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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 3  |  Page : 119-124

Comparative analysis of four methods for enumeration of platelet counts in thrombocytopenic patients


1 Department of Allied Health Sciences, Sultan Qaboos University; Department of Haematology, College of Medicine and Health Sciences, Sultan Qaboos University Hospital, Al Khoudh, Muscat, Sultanate of Oman
2 Department of Haematology, College of Medicine and Health Sciences, Sultan Qaboos University Hospital, Al Khoudh, Muscat, Sultanate of Oman
3 Department of Genetics, College of Medicine and Health Sciences, Sultan Qaboos University, Al Khoudh, Muscat, Sultanate of Oman

Date of Web Publication18-Sep-2015

Correspondence Address:
Boulassel Mohamed-Rachid
Department of Allied Health Sciences, College of Medicine and Health Sciences, Sultan Qaboos University, Room No. 2063, P. O. Box: 35, Postal Code 123, Al-Khoudh, Muscat
Sultanate of Oman
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Source of Support: This work was supported in part by grants from the Oman Research Council (# ORG/HSS/13/002) and the Sultan Qaboos University (#IG/MED/HAEM/14/01)., Conflict of Interest: There are no conflicts of interest.


DOI: 10.4103/1658-5127.165654

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  Abstract 

Background: Even with the most reliable automated blood cell analyzers, obtaining an accurate platelet count in patients with thrombocytopenia is still challenging especially when making decisions for platelet transfusions. The Cell-Dyn Sapphire offers three methods for platelet counting including the optical, impedance, and immunological techniques. Aim: A comparative analysis evaluating the performance of these three methods, along with the manual technique, was performed in thrombocytopenic patients. Materials and Methods: A total of 32 blood samples from patients without a history of chemotherapy and a platelet count <50 ×- 109/l were tested in parallel by four different methods. Results: Compared to other techniques, impedance method provided an overestimation of platelet count (P = 0.0008) and failed to show a result in 15% of cases with low platelet counts (<15 ×- 109/l). Good to excellent correlations and reliability values were evidenced among study methods, but a poor reliability was noticed between the impedance and immunological methods with an intraclass correlation coefficient of 0.49 (confidence interval: 0.15–0.73; P = 0.003). In the bias analysis, the impedance method showed the highest levels of disagreement with other techniques. Summary/Conclusion: Collectively, these results provide evidence that the optical or immunological technique appear to be superior to the impedance method in estimating low platelet counts with the automated analyzer Cell-Dyn Sapphire. As such, the healthcare staff and the physicians must be aware of this limitation, especially in the presence of severe thrombocytopenia, when a decision of platelet transfusion has to be made.

Keywords: Automated analyzer Cell-Dyn Sapphire, manual method, platelet counting, thrombocytopenia


How to cite this article:
Mohamed-Rachid B, Raya AF, Sulaiman AH, Salam AK. Comparative analysis of four methods for enumeration of platelet counts in thrombocytopenic patients. J Appl Hematol 2015;6:119-24

How to cite this URL:
Mohamed-Rachid B, Raya AF, Sulaiman AH, Salam AK. Comparative analysis of four methods for enumeration of platelet counts in thrombocytopenic patients. J Appl Hematol [serial online] 2015 [cited 2023 Feb 4];6:119-24. Available from: https://www.jahjournal.org/text.asp?2015/6/3/119/165654




  Introduction Top


Over the past decade, substantial advances have been made in automated hematology analyzers for diagnostic use and characterization of blood cells. This has progressively diminished the role of manual methods in hematology laboratories for routine testing. Although hematology analyzers usually provide reliable platelet counts, their accuracy has been questioned when enumerating low platelet counts, in the presence of platelet abnormalities or interference of platelet-like fragments.[1]

In patients with severe thrombocytopenia obtaining accurate and precise platelet counts by automated analyzers is still challenging especially when a clinical decision has to be made for platelet transfusion. The recent results of the international Biomedical Excellence for Safer Transfusion Collaborative study clearly indicate that significant inaccuracy exists when counting low levels of platelets using routine hematology analyzers. This inaccuracy might impact on over or under-transfusion of platelet concentrates to patients at high-risk of bleeding.[2] Similarly, using different analyzers, De la Salle et al., reported that the coefficient of variation increases up to 43% as the platelet count decreases, particularly once the platelet counts range between 5 and 10 × 109/l.[3] Therefore, finding the reliable method to assess low platelet counts in thrombocytopenic patients becomes of paramount importance for clinical decisions.

