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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 14
| Issue : 2 | Page : 137-145 |
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A comparative study on determination of erythrocyte sedimentation rate by two automated erythrocyte sedimentation rate analysis techniques-ves matic cube 30 and mixrate X20 in comparison to modified manual westergren method
Seena Susan Itty, PV Priya, CK Anju, S Sankar
Department of Pathology, Government Medical College, Kottayam, Kerala, India
| Date of Submission | 18-Mar-2023 |
| Date of Decision | 29-May-2023 |
| Date of Acceptance | 03-Jun-2023 |
| Date of Web Publication | 27-Jul-2023 |
Correspondence Address:
Dr. P V Priya
Associate Professor, Department of Pathology, Government Medical College, Kottayam, Kerala
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/joah.joah_11_23
BACKGROUND: Erythrocyte sedimentation rate (ESR) is a widely used simple and cost-efficient lab test for the diagnosis and follow-up of many diseases. Even though the Westergren method is considered as gold standard, it has many drawbacks such as long test time duration, infection risk to technician, and need of citrated blood sample and to overcome these limitations, automated ESR analysis techniques have been introduced. This study aimed to compare and assess the agreement as well as to analyze the correlation between the ESR values obtained by two automated ESR analysis techniques– Ves Matic Cube 30 and Mixrate-X20 against the gold standard–Modified Manual Westergren method.
MATERIALS AND METHODS: Four milliliter blood samples were collected from 1174 patients who came to the Central laboratory with complete blood count and ESR test request forms, after taking informed consent. Each of these samples was subjected to ESR test in both Mixrate X20 and Ves Matic Cube 30, followed by ESR analysis through the modified manual Westergren method. All values obtained were recorded and analyzed using SPSS software. Mean, standard deviation, Bland–Altman agreement analysis, Linear regression, and Pearson's correlation coefficient analysis was done.
RESULTS: Of 1174 study samples, 588 were male and 586 were female and major proportion of the study sample was in the age group of 50–59 years. Bland–Altman agreement analysis of manual ESR Vs Ves Matic cube 30, showed a mean difference of −2.44 ± 13.01 with 95% limit of agreement (LOA) between −27.93 and 23.05 whereas it was 0.05 ± 1.68 with 95% LOA between −3.24 and 3.34 for manual ESR vs Mixrate X20. Pearson's correlation coefficient for manual ESR Vs Ves Matic Cube 30 was r = 0.891 whereas r = 0.998 for manual ESR Vs Mixrate X20, indicating a strong positive correlation between ESR values obtained through manual method and both automated ESR analyzers.
CONCLUSION: ESR results from both automated analyzers are agreeable to the reference method with no proportional bias statistically. However, the results from Mixrate X20 ESR analyzer correlate very well with the manual ESR, with close readings that do not affect the clinical interpretation and so can be used in clinical laboratories to optimize workflow and use of human resources.
Keywords: Erythrocyte sedimentation rate, Mixrate X20, Ves Matic Cube 30
How to cite this article:
Itty SS, Priya P V, Anju C K, Sankar S. A comparative study on determination of erythrocyte sedimentation rate by two automated erythrocyte sedimentation rate analysis techniques-ves matic cube 30 and mixrate X20 in comparison to modified manual westergren method. J Appl Hematol 2023;14:137-45 |
How to cite this URL:
Itty SS, Priya P V, Anju C K, Sankar S. A comparative study on determination of erythrocyte sedimentation rate by two automated erythrocyte sedimentation rate analysis techniques-ves matic cube 30 and mixrate X20 in comparison to modified manual westergren method. J Appl Hematol [serial online] 2023 [cited 2023 Sep 27];14:137-45. Available from: https://www.jahjournal.org/text.asp?2023/14/2/137/382408 |
| Introduction | |  |
Erythrocyte sedimentation rate (ESR) is a widely used simple,[1] cost-efficient laboratory test,[2] which is used for diagnosis and follow-up of many diseases, particularly infections, inflammatory diseases, autoimmune diseases, and some malignancies. Although it is not specific,[3] it is particularly a sensitive indicator of silent and chronic inflammation that is the underlying process in many diseases – a sickness indicator.[4] Thus despite the availability of alternative inflammatory parameters such as C-reactive protein level and white blood cell count, it is still a frequently requested parameter by clinicians and probably the most widely measured index of acute phase response.[5]
