Abstract. Hematopathology / Counting in Thrombocytopenic Specimens

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1 Hematopathology / Counting in Thrombocytopenic Specimens The Accuracy of Platelet Counting in Thrombocytopenic Blood Samples Distributed by the UK National External Quality Assessment Scheme for General Haematology Barbara J. De la Salle, FIBMS, 1 Paul N. McTaggart, FIBMS, 1 Carol Briggs, FIBMS, 2 Paul Harrison, FRCPath, 3 Caroline J Doré, 4 Ian Longair, 2 Samuel J. Machin, FRCPath, 2 and Keith Hyde, FRCPath 1 Key Words: Proficiency testing; Thrombocytopenia; Platelet count; Laboratory hematology; Transfusion DOI: /AJCP86JMBFUCFCXA Abstract A knowledge of the limitations of automated platelet counting is essential for the effective care of thrombocytopenic patients and management of platelet stocks for transfusion. For this study, 29 external quality assessment specimen pools with platelet counts between 5 and /L were distributed to more than 1,100 users of 23 different hematology analyzer models. The same specimen pools were analyzed by the international reference method (IRM) for platelet counting at 3 reference centers. The IRM values were on average lower than the all-methods median values returned by the automated analyzers. The majority (~67%) of the automated analyzer results overestimated the platelet count compared with the IRM, with significant differences in 16.5% of cases. Performance differed between analyzer models. The observed differences may depend in part on the nature of the survey material and analyzer technology, but the findings have implications for the interpretation of platelet counts at levels of clinical decision making. Accurate and reproducible platelet counts are essential for the management of thrombocytopenic patients at risk of bleeding, such as patients undergoing cytotoxic therapy for hematologic malignancy. Current UK guidelines recommend a threshold of /L for prophylactic platelet transfusion and suggest that this threshold might be reduced further, to /L, for patients without risk factors. 1 This reduction would conserve valuable stocks of platelets for transfusion and reduce the exposure of patients to the risks associated with transfusion of blood components. The accuracy of platelet counts produced by routine automated hematology analyzers has been questioned, 2-4 and the limitations of platelet counting at these extremely low levels should be understood by people responsible for the care of patients. The methods used for automated platelet counting are impedance, optical scatter, optical fluorescence, and immunologic flow cytometry. The traditional gold standard method was manual phase contrast microscopy, 5,6 although this method is time-consuming and imprecise at low counts. 7 The introduction of the international reference method (IRM) for platelet counting by flow cytometry 7-9 has improved the precision and accuracy of platelet counting at thrombocytopenic levels and offers a suitable comparator for routine platelet counting methods. The UK National External Quality Assessment Scheme for General Haematology [UK NEQAS (H)] is uniquely able to undertake major state-of-the-art comparisons of equipment performance. Unlike many other external quality assessment (EQA) or proficiency testing providers, UK NEQAS (H) distributes the same in-house prepared survey material to all instruments for the performance assessment of full blood count (FBC) parameters, including the platelet Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA 65

2 De la Salle et al / Counting in Thrombocytopenic Specimens count. UK NEQAS (H) has more than 2,000 analyzers from all of the major instrument manufacturers registered in the FBC scheme. The results of 29 EQA specimen pools with all-methods median (AMM) platelet counts of between 5 and /L, distributed since 2006, have been compared with the median IRM platelet count determined at 3 independent sites to assess the performance of automated hematology analyzers at these very low levels for clinical decision making. Materials and Methods Survey Material UK NEQAS (H) FBC survey material was prepared from leukocyte-depleted human whole blood, anticoagulated with CPD-A1, obtained from healthy blood donors who had consented to the use of their blood for EQA purposes, and supplied by the UK National Health Service Blood and Transplant Service. For each pool, 5 to 6 individual donations of ABO group compatible, leukocyte-depleted whole blood, less than 72 hours old, were used. ABO group compatible whole blood and buffy coat residues were added to each p ool to raise the platelet count to the desired level. During the preparation of each specimen pool, the platelet count was assessed using a Sysmex K-4500 hematology analyzer (Sysmex, Kobe, Japan). Specimen pools were partially fixed using glutaraldehyde and formaldehyde, and the antibiotics penicillin and gentamicin were added. 