Is automated platelet counting still a problem in thrombocytopenic blood?

ORIGINAL ARTICLE

Is automated platelet counting still a problem in thrombocytopenic blood?

Raimundo Antônio Gomes Oliveira; Maria Mariko Takadachi; Kimiyo Nonoyama; Orlando César de Oliveira Barretto

Hematology Laboratory, Hospital do Servidor Público, and Hematology Division, Instituto Adolfo Lutz, São Paulo, Brazil

^{Correspondence}

ABSTRACT

CONTEXT: Reliable platelet counting is crucial for indicating prophylactic platelet transfusion in thrombocytopenic patients.

OBJECTIVE: To evaluate the precision and accuracy of platelet counting for thrombocytopenic patients, using four different automated counters in comparison with the Brecher & Cronkite reference method recommended by the International Committee for Standardization in Hematology (ICSH).

TYPE OF STUDY: Automated platelet counting assessment in thrombocytopenic patients.

SETTING: Hematology Laboratory, Hospital do Servidor Público Estadual de São Paulo, and the Hematology Division of Instituto Adolfo Lutz, São Paulo, SP, Brazil.

MAIN MEASUREMENTS: Brecher & Cronkite reference method and four different automated platelet counters.

PARTICIPANTS: 43 thrombocytopenic patients with platelet counts of less than 30,000/µl

RESULTS: The ADVIA-120 (Bayer), Coulter STKS, H1 System (Technicom-Bayer) and Coulter T-890 automatic instruments presented great precision and accuracy in relation to laboratory thrombocytopenic samples obtained by diluting blood from normal donors. However, when thrombocytopenic patients were investigated, all the counters except ADVIA (which is based on volume and refraction index) showed low accuracy when compared to the Brecher & Cronkite reference method (ICSH). The ADVIA counter showed high correlation (r = 0.947). However, all counters showed flags in thrombocytopenic samples.

CONCLUSION: The Brecher & Cronkite reference method should always be indicated in thrombocytopenic patients for platelet counts below 30,000 plt /µl obtained in one dimensional counters.

Keywords: Platelet counting. Automatic counters. Transfusion. Thrombocytopenic patients.

RESUMO

CONTEXTO: A contagem de plaquetas confiável é de grande importância para avaliar a necessidade da transfusão profilática em pacientes plaquetopênicos.

OBJETIVO: Avaliar, em amostras plaquetopênicas, a precisão e exatidão da contagem de plaquetas em quatro contadores automáticos em comparação com método de referência de Brecher & Cronkite recomendado pelo Comitê Internacional de Estandardização em Hematologia.

TIPO DE ESTUDO: Avaliação da contagem automatizada de plaquetas em pacientes trombocitopênicos.

LOCAL: Hospital do Servidor Público Estadual (São Paulo - SP). Instituto Adolfo Lutz.

PARTICIPANTES: 43 pacientes trombocitopênicos com contagens de plaquetas inferiores a 30.000/µL.

VARIAVEIS ESTUDADAS: Método de Brecher & Cronkite como padrão de referência e quatro contadores automáticos.

RESULTADOS: Os contadores automáticos ADVIA-120 (Bayer), Coulter STKS, H1 System (Technicom-Bayer) e Coulter T-890 demonstraram boa precisão e exatidão em amostras plaquetopênicas obtidas em laboratório de hematologia a partir de amostras normais. Apenas o ADVIA-120, que utiliza dois princípios de contagem (volume e índice de refração), demonstrou boa correlação com o método de referência recomendado pelo Comitê Internacional de Estandardização em Hematologia (ICSH, 1984/1988) para as amostras dos pacientes trombocitopênicos (r = 0,947). Entretanto, todos os aparelhos pediram nova contagem de plaquetas (flags) para as amostras trombocitopênicas.

CONCLUSÃO: A utilização do método de referência de Brecher Cronkite deve ser uma conduta rotineira e indispensável em todos pacientes trombocitopênicos com contagens abaixo de 30,000 plaq /µl obtidas em contadores que utilizam-se apenas do volume como princípio de contagem.

Palavras-chave: Contagem. Plaquetas. Automação. Transfusão. Plaquetopenia.