ESR is a measurement of the rate at which the erythrocytes settle out from the plasma and is measured as the number of millimeters of clear plasma present at the top of the column after one h (mm/h).[6] To analyze ESR, the International Council for Standardisation in Haematology (ICSH) recommends the Westergren method as the reference standard method.[7] However, it has certain drawbacks such as long test time duration, infection risk to the technician,[8] vulnerability to environmental contamination, need of citrated plasma,[2] and relatively large amount of blood required.[9] To overcome these limitations, several new automated ESR analysis techniques have been developed in recent years. The advantages of the new techniques are to guarantee safety to operators using automated and closed systems,[5] which automate the measurement itself and optimize the workflow and use of human resources. The new techniques also create a unique workstation for measuring ESR and performing other hematological tests in a single specimen.[10]
One of the automated systems for ESR determination is Ves-Matic Cube 30 which is programmed to determine ESR on whole blood samples anticoagulated with Ethylene Diamine Tetra Acetic acid (EDTA) by means of a specially designed optical system.[11] Sample analysis is executed completely automatically (mixing of the samples and reading of the sedimentation) and the results, obtained in only 15 min of sedimentation, correlate with those obtained with the Westergren Citrate standard method. In this method, samples are placed at 18° slanting position, based on the principle that slanting increases the sedimentation rate of RBCs, and thus, the results are obtained faster. Another one is Mixrate-X20, which uses special 8 mm × 120 mm tubes containing sodium citrate and follows the sedimentation of each samples independently.[12] It is based on the principle that increasing the diameter of ESR measurement tubes increases the rate of sedimentation and the reading is measured using infrared beam. Thus, the present study is undertaken to compare and evaluate the consistency of ESR assay results of these two automated ESR analysis techniques– Ves Matic Cube 30 and Mixrate-X20 in comparison to the gold standard-manual Westergren method.
| Materials and Methods | | |
A hospital-based analytical cross-sectional study was conducted among patients attending the central laboratory with complete blood count (CBC) and ESR test requests irrespective of age and sex, between November 2019 and December 2020. The estimated sample size for Bland–Altman agreement analysis[13] was 1174 based on the study conducted by Mahato and Karkuzhali which compared the ESR values obtained by Vesmatic Cube 30 and Modified Westergren method.
Patients with a requisition for CBC and ESR tests to be done were included and selected continuously for the study. After taking informed consent, whole blood samples were collected under aseptic precautions and under standardized conditions in 4 ml EDTA vacutainers. All blood samples collected were assayed within 4 h of venipuncture, and the blood was mixed carefully before mechanical aspiration, according to the ICSH recommendation. Clotted blood samples were excluded.
In Mixrate X20, specially designed 8 mm × 120 mm ESR measurement glass tubes which contain sodium citrate at 3.8% and which draw 1.2 ml blood were used. From the 4 ml blood received, blood was transferred into the ESR glass tube up to the graduation mark using plastic dropper and was mixed by repeated inversions. After the samples were inserted into the analyzer, samples were automatically mixed for 5 min, following which sedimentation took place. The vacuum tubes were held in a vertical position at ambient temperature. The instrument takes two measurements using infrared light, an initial reading at zero time to determine the fill level in the tube and a final reading after 30 min that reflects the erythrocyte sedimentation levels. A mathematical formula is applied to the observed result to determine the ESR in millimeters per hour, correlated to the manual Westergren method. After 30 min, results are available as a printout as well as displayed on the screen [Figure 1]. | Figure 1: ESR analysis in Mixrate X20. ESR = Erythrocyte sedimentation rate
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At the same time, the EDTA vacutainers with remaining blood were inserted into the sample holding rotor of the Ves Matic Cube 30 automated ESR analyzer. Within the analytical module, the sample was rotated 15 times at a rotation of 120°. After the sample was mixed, it was allowed to stand at 18° slant from vertical and the level of blood at time 0 of sedimentation is read for each sample using an optical system. After 15 min of sedimentation, the final level reached by the settling red cells was read and recorded. The initial reading measured the initial height of the blood sample using a light-emitting diode-based optical system, and the final reading measured the point between the erythrocyte layer and the plasma layer. The decrease in the height of the erythrocyte column was determined and used to mathematically derive the ESR in millimeters per hour. At the end of 33 min, the results were obtained as a printout as well as displayed on the screen [Figure 2]. | Figure 2: ESR analysis in Ves Matic Cube 30. ESR = Erythrocyte sedimentation rate
