Accred Qual Assur (2007) 12:643–645 DOI 10.1007/s00769-007-0323-0
PRACTITIONER’S REPORT
Between-examiner reproducibility in manual differential leukocyte counting Xavier Fuentes-Arderiu Æ Mariona Garcı´a-Panyella Æ Dolors Dot-Bach
Received: 28 June 2007 / Accepted: 8 October 2007 / Published online: 24 October 2007 Springer-Verlag 2007
Abstract In manual–visual differential leukocyte counts, the counting itself may be different depending on the examiner. We estimated the between-examiner (B-E) reproducibility in differential leukocyte counting. During 2 months, daily, after performing manual–visual differential leukocyte counts, two slides with blood films were retained. Next day these two blood films were re-examined by different examiners. For each type of leukocyte the B-E reproducibility was estimated from the 58 pairs of data obtained by four technicians. The B-E coefficient of variation for each type of leukocyte was: basophilocytes, 263.2%; eosinophilocytes, 68.8%; lymphocytes, 32.5%; metamyelocytes, 69.6%; monocytes, 55.0%; myelocytes, 132.5%; and neutrophilocytes, 6.6%. For each type of leukocyte the coefficient of variation is the sought estimate of the average reproducibility due to the ‘‘human factor’’ as a whole, without regard to any examiner in particular. Knowledge of this component of day-to-day imprecision, and its follow-up, may be used to decide corrective actions (training of the examiner, etc.) and to set critical differences to interpret the significance of changes in serial results. Keywords Imprecision Reproducibility Laboratory practice Leukocytes Quality control Statistics
Introduction Automatic instruments for differential leukocyte counting give results with greater precision and trueness that those obtained by manual–visual differential leukocyte counting, especially for the routine 100-cell eyecount leukocyte differential [1, 2]. Notwithstanding, the International Consensus Group for Hematology Review of the International Society for Laboratory Hematology has published a set of criteria for action following automated cell blood and leukocyte differential counts [3], including 41 consensus rules from which 29 have slide review as the final step. This is one of the reasons why in clinical laboratories worldwide, despite all technological improvements, thousands of differential leukocyte counts are done by manual– visual microscopic examination of a blood film. Differential leukocyte counting carried out by manual– visual microscopic examination of a peripheral blood film has an imprecision that may seriously affect clinical decision-making [4]. The day-to-day imprecision of this measurement procedure depends, among other factors, on: • • • • • •
X. Fuentes-Arderiu (&) M. Garcı´a-Panyella D. Dot-Bach Laboratori Clı´nic, IDIBELL––Hospital Universitari de Bellvitge, Feixa Llarga s/n, 08907 L’Hospitalet de Llobregat, Catalonia, Spain e-mail:
[email protected]
•
blood sample collection, mixing (homogeneity) of the blood sample, quality of the blood film, quality of the stain, random distribution of leukocytes (Poisson or Gaussian distribution), area of slide holding the blood film examined for differential counting, and errors of cell identification.
Among all these sources of imprecision, the last two are highly dependent on the human eye and mind. Logically,
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such ‘‘human factors’’ will have more impact when a blood film may be indistinctly examined by one of several examiners. Thus today the blood film of a patient is examined by examiner A but the next blood film of the same patient may be examined by examiner B. This leads to the concept of ‘‘between-examiner reproducibility’’. For each percentage of different types of leukocyte, this reproducibility may be quantitatively expressed by the standard deviation (or coefficient of variation) estimated from replicate examinations of the same blood film by different examiners. In this article we present a real example of estimation of between-examiner (B-E) reproducibility in differential leukocyte counting under routine conditions.
Accred Qual Assur (2007) 12:643–645 Table 1 Coefficients of variation (CV), and mean percentages of cells ð xÞ; corresponding to B-E imprecision in differential leukocyte counting Quantity
xð%Þ
CV (%)
Lkcs (B)—neutrophilocytes; num. fr.
78.15
6.6
Lkcs (B)––lymphocytes; num. fr.
10.78
32.5
4.91 1.88
55.0 68.8
Lkcs (B)––basophilocytes; num. fr.
0.19
263.2
Lkcs (B)––myelocytes; num. fr.
0.83
132.5
Lkcs (B)––metamyelocytes; num. fr.
3.16
69.6
Lkcs (B)––monocytes; num. fr. Lkcs (B)––eosinophilocytes; num. fr.
Quantities corresponding to the differential leukocyte counting are described in accordance with the standards EN 1614 [6] and ISO 15189 [7] Lkcs, leukocytes; B, blood; num. fr., number fraction
Materials and methods For 2 months, daily, after performing routine manual– visual differential leukocyte counts, two slides with stained blood films were retained—one slide from a blood sample with a concentration of leukocytes between 5 · 109 L–1 and 10 · 109 L–1, and the other slide from a blood sample with a concentration of leukocytes between 10 · 109 L–1 and 20 · 109 L–1. The counting results and the name of the technicians involved (the examiners) were recorded daily. Next day these two blood films were re-examined by different examiners who did not know the previous results, and the new counting results and the name of the new technicians involved were also recorded. A total of 58 pairs of results obtained by four technicians of average training and skill were taken into account in the study. Assuming that distributions of differences between examiners are Gaussian, the standard deviation (s) corresponding to the B-E reproducibility was estimated for each quantity from the 58 pairs of data by use of the equation [5]: rffiffiffiffiffiffiffiffiffiffi P 2ffi di s¼ 2n where di is the between-examiners difference and n is the number of blood films examined.
Results For each quantity measured in the differential leukocyte count, the B-E reproducibility estimated from four average trained and skill technicians is shown in Table 1. Coefficients of variation in Table 1 were calculated from the standard deviation and the mean percentage of each type of leukocyte.
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Discussion Without even considering how close to or far from the true number fraction (percentage) the result for each type of leukocyte is, the counting itself may be different depending on the examiner and can be influenced by the subjective nature of this examination procedure. If the same person were to examine the blood film a second time, the percentage of each type of leukocyte would not invariably be the same. This ‘‘human factor’’ includes, but is not limited to, fatigue, psychological pressures, illness, monotony, distractions, lack of motivation, and––generally the most important––lack of appropriate and continuous training. For each quantity, the coefficient of variation corresponding to the B-E reproducibility is the sought estimate of the average reproducibility due to the ‘‘human factor’’ as a whole, without regard to any examiner in particular. The ‘‘human factor’’ is one of the most important weaknesses of the performance quality of routine manual– visual differential leukocyte counts. Errors in leukocyte type misclassification, which occur randomly, despite being systematic in nature, contribute to the day-to-day imprecision of this measurement procedure. However, quantification of imprecision caused by the ‘‘human factor’’, that is, quantification of B-E reproducibility estimated with the group of people doing this job daily, is seldom done. As the B-E reproducibility is a relevant component of the day-to-day imprecision of differential leukocyte counting, every clinical laboratory should know the magnitude of this imprecision. Knowledge of this component of day-to-day imprecision, and its follow-up, may be used to decide strategies for continuous improvement, including corrective action (training of the examiner, motivation, etc.), to diminish the different visual perception (subjectivity) and other sources of variation among examiners. On the other hand,
Accred Qual Assur (2007) 12:643–645
knowledge of the B-E reproducibility, expressed as a variance, jointly with the variance due to random distribution of leukocytes (Poisson distribution) and the variance due to the intra-individual biological variation of the number fraction of different leukocyte types [8], allows setting of critical differences for interpreting the significance of changes in serial results [9] and estimating the uncertainty of measurement.
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