference Difference Mean Mean (x1000/uL) SD of t-value P-value (x1000/uL (x1000/uL) (CI for (CI for mean) (CI for mean) mean) Micro/Schisto* 40.4 35.0 +5.4 2.1 0.06 (19.7 61.1) (16.0 54.0) (-0.2 11.1) 9.8 N = 16

MATERIALS AND METHODS


Over a period of nine weeks, 50 consecutive K3-EDTA anticoagulated samples with potential platelet interferences were selected by careful examination of the platelet and RBC histograms on the Coulter LH750 analyzer (Beckman Coulter, Hialeah, FL). Samples were processed according to Figure 1. Duplicate phase PLT counts performed within 4 hours of each other. Institutional IRB approvals were obtained.

Acknowledgements
This study was done in partial fulfillment of a Masters in Health Sciences at UMDNJ SHRP. We wish to acknowledge Dr. Sanford Kempin, Director of Clinical Research Attending Physician - Medical Oncology at SVCCC, for his thoughtful critique of the study and for providing advice on medically related content.

Morphology criteria used for cell identification: A. Schistocytes are fragments of RBCs; microcytes are very small RBCs. B. Large platelets are approximately the same size as RBCs and giant platelets are larger than RBCs.

Platelet Fitted Curve Algorithm


The algorithm generated a fitted curve in only 2 of 40 samples studied, a failure rate of 95%. In the two platelet counts reported with a fitted curve, the results were lower than the phase reference method (75,000/uL impedence vs 97,300/uL phase; 65,000/uL impedence vs 94,000/uL phase). Large platelets were present in both samples.

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