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After the ESR analysis by Ves Matic Cube 30 is completed, the vacutainer tubes were taken out and mixed well by repeated inversions. In the manual Modified Westergren method, 2 ml of this well-mixed EDTA blood sample was diluted with 0.5 ml of 0.9% NaCl which was then aspirated into 200 mm plastic Westergren pipettes up to 0 mark and put onto a dedicated holder that assured correct vertical position during analysis. A timer was set and the tube was left undisturbed for 1 h and the result was read exactly after 60 min and recorded [Figure 3]. | Figure 3: Manual Modified Westergren method of ESR analysis. ESR = Erythrocyte sedimentation rate
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The ESR values obtained by the two automated ESR analysis techniques as well as by the manual Westergren method were recorded and analysis done using IBM SPSS statistics software. The mean and standard deviation of results derived from all three methods were determined.
Linear regression and Pearson's correlation analysis were utilized for correlation analysis. An “r” value above 0.7 indicates a strong positive correlation. Bland–Altman analysis was used to determine bias and limits of agreement.
| Results | | |
Among the 1174 samples analyzed, 37 samples showed a result of ERROR on Ves Matic Cube 30 and so they were not included for statistical analysis of ESR for manual modified Westergren method versus Ves Matic Cube 30 (Net sample size-1137). Furthermore, for Mixrate X20, 2 blood samples showed a result of >140 and so these two samples were not included in the statistical analysis of ESR for manual versus Mixrate X20 (Net sample size-1172).
The age of patients included in this study ranged from 7 to 88 years old. The maximum number of patients belonged to the age group of 50–59 years (26.66%). There were only 4 patients with age <10 years and 10 patients with age more than 80 years [Table 1].
Among the 1174 study samples, 588 were male and 586 were female. Males (50.1%) were slightly more predominant than females in this study.
In our study, 600 samples (51.1%) had an ESR value between 1 and 20 mm/h, 408 samples with an ESR value between 21 and 60 mm/h, 117 samples with an ESR value of 61–100 mm/h and 49 samples with an ESR of above 100 mm/h [Figure 4]. | Figure 4: Distribution of ESR in the study. ESR = Erythrocyte sedimentation rate
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The mean of the 1174 ESR values obtained through manual Modified Westergren method was 30.33 with a standard deviation of 29.302. The mean value of 1172 ESR results obtained via Mixrate X20 is 30.08 and the standard deviation 28.922. Out of the 1137 ESR results obtained via Ves Matic Cube 30, the mean value of the measurements is 31.44 with a standard deviation of 28.084 [Table 2].
Bland –Altman agreement analysis of manual modified Westergren method versus Ves Matic Cube 30
The mean of the manual ESR and Ves Matic Cube 30 ESR values were plotted against the difference between two measurements. The mean difference between the two methods and 95% limits of agreement at 1 h for whole data were found to be −2.44 ± 13.01 (95% limits of agreement, −27.93–23.05), based on which Bland–Altman Plot was made [Figure 5] [Table 3]. | Figure 5: Bland Altman plot of ESR values from Manual modified Westergren method and Ves matic Cube 3. ESR = Erythrocyte sedimentation rate
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 | Table 3: One sample “t”-test of manual modified Westergren method versus Ves Matic Cube 30
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Bland–Altman agreement analysis of manual modified Westergren method versus Mixrate X20
The mean of the manual ESR and Mixrate X20 ESR values are plotted against the difference between two measurements. The mean difference between the two methods and 95% limits of agreement at 1 h for whole data was found to be 0.05 ± 1.68 (95% limits of agreement, −3.24–3.34) [Figure 6] [Table 4]. | Table 4: One sample t-test of manual modified Westergren method versus Mixrate X20
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 | Figure 6: Bland-Altman Plot of ESR values from Manual Modified Westergren method and Mixrate X20. ESR = Erythrocyte sedimentation rate
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Linear regression analysis-manual modified Westergren method versus Ves Matic Cube 30 [Table 5] | Table 5: Linear regression analysis between manual modified Westergren method versus Ves Matic Cube 30
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Non-significant P value shows no proportional bias (good agreement) between two measures. Here, P value obtained is 0.105, which is more than 0.05. Hence, statistically not significant, and therefore, the ESR values obtained through Ves Matic Cube 30 are agreeable with the gold standard modified manual Westergren method [Table 5].