Currently, most automated hematology analyzers employed two fundamental principles for counting platelets namely electronic impedance and optical density. Recently, some analyzers were also equipped with flow cytometry capabilities for platelet counting.[4] Thus, these analyzers can provide platelet counts by means of optical, impedance, and immunological methods.[4]

The initial reference method recommended by the International Committee for Standardization in Haematology, for assessing platelet count was the manual technique using a hemocytometer and a phase contrast microscope.[5] Since 2001, the International Council for Standardization in Haematology and the International Society of Laboratory Hematology recommended the immunological method by flow cytometry together with a semi-automated single-channel aperture impedance counter, as the reference method for enumerating platelets.[6] The immunological method uses conjugated monoclonal antibodies directed against specific platelet antigens such CD41 and CD61. This method demonstrates reliable and accurate results for low platelet counts, but it is not available in all laboratories.[7]

As automated hematology analyzer Cell-Dyn Sapphire (Abbott Diagnostics, CA, USA) provides simultaneous impedance, optical and immunological techniques, this study was undertaken to compare low platelet counts using the three different methods, along with the manual technique, in blood samples of thrombocytopenic patients.


  Materials and Methods Top


Blood Samples

Blood specimens were collected from the morning batch of samples received at the laboratory hematology, Sultan Qaboos University Hospital, Sultanate of Oman. All samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and selected after routine testing on the basis of a platelet count <50 × 109/l and without a history of chemotherapy. These samples were tested in parallel by different methods within 4 h after phlebotomy. This study was reviewed and approved by the Ethics Committee of the College of Medicine and Health Sciences, Sultan Qaboos University Hospital, Sultanate of Oman.

Microscopic Evaluation of Blood Smear

The stained peripheral blood smears were evaluated for platelet count using the routine microscopy technique. This method was also used to evaluate the presence of platelet aggregates, thrombocyte abnormalities or white cell and red cell fragments that may interfere with platelet count.

Enumeration of Platelets by the Automated Analyzer Cell-Dyn Sapphire

The hematology analyzer Cell-Dyn Sapphire (Abbott Diagnostics, CA, USA) was used to enumerate the platelet count by optical, impedance and immunological (CD61-immunoplatelet) methods. The calibration status, quality control and maintenance procedures were performed daily on this analyzer according to the manufacturer's instructions. In brief, the optical platelet count is obtained through a two-dimensional analysis that estimates the complexity and the density of the platelet represented as a cytogram of the light intensity at 7° and 90° angles. Floating thresholds were used to discriminate between platelets and nonplatelet particles.

The impedance platelet count uses hydrodynamic focusing and single-dimensional histogram analysis to count the platelets based on their size.

The CD61-immunoplatelet method uses a fluorescein isothiocyanate conjugated monoclonal antibody directed against the platelet glycoprotein IIIa. This method is fully automated and discriminates platelets using the fluorescence FL1 channel and 7° and 90° scatter. The platelet absolute count is reported without the need for operator intervention or correction for platelet/red cell coincidence.

Statistical Analysis

Quantitative data were summarized using mean, standard deviation (SD) and range. Differences in platelet counts among study methods were evaluated using analysis of variance (one-way ANOVA). If the results of the ANOVA were statistically significant, then the unpaired nonparametric Mann–Whitney U-test was used to identify differences between methods. Correlations among study methods were evaluated by Spearman's correlation test. Bland–Altman plots were produced, and intraclass correlation coefficients (ICC) were calculated for assessing agreement between measurements and for the reliability of the platelet measurements. All tests were two-tailed with an alpha level of 0·05. Analyses were performed using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA).


