Hematopathology / QUALITY CONTROL OF THE ERYTHROCYTE SEDIMENTATION RATE

Erythrocyte Sedimentation Rate Use of Fresh Blood for Quality Control Mario Plebani, MD, and Elisa Piva, MD Key Words: Erythrocyte sedimentation rate; Westergren method; ICSH recommended method; NCCLS approved standard; ESR automated systems; ESR quality assurance

The erythrocyte sedimentation rate (ESR) remains the most widely used laboratory test for monitoring the course of infections, inflammatory diseases, and some types of cancer. Several test methods have been developed recently, and as a result the safety and reliability of ESR testing procedures have improved. The method recommended by the International Council for Standardization in Haematology and the National Committee for Clinical Laboratory Standards for ESR measurement is based on the traditional Westergren method, using EDTA-anticoagulated samples without dilution. In clinical laboratories, reliable methods for calibration and the use of appropriate control materials are required for monitoring the accuracy and precision of the routine method. We describe and evaluate a procedure for achieving the daily quality control of ESR and for establishing the limits of agreement between working and reference methods. Data from routine patient samples were used to calculate the daily cumulative mean and to monitor its reproducibility over time. Finally, to monitor analytic performance, a comparison was made between results from the measurement of ESR in specimens stored at 4°C for 24 hours and results obtained in fresh samples.

© American Society for Clinical Pathology

Estimation of the erythrocyte sedimentation rate (ESR), now more appropriately referred to as the “length of sedimentation reaction in blood,” has a long-standing history and tradition in clinical laboratories.1 ESR, the most widely used laboratory measure of disease activity in clinical medicine, is still considered useful for monitoring inflammatory diseases, rheumatoid arthritis in particular. 2 Recent reviews and searches using MEDLINE indicate that this test is widely used both for the diagnosis and follow-up of patients with these conditions.3-6 In recent decades, several new techniques for measuring ESR have been developed and introduced in clinical laboratories to address the following needs: (1) to guarantee safety to operators by using automated and closed systems; (2) to automate the measurement itself and optimize the workflow and the utilization of human resources; (3) to create a unique workstation for measuring ESR and performing other hematologic tests (eg, erythrocyte, leukocyte, and reticulocyte concentrations) in a single specimen. This should be done, as suggested by the International Council for Standardization in Haematology (ICSH) and the National Committee for Clinical Laboratory Standards (NCCLS), by using the recommended specimen, undiluted blood with tripotassium (K 3 ) EDTA, which is more reliable than the traditional sodium citrate.7,8 Recently we evaluated and validated a new measurement procedure, the so-called TEST1EC (SIRE Analytical System, Udine, Italy), which allows automated, safe, precise, and accurate measurement of the ESR in specimens collected in both sodium citrate and K3EDTA.9 In an era of quality assurance and accreditation of medical laboratories, great attention should be given to the Am J Clin Pathol 2002;117:621-626

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Abstract

Plebani and Piva / QUALITY CONTROL OF THE ERYTHROCYTE SEDIMENTATION RATE

issue of quality control and quality assurance for ESR measurement. Because the human erythrocyte sedimentation reaction is confined to fresh blood and is transient, reference or control materials of the usual type are not available. Stabilized specimens of human or nonhuman origin cannot be considered acceptable in place of fresh human blood or suitable for use in methods such as the TEST1 EC , which measure the kinetics of RBC sedimentation, not simply the final fall of RBCs after a fixed time. The aim of the present study was to describe and evaluate a procedure based on the use of fresh human whole blood for the daily quality control of ESR in clinical laboratories.

Materials and Methods

The TEST1EC Analyzer The TEST1EC, a closed automated analyzer, determines the ESR in a standard-sized, closed vacuum tube with a perforating stopper. The tubes were placed in appropriate racks, and their content was mixed by slow rotation for about 3 minutes. The sample loader housed 4 racks holding 15 tubes each at the same time. The blood sample, withdrawn directly from the collection tube by a closed aspiration needle, was delivered into a capillary tube where it was accelerated via a “stopped flow” circuit, which causes the sedimentation of erythrocytes. The sensing area was maintained at a temperature of 37°C. The system uses an infrared-ray microphotometer with a light wavelength of 650 nm. The electrical pulses, collected by a photodiode detector, are directly correlated to the concentration of erythrocytes present at each capillary level. The pulses, measured per time unit, then are used to delineate the sedimentation curve for each sample by means of a mathematical algorithm. The mean decrease in the signal per time unit, called medium signal, and the square root of the integral 622