Linear regression analysis-manual modified Westergren method versus Mixrate X20
Here, P value obtained is 0.809, which is more than 0.05. Hence, statistically not significant, and therefore, the ESR values obtained through Mixrate X20 is agreeable with the gold standard modified manual Westergren method [Table 6]. | Table 6: Linear regression analysis between manual modified Westergren method versus Mixrate X20
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Pearson's correlation coefficient (r):
If x and y are the two variables of discussion and n is the sample size, then the correlation coefficient can be calculated using the formula:
Manual modified Westergren method versus Mixrate X20 [Figure 7]: | Figure 7: Comparison of Mixrate X20 and Manual Modified Westergren method by linear regression analysis
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N = 1172
Y = 0.9979x + 0.0128
R2 = 0.9966
r = 0.998
This indicates that there is a very strong positive correlation between ESR values obtained by manual modified Westergren method and the automated ESR analyzer Mixrate X20.
Manual modified Westergren method versus Ves Matic Cube 30 [Figure 8]: | Figure 8: Comparison of Ves Matic Cube 30 and Manual Modified Westergren method by linear regression analysis
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N = 1137
Y = 0.9103x + 5.0388
R2 = 0.7932
r = 0.891
This means that there is a strong positive correlation between manual ESR and ESR value obtained by the automated ESR analyzer Ves Matic Cube 30, but not as much as that obtained with Mixrate X20.
| Discussion | | |
The determination of ESR is one of the most commonly employed laboratory tests worldwide. It is easy to perform, inexpensive, and therefore used as a routine test in various clinical conditions to aid in diagnosis, management, and follow-up. The ICSH has recommended the Westergren method as the reference standard method for ESR determination. However, the introduction of automated ESR analyzers has optimized the workflow and use of human resources. In this study, we compared the ESR values obtained through two automated ESR analysers-Ves Matic Cube 30 and Mixrate X20 against the reference method-Modified Westergren method.
ESR determination was done in 1174 blood samples received in our laboratory. The age of the population included in the study ranged between 7 and 88 years. The maximum number of patient samples belonged to the age group of 50–59 years (26.66%). There were 4 patients under the age of 10 years and 10 patients with age more than 80 years.
The male:female ratio in the present study was 1.004:1. Males slightly predominate in this study. In the study done by Mahato and Karkuzhali[14] and Casadio et al.,[15] the age and sex of the patient samples were not included in the study.
Distribution of erythrocyte sedimentation rate
As per the ICSH recommendations, a minimum of 40 samples each should be tested in 3 different groups of values-1–20 mm/h, 21–60 mm/h and more than 60 mm/h.[16] In this study, an ESR value of 1–20 mm/h was obtained in 600 samples (maximum number of samples-51.11%), 21-60 mm/h in 408 samples, 61–100/h in 117 samples, and a value of >100 mm/h obtained in 49 samples. In the study by Casadio et al.,[15] maximum number of ESR results was in between 21 and 60 mm/h [Table 7]. | Table 7: Comparison of distribution of erythrocyte sedimentation rate with similar studies
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Error result was displayed as the result for 37 blood samples in the Ves Matic Cube 30 automated ESR analyzer whereas Mixrate X 20 analyzer gave results for all 1174 samples with two results being >140 and so was excluded from statistical analysis.