INTRODUCTION

It is widely accepted that automation has afforded high precision and accuracy for platelet counting in normal individuals.^1-5 However, automated counting is still very controversial in the case of samples from thrombocytopenic or other patients in which other small particles could generate electrical or optical signals that are similar to platelets, such as debris and red cell fragments.^4,6-11 Most counters nowadays employ the principle of electrical impedance or optical signals for counting the platelets in peripheral blood, using the particle volume for counting them.¹² On the other hand, the presence of large platelets beyond the upper threshold may lead to underestimation of the platelet counts.^13-15 The use of multiple light scatter parameters rather than impedance alone has improved the ability to discriminate platelets.⁶

Prophylactic platelet transfusions have been successfully employed in hematological patients under chemotherapy when the platelet levels drop to lower than 20,000/µL. Nevertheless, in an attempt to lower the risks in platelet transfusions in bone marrow transplants, as well as reducing the cost, there is a great tendency to use 10,000/µL,^16-21 or even 5,000/µL as advocated by Gmür et al.,²² as the threshold for prophylactic or therapeutic platelet transfusions. Thus, higher precision and accuracy in platelet counting is required.⁶ In fact, the Consensus Conference on Platelet Transfusion Therapy of the National Institute of Health,²³ reported that there was a lack of reproducibility and a variability in platelet counts at low levels. This fact is a great problem in recommending a standard threshold for platelet transfusion in thrombocytopenic patients.

Manual platelet counting in the Neubauer chamber, by means of a phase-contrast microscope,^24,25 has been recommended as the reference method for assessing the platelet number by the International Committee for Standardization in Hematology (ICSH -1984).²⁶ Quite recently, the International Council for Standardization in Hematology and the International Society for Laboratory Hematology²⁷ have recommended the use of labeled platelets in a fluorescence-flow cytometer, together with a semiautomated, single-channel aperture-impedance counter as the reference method for platelet counting, but few centers are able to afford this.

This investigation was thus carried out with the objective of studying the accuracy and precision of automated instruments and comparing these with the recommended manual method (ICSH 1984) for low platelet counts. Different instruments based on different technical characteristics, such as refraction index and platelet size, were used.

METHODS

Two different materials were employed:

1. Blood samples from four normal individuals were diluted with isotonic solution in order to make target low- platelet suspensions (30,000; 20,000; 10,000 and 5,000 platelets per µL), in accordance with Lawrence et al.¹⁶ Every target sample was counted 9 times (3 dilutions in triplicate).

2. Blood samples from 43 thrombocytopenic patients presenting less than 30,000 platelets per µL, 33 of them presenting leukemia and 10 with several diseases such as idiopathic thrombocytopenic purpura, myelodysplastic syndrome and pancytopenia, from the Hematology Laboratory of Hospital do Servidor Público Estadual de São Paulo (HSPE), São Paulo, were also studied.

Four automated hematology analyzers were studied: ADVIA^TM 120 Hematology System (Bayer, Tarrytown, New York, USA),^10,15 H1 Technicon System (Technicon Instrument Corporation/ Tarrytown, New York),^28-30 Coulter STKS (Coulter, USA),³¹ and Coulter T-890 (Coulter, USA),³² as well as the reference method recommended by the International Committee for Standardization in Hematology (1984): the Brecher & Cronkite method.^24,25

The precision and accuracy of all blood cell counters were assessed daily in comparison with standards provided by the manufacturers. All blood samples from thrombocytopenic patients were processed within 1 hour after blood draw for automated methods, and up to 3 hours for the manual counts, at room temperature. All counts were performed in triplicate. For the reference method (ICSH 1984), a minimum of 200 cells was counted in the Neubauer chamber.

Every instrument was compared with the reference method by a linear correlation test. The Student "t" test was employed for comparisons between all instrument data and for data obtained using the reference method as well, with a significance level of 5%.

RESULTS

Laboratory thrombocytopenic samples from normal donors

The ADVIA, STKS and H1 counters showed variable differences between the obtained mean values and the target values, ranging from 1.4% to 5.4% for the 5,000 target group, from 2.3% to 0.3% for the 10,000 target group, from 4.5% to 1.2% for the 20,000 target group, and from 1.6% to 0.1% for the 30,000 platelets per µL target group. The T-890 counter, however, showed mean values from 11 to 16.5% lower than the target values, for the 10,000 to 30,000 platelets per µL target groups. For the 5,000-target group, the results were 19.05% lower than the target value (Tables 1 and 2).

The coefficients of variations shown by the groups, for all the counters, were lower than 9.5% for the 5,000-target group, lower than 7.8% for the 10,000-target group, lower than 5.8% for the 20,000-target group and lower than 4.6% for the 30,000 platelets per ml target group (Table 1).

The dilutions of the platelet suspensions were checked by the linear correlation test and showed values of r > 0.99 for all counters. The "y" axis intercepts, which represent the number of platelets per µl, were close to zero for all counters. The slope was close to 1, except for T-890 (slope = 0.88).