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Comparison Between the Routine and the Westergren Method Blood samples with TEST1EC ESR values ranging from 2 to 120 mm were selected for comparison studies. The reference technique used for this was the Westergren method, which was performed according to the ICSH recommendation.7 Briefly, 250-mm-long plastic tubes with a bore of 2.55 mm were used; all specimens, chosen from the routine samples, were EDTA-anticoagulated. The test was performed within 4 hours from venipuncture, and the blood was mixed carefully before mechanical aspiration. We then determined the bias, the agreement limits, and the 95% confidence interval (CI) of the routine method (TEST1EC) in comparison with the ICSH method (Westergren). The 95% CI of the mean bias was calculated for 10 representative groups of ESR values. We also compared the 2 methods using samples as the “reference materials.” Samples were prepared following ICSH standardized recommendations (reference method), using patient blood samples with hematocrit values between 33% (0.33) and 35% (0.35). Comparison Between Fresh and Stored Samples (Day-to-Day Control) Throughout the study period, ESRs were measured in each sample within 4 hours after the blood collection and 24 hours after storage at 4°C. In 10 different representative groups of ESR values, we evaluated the bias, the agreement limits, and their 95% CIs. Cumulative Mean Values Every day for 20 days, we calculated the mean values of the average of data from 100 patients after which the mean, SD, and variation coefficient of the cumulative mean were calculated. Statistical Analysis The statistical analysis of data was carried out using MS Excel ’98 software (Microsoft, Seattle, WA) to estimate the correlation factor, the linear regression, and the correlation between TEST1EC and the Westergren method. The method comparison and the differences between ESR values from day to day were performed by using Bland-Altman analysis with the Astute package (Diagnostic Development Unit, © American Society for Clinical Pathology

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Subjects In this study, subjects were selected randomly from the entire population of both hospitalized and ambulatory patients. Blood samples were obtained and processed for analysis during routine work. Undiluted blood specimens anticoagulated with K3EDTA (Becton Dickinson System Europe, Meylan Cedex-France) were used for analysis. The samples were obtained in standardized conditions (collection in the morning after a night’s fasting) and tested within 4 hours of venipuncture, according to ICSH recommendations. In each specimen, the concentrations of erythrocytes and leukocytes, the hemoglobin concentration, and the volume fraction of erythrocytes were further measured using the Coulter GenS System (Coulter, Miami, FL).

signal were transformed to comparable Westergren values by a linear regression model. Improvements recently were made to the original technique. Specifically, the rotation speed was increased from 16 to 60 rpm to achieve better, more complete mixing of blood. The system operates at a rate of 180 samples per hour in continuous loading, providing a result about every 20 seconds, and requires 150 µL of blood for each sample. The TEST1EC plots the daily and the cumulative means of ESR values for 30 days before operation. Results are expressed in millimeters.

Hematopathology / ORIGINAL ARTICLE

120 TEST1EC Method (mm)

TEST1EC Method (mm)

160 140 120 100 80 60 40 20 0

100 80 60 40 20 0

0

20

40

60

80

100

120

140 160

0

20

Westergren Method (mm)

40

60

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Westergren Method (mm)

❚Figure 2❚ Relationship between the reference (Westergren) and the routine (TEST1EC) methods for measuring the erythrocyte sedimentation rate. Samples (n = 75) were collected in tripotassium EDTA anticoagulated with a hematocrit value between 33% (0.33) and 35% (0.35), as recommended by the International Council for Standardization in Haematology procedure to prepare a “reference material” (y = –3.38 + 1.094x; r = 0.92).

University of Leeds, Leeds, England). The statistical analysis of the CI of data was achieved using the CIA (Confidence Interval Analysis) software, as presented by Gardner et al.10

different TEST1EC analyzers with respect to the initial value: the mean differences were 2.86 (95% CI, 2.413.31) and 2.28 (95% CI, 1.90-2.65), respectively. This can be translated into mean percentage decreases of 9% and 11%, respectively, from one day to the next. By analyzing these data, we identified 10 groups of samples with different ESR values, and we obtained the bias, the agreement limits, and the 95% CI for each subgroup ❚Table 3❚. Moreover, we calculated the limits of acceptability for stored samples ❚Table 4❚, thus permitting the laboratory to easily monitor the day-to-day reliability of the assay system.