Comparison of the techniques of Mixrate X20 and Ves Matic Cube 30 with manual modified Westergren method
Even though manual Modified Westergren method is considered the gold standard for ESR analysis, it has certain disadvantages like infection risk to the laboratory technician by blood-borne pathogens such as Hepatitis B and HIV since it is an open method in which the blood collection tubes have to be opened for dilution with normal saline and aspirated into ESR pipettes. In addition, it requires long test time duration (1 h) and relatively large amount of blood (minimum 2 ml). But for both the automated ESR analysers-Ves Matic Cube 30 and Mixrate X20, a simplified technique with unopened blood collection tubes that avoid sample handling and reduce the operator's risk of infection is utilized. To perform the analysis, the operator has to simply place the samples into the instrument in which automated mixing process takes place. Both require lesser time when compared to manual ESR method –30 samples can be analyzed at a time in Ves Matic Cube 30 with results obtained within 34 min whereas 20 samples can be analyzed at a time in Mixrate X20 with results obtained within 30 min including mixing of samples. Furthermore, the results are obtained as a printout as well as displayed on screen and can be stored in inbuilt memory for later reference. However, the added advantage of Ves Matic Cube 30 over Mixrate X20 is that, in Ves Matic Cube 30, ESR analysis can be done in the original EDTA tubes used for CBC and therefore not necessary to use a dedicated Citrate tube. But for Mixrate X20, specially designed 8 mm × 120 mm glass tubes that already contain 0.32 ml 3.8% sodium citrate are required for ESR analysis which provides correct dilution of anticoagulant.
Bland Altman agreement analysis of erythrocyte sedimentation rate
In the present study, the results obtained from Ves Matic Cube 30 analyzer and Mixrate X20 were compared with the manual modified Westergren method using the agreement analysis of Bland and Altman. Agreement analysis is a more sensitive method than correlation coefficient for comparison between two methods.[17] The use of Bland and Altman analysis for evaluating the agreement between two methods assesses the mean of the differences between the two methods (i.e., bias) and also the limits of agreement by calculating the standard deviation of the differences. Only when the 95% limit of agreement calculated as mean of differences ± (1.96 × standard deviation), does not affect the clinical interpretation, the two methods can be used interchangeably. In our study, the mean difference between manual ESR and Ves Matic Cube 30 was −2.4389 ± 13.0056 with 95% limits of agreement, −27.930–23.052 whereas in the previous study by Mahato and Karkuzhali,[14] the mean difference between manual ESR and Ves Matic Cube 30 was only 1.2 ± 3.032, with 95% confidence interval (CI) between 4.742 and 7.142. But when we compared manual ESR with another automated ESR analyser-Mixrate X20, the mean difference between manual ESR and Mixrate X20 ESR measurements was 0.0503 ± 1.680 with 95% CI between 3.24 and 3.34. Thus, this study indicates that even though both the automated methods are agreeable with the reference manual modified Westergren method based on statistical analysis, the results obtained from Mixrate X20 agree well with manual ESR measurements, with close readings and the difference obtained in the values (d ± 1.96SD) does not affect the clinical interpretation of ESR. This may be due to the use of citrated blood samples in specially designed ESR glass tubes and which follows the sedimentation of each sample independently in Mixrate X20 analyzer whereas EDTA anticoagulated blood samples were used for ESR analysis in Ves Matic Cube 30 [Table 8]. | Table 8: Comparison of Bland Altman agreement analysis of manual erythrocyte sedimentation rate versus Mixrate X20 and manual erythrocyte sedimentation rate versus Ves Matic Cube 30 with similar studies
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Pearsons correlation coefficient analysis of erythrocyte sedimentation rate [Table 9] | Table 9: Comparison of Pearson's correlation coefficient analysis of manual erythrocyte sedimentation rate versus Mixrate X20 and manual erythrocyte sedimentation rate versus Ves Matic Cube 30 with similar studies
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In our study, we found a strong correlation between the manual modified Westergren method and both the automated ESR analysers-Mixrate X20 and Ves Matic cube 30 since a value of more than 0.7 for correlation coefficient (r) indicates a strong correlation. However, the r value is higher for Mixrate (r = 0.998) when compared with Ves Matic Cube 30(r = 891), which indicates that a very strong positive correlation exists between ESR values obtained by Mixrate X20 and manual modified Westergren method. In a similar study by Casadio et al.[15] also, there is a very strong correlation between Mixrate X 20 and Manual ESR (r = 0.987) [Table 9].
