The absolute recommendation of chamber Neubauer method for platelets counting instead of indirect methods in severe thrombocytopenic patients

unitermos abstract Accurate and precise platelet counting is crucial for recommending platelets transfusion for thrombocytopenic patients, principally when platelet counts are bellow 30,000/μl. As most laboratories still use the indirect methods for confirming low automated platelet counts, this work compared two indirect methods used in practice (Fonio and Nosanchunk et al.) with the International Committee for Standardization in Hematology recommended direct method (Brecher and Cronkite). The obtained data show that the indirect methods present low precision and accuracy, and that the direct method should always be employed in severe thrombocytopenic samples thanks to its high precision. platelet counts

show that the indirect methods present low precision and accuracy, and that the direct method should always be employed in severe thrombocytopenic samples thanks to its high precision.

Introduction
Platelet counting is an important tool used in clinical investigation to avoid bleeding risks.There is a general agreement that automation brought precision and accuracy for platelet counts in healthy individuals.Nevertheless, reliable platelet counts in thrombocytopenic patients is still controversial (6,11,15,19).
Based on the studies of Gaydos et al. (1962), most of the institutions use 20.000/µl (7) as threshold for platelet transfusion, whereas others employ 10.000/µl (3, 9, 10, 14, 16, 17).The indirect platelet counting methods were performed in triplicate.Each count was obtained as the mean of 10 oil immersion fields (1000x amplification), in a total of 30 fields.
The ICSH method was also analyzed by triplicate counts.For every counting a sample was diluted in triplicate.The minimum of 200 platelets at Neubauer chamber was taken as standard procedure.The mean, standard deviation, and coefficient of variation for the manual (direct and indirect) platelet counts were compared with the direct method.Analysis of variance statistical test was employed.

Results
The results are shown in Tables 1 and 2. They indicate that indirect manual methods with variation coefficients higher than the upper acceptable threshold (15%) have lower precision and accuracy when compared to the direct reference method (Tables 1 and 2).The variable is the coefficient of variation (CV) in patients with less than 30,000plt/µl (n = 43).

Discussion
According to Berkson (2), the specific error of hemocytometer depends on the number of cells which are counted in each area of the reticulum.Using the formula proposed by this author in similar conditions to this study, the chamber error itself would be of CV = 7.59%.Therefore, as 200 cells were always counted, these experiments disclosed CV = 10.49% for platelet counts less than 30,000/µl, and are similar to the data obtained by Dickerhof et al. (6) for platelets around 10,000/µl (CV = 14.7%).Hanseler et al. (11) results show that the correct use of the Neubauer chamber presents a satisfactory precision (CV < 15%) in counts above 4,000plt/µl.The results listed in Tables 1 and 2 indicate that the direct hemocytometer method is much more precise than the indirect methods in thrombocytopenic patients.For levels lower than 40,000plt/µl, the results obtained by Lawrence ( 16) (CV = 30.4%,p < 0.001) confirm the lower precision of indirect methods.
Based on the threshold established for prophylactic platelet transfusion (10,000 or 20,000plt/µl), the results obtained by the indirect methods show that even triplicate counts, of 10 fields each, may jeopardize the clinical decisions when prophylactic or even therapeutic platelet transfusion are in discussion.

Conclusion
The direct Neubauer chamber method for levels lower than 30,000plt/µl seems to be the most accurate and precise method for patients presenting bleeding risks.

Laboratorial
When precision is required for taking decisions regarding platelet transfusion indication, the blood smear methods are not helpful and may even impair them.