Results ❚Figure 1❚ and ❚Figure 2❚ show the comparison between

the reference method (Westergren) and TEST1EC results in unselected specimens obtained during routine clinical sampling and using reference materials (fresh human blood specimens with a hematocrit between 33% [0.33] and 35% [0.35]). A significant difference was found between the intercept value of the 2 groups (3.54; 95% CI, 1.85 to 5.23; P = .0001; and –3.38; 95% CI, –8.87 to 2.09; P = .22). ❚Table 1❚ shows results of the comparison between the data obtained by the Westergren and the TEST1EC methods in 509 specimens grouped into 10 classes on the basis of ESR values. The relative bias estimate, agreement limits, and 95% CI are reported. No significant bias was found for any of the groups with clinically relevant ESR values ❚Table 2❚. ❚Figure 3❚ shows the data obtained by calculating the mean daily value for routinely collected specimens over time and the corresponding SDs and by reporting these data on a traditional quality control chart. The individual daily means ranged around the average mean (26.57 ± 3.81) and were always within ± 2 SDs. The storage of 1,140 whole blood samples at 4°C for 24 hours caused a decrease in ESR values obtained by 2 © American Society for Clinical Pathology

❚Table 1❚ Bias, Upper and Lower Limits, and 95% Confidence Interval of the Bias for Different Values of ESR Results Obtained by the TEST1EC and the Westergren Methods (n = 509)* ESR Values (Range, mm) 2-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100

Bias

Upper and Lower Limits of the Bias

95% Confidence Interval of the Bias

1.77 –0.94 –1.43 –1.35 3.42 2.29 1.7 4.4 4.41 –1.7

–6.10 to 9.65 –15.94 to 14.05 –14.96 to 12.10 –15.63 to 12.92 –17.76 to 24.61 –26.46 to 31.05 –28.32 to 31.72 –21.32 to 30.12 –31.18 to 40 –46.63 to 43.23

0.90 to 2.64 –2.56 to 0.67 –3.24 to 0.38 –3.54 to 0.83 –0.03 to 6.88 –2.59 to 7.18 –3.19 to 6.59 –0.50 to 9.30 –4.92 to 13.75 –18.09 to 14.69

ESR, erythrocyte sedimentation rate. * For proprietary information, see the text.

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❚Figure 1❚ Relationship between the reference (Westergren, according to the International Council for Standardization in Haematology recommendation) and the routine (TEST1EC) methods for measuring the erythrocyte sedimentation rate. Samples (n = 509) were collected in tripotassium EDTA with a different range of hematocrit values (y = 3.54 + 0.92x; r = 0.93).

Plebani and Piva / QUALITY CONTROL OF THE ERYTHROCYTE SEDIMENTATION RATE

Discussion

❚Table 2❚ Acceptability Limits (2.5th and 97.5th Percentiles) of ESR Results Obtained With the TEST1EC* ESR Value (Westergren Method, mm)

Mean ESR Value ± SD (TEST1EC Method, mm)

20 30 40 50 60 70 80 90 100 110 120

20.5 ± 4.3 25.3 ± 11.4 37.8 ± 5.9 57.5 ± 13.4 68 ± 20.9 69 ± 14.4 75.12 ± 24.8 80.25 ± 23.2 100.18 ± 13.0 95.26 ± 16.1 103 ± 19.4

2.5th and 97.5th Percentiles 10-33 10-42 30-50 41-83 50-98 51-93 52-117 53-115 72-117 73-120 75-120

ESR, erythrocyte sedimentation rate. * For proprietary information, see the text.

ESR Mean Daily Value (mm)

+3 SD

40

+2 SD 35

+1 SD

30 25 20

1 SD

15

2 SD 3 SD

10 5 0 1

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37

Days

❚Figure 3❚ Levey-Jennings chart of daily cumulative mean for erythrocyte sedimentation rate (ESR) patient data. The cumulative mean ± SD value, calculated from routine data, was 26.57 ± 3.81 mm (n = 20).

❚Table 3❚ Bias, Upper and Lower Limits, and 95% Confidence Interval of the Bias for Different ESR Values When Comparing Fresh and Stored Specimens* ESR Value (Range, mm) Bias 2-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 >90

0.32 1.05 1.92 4.32 4.18 4.14 5.83 9.38 10.17 9.55

Upper and Lower Limits of the Bias –3.18 to 3.82 –5.74 to 7.85 –13.29 to 17.14 –5.85 to 14.5 –8.83 to 17.2 –12.84 to 21.13 –13.67 to 25.33 –15.28 to 34.04 –12.35 to 32.70 –6.32 to 25.43

ESR, erythrocyte sedimentation rate. * Stored for 24 hours.