  Results Top


A total of 32 consecutive blood samples with platelet counts <50 × 109/l were evaluated in this study. The distribution of the platelet counts according to the optical method, which was set by “default” on the automated analyzer Cell-Dyn Sapphire is summarized in [Table 1].
Table  1: Distribution of platelet counts obtained using the reference method

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During the microscopic enumeration of platelets on blood smears, no platelet clump or giant platelets, nor white cell and red cell fragments were observed. The impedance method fails to provide platelet count in five samples (15%) because the analyzer gave a blank result. Of note, these samples showed low platelet counts (<15 × 109/l) by the optical method.

The mean ± SD and range values of platelet counts were 23 ± 13 × 109/l (1–50), 37 ± 17 × 109/l (4–96), 21 ± 12 × 109/l (2–43), and 22 ± 13 × 109/l (2–54) for the optical, impedance, immunological and manual methods, respectively. No significant differences in platelet counts were found among optical, immunological and manual methods. However, impedance method showed significantly elevated numbers of platelet count as compared to optical (P = 0.0008), immunological (P = 0.0004) and manual (P = 0.0001) methods [Figure 1].
Figure 1: Comparisons of platelet counts among optical, impedance, immunological and manual methods. The medians and P values are shown

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As shown in [Figure 2], a high degree of correlation was observed among the three methods included in the automated analyzer Cell-Dyn Sapphire and the manual method. The optical method showed a stronger correlation with impedance (n = 27, r = 0.91, P < 0.0001) or immunological (n = 32, r = 0.91, P < 0.0001) techniques than did the manual method (n = 32, r = 0.88, P < 0.0001). Of note, moderate associations were also evidenced between impedance technique and immunological (n = 27, r = 0.73, P < 0.001) or manual (n = 27, r = 0.83, P < 0.001) methods. Similarly, a statistically significant correlation was noticed between immunological technique and manual method (n = 32, r = 0.80, P < 0.0001).
Figure 2: Linear regression analyses of platelet counts among optical, impedance, immunological and manual methods. The dotted lines showed the 95% confidence band of the best-fit line. The coefficient of correlations and P values are shown

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Although the ICC values, which measure the degree of similarity among study methods, were statistically highly significant, they were slightly smaller than the Spearman correlation coefficients [Table 2]. The estimated reliability among study methods ranged from 0.49 to 0.90. Of interest, the lower ICC reliability was evidenced between impedance and immunological methods. In line with these observations are degrees of agreement among study methods [Figure 3]. The optical method showed an average bias of −12.5, 1.8 and 0.91 with impedance, immunological and manual techniques, respectively. The average bias between impedance and immunological methods was 14.7 while it was 13.5 with manual technique. Interestingly, the average bias between immunological and manual method was 0.97, indicating that the two techniques are producing closely the same platelet results.
Table  2: Reliability analyses among study methods

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Figure 3: Bland and Altman plots depicting degrees of agreement among study methods. The solid line represents zero difference between the two methods. The dotted lines represent the upper and lower limits of agreement between the two methods as measured by mean ± two standard deviations

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  Discussion Top


Obtaining accurate platelet counts in patients with thrombocytopenia using automated hematology analyzers is still challenging even when the most expensive and precise analyzers are used. Our results showed that the impedance method overestimated the platelet counts and also failed to provide any result in 15% of samples. These findings are in agreement with those reported by other groups.[7],[8] Cid et al., showed that impedance method provided higher platelet counts when compared to the optical or immunological methods and also failed to give platelet measurements in the lower thrombocytopenic range (<20 × 109/l).[7] Similarly, Arroyo et al., showed that the impedance method provided an overestimation of platelet counts compared to other methods. They also reported that in 21 of 47 blood samples from patients with postchemotherapy thrombocytopenia, the impedance method was unable to provide the platelet count results.[8] Collectively, these findings clearly suggest that impedance method will not give the best platelet count at low levels and in a number of cases the counts would be falsely high, which may possibly affect the clinical decisions of platelet transfusions when they are indicated. Therefore, the optical or immunological method is the most accurate for estimating low platelet counts when the automated analyzer Cell-Dyn Sapphire is used.