10 Specimen pools were dispensed and bottled using a bespoke Hook and Tucker Zenyx Beeline dispensing machine (Hook and Tucker Zenyx. Croydon, England). Specimen homogeneity was maintained during bottling using a bespoke mixing system. Homogeneity and Stability of Specimens Survey material homogeneity and stability was determined according to UK NEQAS (H) standard protocols for UK NEQAS (H) FBC survey material. For the study, 10 specimens selected from each pool were analyzed in triplicate for FBC parameters using a Sysmex K-4500 analyzer immediately after bottling, on survey distribution day, and on survey closing day. Within- and between-batch analysis was used to confirm the stability and the homogeneity of the batch for WBC count, hemoglobin concentration, and platelet count. Specimen Distribution This study includes data from 29 specimen pools with AMM values between 5 and /L, distributed as part of the UK NEQAS (H) FBC surveys 0603FB to 0904FB. Specimens were distributed to all registered participants in the UK NEQAS (H) FBC scheme by first class post within the United Kingdom and by door-to-door courier delivery outside the United Kingdom. Transit time varied from 1 to 4 days on average, and the closing date for return of results was 7 days from the distribution date. Data from a further 6 thrombocytopenic specimen pools distributed during the same period were excluded from the study because the data failed the statistical criterion for inclusion, which was that less than 50% of the instrument groups should show a statistically significant difference (P <.01) between the submethod (ie, instrument group) median platelet count for the specimen pool and the IRM. Instrument Grouping Within the UK NEQAS (H) FBC EQA scheme, participants are grouped into 15 instrument types for analysis according to analyzer technology. Each grouping may contain more than 1 analyzer model. For the purposes of this study, results were analyzed by individual analyzer model. The target minimum number of analyzers in each group was 20; data from 2 groups with 18 instruments were included for 2 specimen pools. Several analyzers have more than 1 platelet counting method available: optical, impedance, and, for 1 manufacturer, immunologic. For most analyzers, platelet counts by any of these modes may be reported to UK NEQAS (H). However for Sysmex XE-2100 instruments, previous UK NEQAS (H) FBC survey data has indicated a wider variation for optical compared with impedance platelet counts. Expert advice from the UK NEQAS (H) Steering Committee s General Scientific Advisory Group has suggested that this variation results from platelet activation during the preparation of survey material, making performance assessment difficult. On the advice of the scheme s scientific advisors, and in agreement with the instrument manufacturer, UK NEQAS (H) specifically requests that only the impedance count be reported from Sysmex XE-2100 instruments. Target Platelet Count and Acceptable Range The target platelet count on each specimen was assayed by the IRM 9 at 3 reference centers during the time that the survey was open. Each center assayed 2 specimens from each survey material pool in triplicate, giving a total of 6 assay values for each pool from each site. The 3 reference centers were UK NEQAS (H), University College Hospital London (UCLH, London, England), and the Oxford Haemophilia Centre (Oxford, England), equipped with the FACSCalibur [Becton Dickinson, Oxford, England; for UK NEQAS (H) and Oxford] or the Epics XL (Beckman Coulter, Miami, FL; for UCLH). The comparability of the methods at the 3 centers was validated at the start of the study by counting 10 fresh, whole blood, EDTA-anticoagulated specimens in triplicate at each site on the same day Table Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA

3 Hematopathology / Original Article The acceptable range to ensure that platelet counts are clinically reliable in EQA may be set at 10% to 15% of the target or assigned value, 11 which would give an approximate range of ± /L in absolute terms for the accuracy of a target platelet count of between 35 and /L and an approximate absolute range of ± 3 for a target platelet count of 20 to /L. At target values less than /L, a percentage range is of less value because the range becomes so narrow in absolute terms (< /L) as to be meaningless in practice. For this reason, an acceptable EQA performance range in absolute terms was calculated for each specimen pool as the IRM ± /L for specimen pools with IRM platelet counts of /L or more and the IRM ± 3 for pools with IRM platelet counts of /L or less. The mean IRM counts from each reference center for the 29 survey material pools are shown in Figure 1 : 5 mean counts (of 80) fell outside the acceptable EQA performance Mean Platelet Count ( 10 9 /L) FB2 0605FB1 0606FB1 0607FB1 0608FB1 0609FB1 0611FB1 0701FB2 0704FB1 0706FB1 0707FB2 0708FB1 0709FB1 0711FB1 0712FB1 0801FB2 Table 1 Comparison of the IRM for Platelet Counting at Three Reference Sites * Specimen Pool Mean IRM Platelet Count ( 10 9 /L) Specimen Pool Oxford UCLH UK NEQAS (H) Clotted IRM, international reference method. * The Oxford Haemophilia Centre, Oxford, England; UCLH (University College Hospital London, London, England); and the UK NEQAS (H) (UK National External Quality Assessment Scheme for General Haematology). For the study, 10 fresh, whole blood specimen pools of varying platelet counts were tested in triplicate at each center on the same day. UK NEQAS (H) Oxford UCLH 0802FB1 0802FB2 0803FB1 0803FB2 0804FB2 0805FB1 0807FB1 0807FB2 0808FB1 0809FB1 0811FB1 0812FB1 0904FB2 Figure 1 Comparison of the mean international reference method platelet counts from the 3 reference centers: UK NEQAS (H) (UK National External Quality Assessment Scheme for General Haematology, Watford, England); the Oxford Haemophilia Centre, Oxford, England; and UCLH (University College Hospital London, London, England) for the 29 specimen pools distributed in the study. Counts were provided by all 3 centers for 22 of 29 specimen pools and by 2 of 3 centers for the remainder. The counts shown represent the mean of 6 counts on each specimen pool for each center. Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA 67

4 De la Salle et al / Counting in Thrombocytopenic Specimens range for the individual specimen pools. Of a total of 87 possible mean counts, 7 were not submitted owing to staffing shortages or reagent supply problems at the individual reference centers. Statistical Analysis Statistical analysis was performed using Stata 11 statistical analysis software (StataCorp, College Station, TX). Differences were evaluated by using the independent Student t test or Mann-Whitney U test, as appropriate. Median, estimated SD, 12 and coefficient of variation (CV) values were produced for all methods data and individual instrument group data. Cumulative Relative Frequency Distribution Curves Cumulative relative frequency distribution curves were drawn for each analyzer model and each specimen pool. The proportion of these curves that fell within the acceptable EQA performance range for each specimen pool was determined as an indicator of the accuracy of the results for each analyzer model, compared with the IRM, since the greater the proportion of the curve within the acceptable range, the closer the distribution of the results around the target IRM value. For the purpose of this study, instruments were assessed by the percentage of the curve that fell within the acceptable EQA performance range for each specimen. Reproducibility Study Usin g Individual Instruments A follow-up reproducibility study was undertaken with individual Abbott CELL-DYN 4000 (optical and impedance methods; Abbott Diagnostics, Santa Clara, CA), Siemens ADVIA 120 (Siemens Diagnostics, Tarrytown, NY), and Beckman Coulter LH 750 analyzers, using 4 specimen pools (designated A, B, C, and D) with platelet counts of approximately 5 to /L (pool A), 10 to /L (pool B), 20 to /L (pool C), and more than /L (pool D). Three specimens from each pool were tested 10 times on 3 occasions on the same day, with a 3-hour interval between each of the testing episodes. This approach provided 3 lots of 30 analyses from each of the 4 pools for overall reproducibility analysis for each analyzer. This exercise was repeated with a different set of 4 specimen pools (pools W, X, Y, and Z of approximately the same levels as pools A-D) using a Sysmex XE-2100 analyzer (impedance method) at a later date. The delay was due to a technical problem that made it impossible to complete the exercise on the Sysmex XE-2100 at the same time as the other 3 analyzers. Table 2 Make and Number of Each Analyzer Model and the Number of Specimen Pools Analyzed by Each * No. of Instruments Manufacturer/Model Minimum Maximum No. of Specimen Pools Analyzed (N = 611) Beckman Coulter (Miami, FL) Ac*T 5 Diff Ac*T Diff Gen-S HmX LH LH LH Abbott (Santa Clara, CA) CELL-DYN CELL-DYN CELL-DYN CELL-DYN Sapphire Horiba ABX (Montpellier, France) DX Pentra Pentra Pentra Siemens (Tarrytown, NY) ADVIA ADVIA Sysmex (Kobe, Japan) KX poch-100i SF XE XT-1800i XT-2000i * The number of analyzers in each group varied during the course of the project; the minimum and maximum numbers are given. 68 Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA

5 Hematopathology / Original Article Results Analyzers The results of 23 analyzer models from 5 manufacturers were compared in this study, as listed in Table 2. Data were included for an analyzer model only if 20 (18 in 2 cases) or more instruments returned results for any specimen pool. Of the analyzer groups, 22 analyzed between 23 and 29 specimen pools each. The predominant instruments were the Beckman- Coulter LH 750, Siemens ADVIA 120 and 2120, and Sysmex XE One analyzer type, the Beckman Coulter LH 780, analyzed just 4 specimen pools, since there were insufficient numbers of these analyzers registered in the FBC scheme in the early distributions of the study. With the exception of the LH 780 group, all instruments analyzed specimen pools across the entire range of specimen values distributed. Platelet Counts From each survey material pool, 1,194 to 1,577 individual results were returned across the 23 instrument groups, giving a total of 611 median platelet counts. The AMM platelet count for each specimen pool ranged from 6 to /L. The median IRM count ranged from 5 to /L and was on average /L lower than the AMM result for each specimen pool. The median IRM counts and the acceptable EQA performance range for each specimen pool are shown in Table 3. The CV increased as the platelet count decreased Table 4, ranging from an all-methods value of about 32% at platelet counts of 5 to /L to about 10% at counts of 36 to /L. This same relationship was seen for all analyzer models, with CV values 3 to 4 times greater at counts of 5 to /L (CV range, 15%-43%) compared with counts of 36 to /L (CV range, 5%-11%). Bias Comparison of the 611 median platelet counts with the IRM values showed that 70 (11.5%) of the median counts were within ± /L of the IRM value; 304 (49.8%) showed a positive bias of more than /L higher than the IRM, with 101 (16.5%) showing a statistically significant positive bias (P <.01). 136 median counts were more than /L lower than the IRM, with just 2 (0.3%) showing a statistically significant negative bias (P <.01). Comparison of the median platelet counts with the IRM by instrument type showed that no instrument returned the same value as the IRM for more than 8 (28%) of the 29 specimen pools. Four Table 3 AMM and IRM Median Platelet Counts for Specimen Pools 0603FB to 0904FB and the Acceptable EQA Performance Range * Acceptable EQA Range ( 10 9 /L) Specimen Pool No. of Results AMM ( 10 9 /L) IRM Median ( 10 9 /L) Minimum Maximum 0603FB2 1, FB1 1, FB1 1, FB1 1, FB1 1, FB1 1, FB1 1, FB2 1, FB1 1, FB1 1, FB2 1, FB1 1, FB1 1, FB1 1, FB1 1, FB2 1, FB1 1, FB2 1, FB1 1, FB2 1, FB2 1, FB1 1, FB1 1, FB2 1, FB1 1, FB1 1, FB1 1, FB1 1, FB2 1, AMM, all-methods median; EQA, external quality assessment; IRM, international reference method. * Derived from the IRM ± /L where the IRM median was /L or less and the IRM ± /L where the IRM median was /L or more. Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA 69

6 De la Salle et al / Counting in Thrombocytopenic Specimens instrument models, all from the same manufacturer, returned 63 of the 101 results showing a statistically significant positive bias. Six instrument models showed no significant bias in any of the specimen pools. In general, small bench-top or point-of-care instruments did not perform less well than large, top of the range analyzers Table 5. Accuracy Cumulative relative frequency distribution curves were constructed for each analyzer and specimen pool, and an example of these is shown in Figure 2 for the most common analyzer model from each manufacturer in the study. These curves were used to compare the results of each analyzer group with the IRM value and the acceptable EQA performance range. The cumulative relative frequency curves for each instrument model were analyzed to determine the proportion of each curve that fell within the acceptable range for each specimen pool. Figure 3 shows the mean percentage of the cumulative relative frequency distribution curves, Table 4 Mean CV for Each Instrument Group for Specimen Pools With IRM Values in Various Ranges IRM Median Platelet Count ( 10 9 /L) All methods CV (%) Mean CV (%) by instrument Beckman Coulter (Miami, FL) Ac*T 5 Diff Ac*T Diff Gen-S HmX LH LH LH Abbott (Santa Clara, CA) CELL-DYN CELL-DYN CELL-DYN CELL-DYN Sapphire Horiba ABX (Montpellier, France) DX Pentra Pentra Pentra Siemens (Tarrytown, NY) ADVIA ADVIA Sysmex (Kobe, Japan) KX poch-100i SF XE XT-1800i XT-2000i CV range (%) CV, coefficient of variation; IRM, international reference method. ie, the proportion of counts that fell within the acceptable EQA performance range for each instrument type. This value