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95% Confidence Interval of the Bias 0.13 to 0.50 0.60 to 1.51 0.56 to 3.3 3.23 to 5.42 2.37 to 6.0 1.16 to 7.12 2.04 to 9.61 4.59 to 14.16 4.26 to 16.08 6.81 to 12.96

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The ESR is still used widely in clinical practice as an indicator of inflammation, infection, trauma, or malignant disease, and several billion tests are performed every day in clinical laboratories throughout the world. Although Westergren developed the ESR method in the early 20th century, it has retained its status throughout the years, and the current reference method uses Westergren-type glass or plastic tubes and undiluted and nonhemolyzed blood specimens collected with the K3EDTA anticoagulant. Recently, we evaluated and validated a new automated measurement procedure, the socalled TEST1EC, that allows automated, safe, precise, and accurate measurement of the ESR in clinical practice. The advantages of using this technique in clinical laboratories include safety for operators, full automation, reduced turnaround time, and limited analytic imprecision. However, the major advantage is, in our opinion, its use of undiluted K3EDTA-anticoagulated samples. It is well known that ESR is affected mainly by RBC aggregation or rouleau formation. The collection of specimens with K3EDTA enhances blood cell stability, thus favoring rouleau formation, preserving morphologic features, and obviating unphysiologic effects on the cells, and this is of crucial importance in the ESR reaction. As demonstrated by our findings, this type of anticoagulant and the premixing procedure used by TEST1 EC improve sample stability, thus allowing the measurement of the ESR after the time of 4 hours recommended by the NCCLS and simplifying the handling and transport of specimens. As the EDTA sample is used in most hematologic measurements, ESR analysis from the same sample would lead to further improvement: a smaller sample volume would be required (particularly useful in newborns, children, and oncology patients), sample collection and handling costs would be reduced, and hematologic estimations with ESR could be analyzed with a unique workstation, thus contributing to optimizing the workflow in clinical laboratories. Last, but not least, undiluted specimens anticoagulated with EDTA reduce the risk of preanalytic mistakes due to a partially coagulated specimen or to small clots, an altered blood/sodium citrate ratio, and problems linked to the final volume, inherent mainly in techniques using special tubes for both specimen collection and ESR measurement.11,12 While it is stated in textbooks that “the ESR procedure is more likely to be performed without quality control than any other common hematology test,”13 and the NCCLS states that “although the ESR procedure cannot be calibrated, it is inherently quite stable,” in an era of quality assurance, certification, and accreditation, clinical laboratories must document the quality of ESR measurements. However, because of the nature of the human erythrocyte sedimentation reaction, reference or control materials of the usual type are not available

Hematopathology / ORIGINAL ARTICLE

❚Table 4❚ Acceptability Limits (2.5th and 97.5th Percentiles) in Quality Control Practice for Specimens Stored for 24 Hours ESR Value (Fresh Samples, mm)

2.5th and 97.5th Percentiles

9 ± 2.80 14 ± 2.48 19 ± 3.73 22 ± 4.62 28 ± 4.12 31 ± 2.77 38 ± 4.74 42 ± 3.19 43 ± 2.69 52 ± 6.13 57 ± 4.6 64 ± 16.7 64 ± 5.5 72 ± 11.3 76 ± 7.3 82 ± 10.1 93 ± 5.5 96 ± 2.5 115 ± 4.5

5-19 11-21 15-32 16-34 23-40 26-40 33-46 38-48 40-51 45-63 51-64 51-97 52-80 65-80 60-90 61-106 85-101 93-115 107-120

ESR, erythrocyte sedimentation rate.

for the ESR test, the sedimentation reaction being transient and confined to fresh blood. Although several stabilized specimens of human origin are available on the market and have been introduced in clinical laboratories, their kinetic properties, which are different from those of fresh blood erythrocytes, preclude their use for ESR determination using techniques such as the TEST1EC, which assess the sedimentation rate rather than simply measuring the final fall of erythrocytes after 60 minutes. The NCCLS document recommends the verification of the working routine method against the reference method at a frequency determined by the laboratory standard operating procedure, particularly when any changes in pipettes, personnel, or other such variables are introduced. In addition, it suggests that currently the only feasible way of providing a control material is to specify a method for the production of such material in the laboratory where it is to be used.8 We therefore evaluated a quality control procedure that uses fresh whole blood specimens. It is inexpensive and easy to perform in clinical laboratories. Because of the similar relationship observed between the reference method (Westergren) and the working technique (TEST1EC) in unselected specimens and in reference specimens (samples with a hematocrit of