Samples from thrombocytopenic patients

The mean value of the platelet counts performed in triplicate by the Brecher & Cronkite reference method was 18,040 platelets per µl. As can be observed in Table 3, ADVIA and STKS showed little deviation, but H1 and T-890 exhibited greater deviation.

The linear correlation test between every counter and the reference method for thrombocytopenic patients are shown in Figures 1, 2, 3 and 4. The ADVIA counter exhibited the highest correlation (r = 0.947).

DISCUSSION

With regard to the laboratory targets for platelet counting, the different counters used indicated great accuracy and precision. However, the Coulter T-890 exhibited 11 to 19.5% of the data lower than the desired target values (Tables 1 and 2), similar to what was obtained by Lawrence¹⁶ using a counter that also employed the impedance principle.

When dealing with the thrombocytopenic patient samples, comparative determinations between the automated methods and the reference method suggest that the two-dimensional counting system employed by the ADVIA counter demonstrates higher accuracy in differentiating between platelet and non platelet particles, in comparison with the one-dimensional system used by H1, STKS and T- 890 (Figures 1, 2, 3 and 4). The data herein presented are similar to those obtained by Kunicka et al.¹⁰Dickerhoff and von Ruecker⁴ also showed lower correlation between the H1 counter and flow cytometry (FC) with monoclonal anti-platelet antibodies, when using thrombocytopenic samples of lower than 50,000 platelets per µL. Only FC and the Brecher & Cronkite method showed significant correlation. Interestingly, these are the two reference methods recommended by the ICSH.

Hanseler et al.¹¹ using the H1 counter, claimed that for counts of less than 30,000 platelets per µL, the automated counting should be replaced by the manual chamber procedure. Our data obtained with thrombocytopenic patients also suggest the same for the one-dimensional STKS, H1 and T-890 counters.

The data from Ault⁶ and Kunicka et al.,¹⁰ as well as the data obtained in this investigation for H1, STKS and T-890, suggest that the one-dimensional platelet counters present a tendency to overestimate the platelet counts when other particles with the same platelet size are contaminating the sample. However, all counters showed flags in thrombocytopenic samples.

CONCLUSION

Our results suggest that for platelet counts below 30,000 platelets per µl obtained in one-dimensional counters, the counting method should be replaced by the reference manual procedure, i.e. the Brecher & Cronkite method.

Sources of funding: None

Conflict of interest: None

Date of first submission: March 23, 2002

Last received: August 27, 2002

Accepted: September 30, 2002

PUBLISHING INFORMATION

Raimundo Antônio Gomes Oliveira, MsPhar. Assistant Professor in Clinical Hematology and Biochemist-Pharmacist, Pharmacy Department and University Hospital, Universidade Federal do Maranhão, Maranhão, Brazil.

Maria Mariko Takadachi. Biochemist-Pharmacist, Hematology Laboratory, Hospital do Servidor Público Estadual de São Paulo, São Paulo, Brazil.

Kimiyo Nonoyama, MsPhar. Hematology Division, Instituto Adolfo Lutz, São Paulo, Brazil.

Orlando César de Oliveira Barretto, MD, PhD. Associate Professor, Faculdade de Medicina, Universidade de São Paulo, Laboratório de Investigação Médica-23, São Paulo, Brazil.