ranged from 9% to 80%. Of the predominant instrument groups, the Sysmex XE-2100 (impedance method) and the Beckman Coulter LH 750 achieved more than 70% of counts on average within the acceptable EQA performance range, and the Siemens ADVIA 120/2120 analyzers were within the 50% to 60% range seen with the majority of analyzer models. The 4 analyzer models that showed the greatest number of median counts with a statistically significant bias from the IRM (Abbott CELL-DYN 3200, 3700, 4000, and Sapphire) showed the lowest proportion of counts within the acceptable EQA performance range. Reproducibility Study Using Individual Instruments The results of the overall reproducibility study on individual analyzers Table 6 showed CV values up to 79% lower for 3 of the 4 analyzer models compared with the method group CV values (Table 4). These differences were Table 5 Statistical Significance of the Bias Observed in the Platelet Counts Compared With the International Reference Method Count by Instrument Type * No. With Significant Bias (P <.01) No. of Specimen Pools Positive Negative Beckman Coulter (Miami, FL) Ac*T 5DIFF Ac*T Diff Gen-S HmX LH LH LH Abbott (Santa Clara, CA) CELL-DYN CELL-DYN Cell-DYN CELL-DYN Sapphire Horiba ABX (Montpellier, France) DX Pentra Pentra Pentra Siemens (Tarrytown, NY) ADVIA ADVIA Sysmex (Kobe, Japan) KX poch-100i SF XE XT-1800i XT-2000i Total * In general, small bench-top analyzers did not perform less well than large, top of the range analyzers. 70 Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA

7 Hematopathology / Original Article Cumulative Frequency % 80% 70% 60% 50% 40% 30% 20% 10% 0% Platelet Count ( 10 9 /L) ABX Pentra 120 ADVIA 120 CELL-DYN 4000 Coulter LH 750 Sysmex XE Figure 2 Cumulative relative frequency distribution curves for each analyzer type and specimen pool, as shown for the most common analyzer models from each manufacturer for specimen pool 0712FB1, were used to determine the proportion of counts for each analyzer group that fell within the acceptable external quality assessment (EQA) performance range for the specimen pool. The greater the proportion of the curve that fell within this range, the closer the observed results were to the international reference method (IRM). The IRM result and acceptable EQA performance range for specimen pool 0712FB1 was 7 ± /L. Note that the lines for the Sysmex XE-2100 and Coulter LH 750 instruments largely overlay each other. Manufacturer locations are as follows: ABX Pentra 120, Horiba ABX, Montpellier, France; ADVIA 120, Siemens, Tarrytown, NY; CELL-DYN 4000, Abbott, Santa Clara, CA; Coulter LH 750, Beckman Coulter, Miami, FL; and Sysmex XE-2100, Sysmex, Kobe, Japan. Plt-I Plt-O Plt-I, Plt-O, Plt-Imm Beckman Coulter Ac*T5 Diff Beckman Coulter Ac*T Diff Beckman Coulter Gen-S Beckman Coulter HmX Beckman Coulter LH 500 Beckman Coulter LH 750 Beckman Coulter LH 780 Abbott CELL-DYN 3200 Abbott CELL-DYN 3700 Abbott CELL-DYN 4000 Abbott CELL-DYN Sapphire Horiba ABX DX 120 Horiba ABX Pentra 120 Horiba ABX Pentra 60 Horiba ABX Pentra 80 Siemens ADVIA 120 Siemens ADVIA 2120 Sysmex KX-21 Sysmex poch-100i Sysmex SF-3000 Sysmex XE-2100 Sysmex XT-1800i Sysmex XT-2000i Figure 3 The mean percentage of counts for each specimen pool that fell within the acceptable external quality assessment performance range is shown for each analyzer type, together with the counting method offered by the analyzer platform: impedance (Plt-I), optical (Plt-O), or immunological (Plt-Imm). Sysmex XE-2100 instruments offer an optical and an impedance method but only impedance counts are collected in UK NEQAS (H) (UK National External Quality Assessment Scheme for General Haematology) surveys. Manufacturer locations are as follows: Abbott, Santa Clara, CA; Beckman Coulter, Miami, FL; Horiba ABX, Montpellier, France; Siemens, Tarrytown, NY; and Sysmex, Kobe, Japan. Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA 71

8 De la Salle et al / Counting in Thrombocytopenic Specimens Table 6 Reproducibility Study Showing Mean Platelet Count and Between Replicate CV Values for Single Analyzers of the Most Commonly Represented Models * Abbott CELL-DYN 4000 Sysmex XE-2100 Beckman Coulter Siemens (Impedance) LH 750 ADVIA 120 Impedance Optical Specimen pool W A A A A Platelet count ( 10 9 /L) Between replicate CV (%) Specimen pool X B B B B Platelet count ( 10 9 /L) Between replicate CV (%) Specimen pool Y C C C C Platelet count ( 10 9 /L) Between replicate CV (%) Specimen pool Z D D D D Platelet count ( 10 9 /L) Between replicate CV (%) CV, coefficient of variation. * The reproducibility study undertaken with the Sysmex XE-2100 analyzer used specimen pools different from those used with other instruments as a result of