We also did a correlation study and agreement analysis between the two automated ESR analysers-Ves Matic Cube 30 and Mixrate X20 which showed a fairly strong linear positive correlation with an r = 0.892. Bland–Altman agreement analysis showed a mean difference between two methods and 95% CI of −2.5031 ± 12.932 (95% limits of agreement, −27.8506–22.8443), which means that ESR value obtained by both methods are agreeable.
Age and erythrocyte sedimentation rate [Table 10] | Table 10: Comparison of the correlation between age and erythrocyte sedimentation rate values obtained via Manual method, Mixrate X20 and Ves Matic Cube 30
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In our study, when we compared the relationship between age and ESR value, we found that there is a weak positive, but significant correlation between age with ESR value obtained through all the 3 methods as depicted in the table below. In the previous studies by Mahato and Karkuzhali[14] and Casadio et al.,[15] the age of the patients whose blood sample was taken for ESR measurement was not recorded and analyzed [Table 10].
Gender and erythrocyte sedimentation rate
In our study, there also existed a statistically significant difference in the ESR values of males and females measured through all the three methods – manual modified Westergren method, Ves Matic Cube 30, and Mixrate X20 with a P value of 0.000 (<0.05). The gender of the blood samples analyzed were not recorded and analyzed by the previous studies by Mahato and Karkuzhali[14] and by Casadio et al.[15]
The discrepancy between the ESR values obtained through automated analyzers and the reference Modified Westergren method can lead to misinterpretation of ESR and can affect clinical diagnosis as well as management and follow-up. Hence, for optimal patient care and quality control, it is very well essential to make sure that the ESR values obtained from automated analyzers are very well agree with the reference method.[18] Recent studies indicate that the automated analysis of ESR has high comparability with the Westergren method.[19],[20] From our study also, it has been found that ESR results from both Mixrate X 20 and Ves Matic Cube 30 are agreeable to the reference method with no proportional bias statistically. However, the results obtained from Mixrate X20 correlate very well with the manual ESR values with close readings that do not affect the clinical interpretation. This may be due to the fact that in Mixrate X20 automated analyzer, citrated blood samples are used in specially designed 8 mm × 120 mm glass tubes with an increased diameter than the conventional Westergren tubes for analysis of ESR.
| Conclusion | | |
On comparing the techniques of Ves Matic Cube 30 and Mixrate X20 against modified manual Westergren method, both automated analyzers have a simplified technique with a closed method and inbuilt mixing process, which avoids sample handling and reduced infection risk to operator. Both require less time for ESR analysis, but the added advantage of Ves Matic Cube 30 over Mixrate X20 is that the same EDTA samples collected for CBC can be utilized for ESR analysis in Ves Matic Cube 30, whereas custom-made glass tubes containing sodium citrate are required for ESR analysis through Mixrate X20.
From our study, it has been found that ESR results obtained from both the automated ESR analyzers are agreeable to the reference method with no proportional bias statistically. However, the results obtained from Mixrate X20 ESR analyzer correlate very well with the manual ESR values, with close readings that do not affect the clinical interpretation. Hence, the principle of increasing the diameter of ESR measurement tubes as well as correct dilution with sodium citrate anticoagulant is better than increasing the slanting/sloping of tubes and undiluted samples for increasing the rate of sedimentation, that could be utilized in automated ESR analyzers.
Limitations
Only 4 ml of blood was available for performing the ESR via 2 automated ESR analyzers as well as by the manual modified Westergren method. The blood sample utilized for ESR analysis by Ves Matic Cube 30, was again used after proper mixing for estimation of ESR through manual modified Westergren method. Hence, there was a delay of 34 min between ESR analysis via automated methods and manual Modified Westergren method.
Acknowledgment
Department of Pathology, Government Medical College, Kottayam, Kerala, India.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]
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