COPYRIGHT©2003, Associação Paulista de Medicina

• 1- Ross DW, Bentley SA. Evaluation of an automated hematology system (Technicon H-1). Arch Pathol Lab Med 1986;110(9):803-8.
• 2- Cornbleet PJ, Myrick D, Judkins S, Levy R. Evaluation of the CELL-DYN 3000 differential. Am J Clin Pathol 1992;98(6):603-14.
• 3- Arkin CF. Quality control and standardization in the hematology laboratory. In: Bick RL, editor. Hematology: clinical and laboratory practice. St Louis: Mosby; 1993.p.17-37.
• 4- Dickerhoff R, Von Ruecker A. Enumeration of platelets by multiparameter flow cytometry using platelet-specific antibodies and fluorescent reference particles. Clin Lab Haematol 1995;17(2):163-72.
• 5- Jones RG, Faust AM, Matthews RA. Quality team approach in evaluating three automated hematology analyzers with five-part differential capability. Am J Clin Pathol 1995;103(2):159-66.
• 6- Ault KA. Platelet counting: Is there room for improvement? Lab Hematol 1996;2:139-43.
• 7- Mayer K, Chin B, Magnes J, Thaler HT, Lotspeich C, Baisley A. Automated platelet counters: a comparative evaluation of latest instrumentation. Am J Clin Pathol 1980;74(2):135-50.
• 8- Ross DW, Ayscue L, Gulley M. Automated platelet counts: accuracy, precision, and range. Am J Clin Pathol 1980;74(2):151-6.
• 9- Kjeldsberg CR. Principles of hematologic examination. In: Lee GR, Bithell TC, Foerster J, et al., editors. Wintrobe's Clinical Hematology. 9^th ed. Philadelphia: Lea & Febiger; 1993.p.11-12.
• 10- Kunicka JE, Fischer G, Murphy J, Zelmanovic D. Improved platelet counting using two-dimensional laser light scatter. Am J Clin Pathol 2000;114:283-9.
• 11- Hanseler E, Fehr J, Keller H. Estimation of the lower limits of manual and automated platelet counting. Am J Clin Pathol 1996;105(6):782-7.
• 12- Klee GG. Performance goals for internal quality control of multichannel haematology analysers. Clin Lab Haematol 1990;12(Suppl 1):65-74.
• 13- Rowan RM. Platelet counting and the assessment of platelet function. In: Keopke JA, editor. Practical Laboratory Hematology. New York: Churchill Livingstone; 1991.p.157-70.
• 14- Bode AP. The use of flow cytometry in the study of blood platelets. In: Riley RS, Mahin EJ, Ross W. Clinical applications of flow cytometry. New York: Igaku-Shoin; 1993.p.613-33.
• 15- Stanworth SJ, Denton K, Monteath J, Patton WN. Automated counting of platelets on the Bayer ADVIA 120 analyser. Clin Lab Haematol 1999;21(2):113-7.
• 16- Lawrence JB, Yomtovian RA, Dillman C, et al. Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding. Am J Hematol 1995;48(4):244-50.
• 17- Gil-Fernández JJ, Alegre A, Fernández-Villalta MJ, et al. Clinical results of a stringent policy on prophylactic platelet transfusion: non-randomized comparative analysis in 190 bone marrow transplant patients from a single institution. Bone Marrow Transplant 1996;18(5):931-5.
• 18- Sherrill JS, Corash L, Shiffer C, et al. Newer approaches to platelet transfusion therapy. In: Transfusion Medicine Education program of The American Society of Hematology, Orlando, 1996. Proceedings. Philadelphia: ASH; 1996.p.119-31.
• 19- Beutler E. Platelet transfusions: the 20,000/microL trigger. Blood 1993;81(6):1411-3.
• 20- Finazzi G. Prophylactic platelet transfusion in acute leukemia: which threshold should be used. Haematologica 1998;83(11):961-2.
• 21- Navarro JT, Hernández JA, Ribera JM, et al. Prophylactic platelet transfusion threshold during therapy for adult acute myeloid leukemia: 10,000/microL versus 20,000/microL. Haematologica 1998;83(11):998-1000.
• 22- Gmür J, Burger J, Schanz U, Fehr J, Schaffner A. Safety of stringent prophylactic platelet transfusion policy for patients with acute leukemia. Lancet 1991;338(8777):1223-6.
• 23- Consensus Development Conference. Platelet transfusion therapy. JAMA 1987;257(13):1777-80.
• 24- Brecher G, Cronkite EP. Morphology and enumeration of human blood platelets. J Appl Physiol 1950;3:365-77.
• 25- Brecher G, Schneiderman MA, Cronkite EP. The reproducibility and consistency of the platelet count. Am J Clin Pathol 1953;23:15-26.
• 26- England JM, Rowan RM, van Assendelft OW, et al. Protocol for evaluation of automated blood cell counters. International Committee for Standardization in Haematology (ICSH). Clin Lab Haematol 1984;6(1):69-84.
• 27- Platelet counting by the RBC/platelet ratio method: A reference method. Am J Clin Pathol 2001;115(3):460-4.
• 28- Tycko DH, Metz MH, Epstein EA, Grinbaum A. Flow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentration. J Applied Optics 1985;24:1355-65.
• 29- Technicon H*1 System Information Bulletin Technical Publication N⁰ TN8-8588-22. Tarrytown, NY: Technicon Instrument Corp; December, 1988:3-4.
• ³⁰
  30- Technicon H*1 System: Operator's Guide. International Division ed. Tarrytown, NY: Technicon Instrument Corp; 1985: 2/56-57.
• 31- Coulter STKS Analyser with reticulocyte analysis: product reference manual n. 4237182B. Miami: Coulter; 1995.
• ³²
  - Coulter T Series with differential capabilities: product reference manual n. 4235880C. Miami: Coulter; 1993.

Publication Dates

• Publication in this collection
  04 July 2003
• Date of issue
  2003

History

• Reviewed
  27 Aug 2002
• Received
  23 Mar 2002
• Accepted
  30 Sept 2002