technical problems at the XE-2100 testing site that prevented testing to the original schedule. Manufacturer locations are as follows: Abbott, Santa Clara, CA; Beckman Coulter, Miami, FL; Siemens, Tarrytown, NY; and Sysmex, Kobe, Japan. lowest in absolute terms for pool D (platelet count approximately /L). The individual analyzer CV values for these 3 analyzers approached the typical CVs of less than 3% that have been demonstrated in other evaluations, 13 indicating that the reproducibility of results is better on an individual analyzer than between analyzers of the same type at different laboratory locations. The CV values obtained from the Sysmex XE-2100 showed the same trend as the method group CV values but did not demonstrate the same reduction in CV seen with the other 3 analyzer types. The XE-2100 had been tested with a different set of specimen pools (W, X, Y, and Z) owing to an unforeseen technical problem; it is suggested that further work would be required to determine the reasons for the difference with this analyzer. Discussion In addition to the performance monitoring of individual laboratories, an important function of EQA is to improve the harmonization of laboratory results. UK NEQAS (H) is in a unique position to make a comparison of results from a substantial number of the analyzer models in most predominant use. This was a comprehensive study, covering a full range of thrombocytopenic specimen values, with a representative range and statistically robust number of analyzers. This study has shown that for thrombocytopenic EQA specimens, platelet counts using automated analyzers are overestimated in 66.3% of specimens (405/611) and significantly overestimated in 16.5% of specimens (101/611) when compared with the IRM platelet count. This observation is consistent with a multicenter study undertaken with EDTA blood samples from thrombocytopenic patients, demonstrating that the samples used in this study were valid. 7 No instrument matched the IRM for more than 28% (8/29) of specimen pools. Overestimation of thrombocytopenic platelet counts may result in the substantial undertransfusion of platelets in highrisk patients in need of platelet transfusion. The current suggested limit for administration of platelet concentrate is /L according to the British Committee for Standards in Haematology guidelines, and any consideration to reduce that limit to /L must take into account the performance limits of hematology analyzers. The CV increases as the platelet count decreases, particularly once the platelet count falls below /L (J. Parker-Williams, unpublished data, 2003). Previous EQA data 14 showed CV values of 22% to 66% at platelet counts of /L; CV results for specimens between 5 and /L in this study ranged from 15% to 43% for all analyzer types. Although variation was seen for different analyzers, the CV was inversely proportional to the platelet count for all analyzer models. The highest CV occurred at the lowest platelet counts, indicating that the poorest interanalyzer precision occurs with the most extreme thrombocytopenic specimens. The reproducibility of platelet counts in thrombocytopenic specimens analyzed on an individual analyzer is better, suggesting that within a single institution, less variability will be seen. This will allow a better demonstration of cumulative trends in a patient but does not indicate that results will be any more accurate. The reasons for inaccuracies in automated platelet counting have been well documented. Spuriously overestimated counts may arise from the failure of analyzers to discriminate red cell fragments and other nonplatelet particles from 72 Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA

9 Hematopathology / Original Article platelets 2,15 ; conversely, underestimation may occur when large or giant platelets are not discriminated from red cells and are excluded from the platelet count. 16 The study showed a range of EQA performance between analyzers, with some demonstrating apparently greater accuracy than others. When assessed in terms of the proportion of counts within the acceptable EQA performance range, the Beckman Coulter LH 750 and Sysmex XE-2100 (impedance method) analyzers had more than 70% of counts within the acceptable EQA performance range, compared to fewer than 10% for the Abbott CELL-DYN 3200 and 4000 analyzers. However, the influence of the nature of the survey material may be a factor in this observation, even though results were similar to previous studies in EDTA-anticoagulated blood samples. 