The results of this study also extend to investigations on the associations among optical, impedance, immunological, and manual methods for evaluating low platelet counts. They confirm other reports showing good positive correlations among study methods.[7],[8],[9] However, the same associations were not observed with the ICC, which determines the reliability of the study methods to yield the same or compatible platelet results. Overall, good to excellent reliability values were displayed, but poor reliability was also noticed between the impedance and immunological methods with a quite wide 95% confidence interval, although the ICC value was statistically significant. The low value of ICC could be attributed to the fact that there are substantial differences between these two methods. This assumption is further supported by the degree of agreement between these two methods in Bland–Altman plot where a high positive bias was evidenced. Additionally, the impedance method was the one showing the highest levels of bias with other techniques indicating that this method was producing different platelet counts, thereby showing none negligible degree of inaccuracy. Therefore, physicians should rely on the platelet results obtained by other techniques rather than the impedance method to base their therapeutic decisions for giving platelet transfusions. Since the immunological method is not available for routine applications in all hematology laboratories, an optical method along with the manual technique by microscopic examination of peripheral blood smear could be used to accurately evaluate low platelet counts. In our laboratory settings, the platelet counts estimated by the manual method correlated well with the optical and immunological methods and further showed a high degree of agreement with both methods.


  Conclusion Top


The results of this study provide evidence that the optical and immunological techniques appear to be superior to the impedance method in estimating low platelet counts with the automated analyzer Cell-Dyn Sapphire. Despite good correlations among study techniques, impedance methods provided an overestimation of platelet counts, and also showed some disagreement with all methods in the bias study. As such, the healthcare staff and the physicians must be aware of this limitation, especially in the presence of severe thrombocytopenia, when a decision of platelet transfusion has to be made.

Financial Support and Sponsorship

This work was supported in part by grants from the Oman Research Council (# ORG/HSS/13/002) and the Sultan Qaboos University (#IG/MED/HAEM/14/01).

Conflicts of Interest

There are no conflicts of interest.



 
  References Top

1.
Zandecki M, Genevieve F, Gerard J, Godon A. Spurious counts and spurious results on haematology analysers: A review. Part I: Platelets. Int J Lab Hematol 2007;29:4-20.  Back to cited text no. 1
    
2.
Lozano M, Mahon A, van der Meer PF, Stanworth S, Cid J, Devine D, et al. Counting platelets at transfusion threshold levels: Impact on the decision to transfuse. A BEST Collaborative-UK NEQAS(H) International Exercise. Vox Sang 2014;106:330-6.  Back to cited text no. 2
    
3.
De la Salle BJ, McTaggart PN, Briggs C, Harrison P, Doré CJ, Longair I, 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.  Back to cited text no. 3
    
4.
Briggs C. Quality counts: New parameters in blood cell counting. Int J Lab Hematol 2009;31:277-97.  Back to cited text no. 4
    
5.
England JM, Rowan RM, van Assendelft OW, Coulter WH, Groner W, Jones AR, et al. Protocol for evaluation of automated blood cell counters. International Committee for Standardization in Haematology (ICSH). Clin Lab Haematol 1984;6:69-84.  Back to cited text no. 5
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6.
International Council for Standardization in Haematology Expert Panel on Cytometry; International Society of Laboratory Hematology Task Force on Platelet Counting. Platelet counting by the RBC/platelet ratio method. A reference method. Am J Clin Pathol 2001;115:460-4.  Back to cited text no. 6
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7.
Cid J, Nascimento JD, Vicent A, Aguinaco R, Escoda L, Ugarriza A, et al. Evaluation of low platelet counts by optical, impedance, and CD61-immunoplatelet methods: Estimation of possible inappropriate platelet transfusion. Transfusion 2010;50:795-800.  Back to cited text no. 7
    
8.
Arroyo JL, García-Marcos MA, López A, Menéndez P, Tabernero MD, Sánchez-Abarca LI, et al. Evaluation of a CD61 MoAb method for enumeration of platelets in thrombocytopenic patients and its impact on the transfusion decision-making process. Transfusion 2001;41:1212-6.  Back to cited text no. 8
    
9.
Trabuio E, Valverde S, Antico F, Manoni F, Gessoni G. Performance of automated platelet quantification using different analysers in comparison with an immunological reference method in thrombocytopenic patients. Blood Transfus 2009;7:43-8.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]


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