4 Several analyzer platforms offer more than 1 platelet counting method, depending on (among other factors) the platelet count. Abbott CELL-DYN 4000 and Sapphire analyzers have 3 methods available: impedance, optical, and immunologic; Sysmex XE-2100 instruments offer optical and impedance counting; Abbott CELL-DYN 3200 and Siemens ADVIA 120 and 2120 analyzers offer optical platelet counting only. The preparation of UK NEQAS (H) FBC survey material requires manipulation and fixation of donor blood. This in vitro manipulation may cause platelet activation, which has been cited as a cause of intermethod variation in platelet counts with greater impact on optical than impedance or immunologic methods. 17,18 UK NEQAS (H) has for some time requested the return of impedance counts only for Sysmex XE-2100 instruments, after consultation with the scheme scientific advisors and the instrument manufacturers. This instruction was in place for the entire period of this study. Although the platelet counting method used by Abbott CELL-DYN users was not gathered for every specimen in this study, the question was asked for surveys 0711FB to 0904FB. Not all participants responded to the question; however, 92% of Abbott CELL-DYN 3200, 4000, and Sapphire users who responded indicated that they used the optical platelet counting method for specimens in the study. By contrast, 89% of Abbott CELL-DYN 3700 users reported using the impedance method. No Abbott CELL- DYN user indicated use of the immunocount. These observations could account for the very wide variability in results and the apparent inaccuracy of the results returned by these instruments. An evaluation of the methods used by these analyzers using fresh, EDTA-anticoagulated blood specimens 19 showed all 3 methods available on the Abbott CELL-DYN platforms (optical, impedance, and immunologic) correlated well with the IRM count, although the immunologic method, which showed the best correlation, is not used routinely. Although the manipulation of the survey material used in this study may have been an artifactual cause of intermethod variation, the potential for discrepancy between platelet counting methods in the presence of marked platelet activation or platelet size differences has implications for clinical decision making in conditions such as disseminated intravascular coagulation 18 and idiopathic thrombocytopenic purpura. 20,21 This study highlights the need for a single material that can be used to assess performance across all technologies and to challenge automated cell counters at the difficult levels of critical clinical decision making. It is essential that pathology service providers and clinicians who rely on these services understand the limitations of the instrumentation in use and the measurement uncertainty of automated platelet counters. We hope that this analysis of performance of platelet counts at highly challenging levels will encourage dialogue among persons responsible for the development of policy, the service providers, the bodies responsible for performance monitoring, and the instrument manufacturers. The overestimation of platelet counts shown in previous work with fresh EDTA specimens 4 and confirmed using stabilized specimens in this study could indicate problems with instrument calibration at thrombocytopenic levels. Addressing this issue may improve platelet counts in thrombocytopenia in the future. From the 1 UK National External Quality Assessment Scheme for General Haematology, Watford, England; 2 Department of Haematology, University College Hospital, London, England; 3 The Oxford Haemophilia & Thrombosis Centre, The Churchill Hospital, Oxford, England; and 4 Medical Research Council Clinical Trials Unit, London. Address reprint requests to Barbara De la Salle: UK NEQAS (H), PO Box 14, Watford, WD18 0FJ, United Kingdom. Paul Harrison is a consultant for Sysmex UK. The University College Hospital London has received an unrestricted educational grant from Sysmex Europe in the last 12 months. Acknowledgment: We thank Anne Mahon for expert technical assistance throughout the study. References 1. British Committee for Standards in Haematology. Guidelines for the use of platelet transfusions. Br J Haematol. 2003;122: Ault KA. Platelet counting: is there room for improvement? Lab Haematol. 1996;2: Norris S, Pantelidou D, Smith D, et al. Immunoplatelet counting: potential for reducing the use of platelet transfusions through more accurate platelet counting. Br J Haematol. 2003;121: Segal HC, Briggs C, Kunka S, et al. Accuracy of platelet counting haematology analysers in severe thrombocytopenia and potential impact of platelet transfusion. Br J Haematol. 2005;128: Brecher G, Schneidermann M, Cronkite EP. The reproducibility of the platelet count. Am J Clin Pathol. 1953;23: Am J Clin Pathol 2012;137: DOI: /AJCP86JMBFUCFCXA 73

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