RRMechatronics WhitePaper ESR
RRMechatronics WhitePaper ESR
RRMechatronics WhitePaper ESR
Executive Summary
Erythrocyte Sedimentation Rate (ESR) is a sensitive, non-specific marker of
inflammation. ESR is used as a “general physical condition” marker, in
combination with clinical history, physical examination and other standard
laboratory tests. It can serve as a guide to aid diagnosis, management and
follow-up of different autoimmune diseases, acute and chronic infections and
tumors.
Modern and fully automated instruments, like the Starrsed, have made the ESR
test even more accurate and safe in comparison with the manual Westergren
version. Several published studies highlight that alternatives to the ICSH and
CLSI declared gold standard ESR method, that use a test principle that is very
different from Westergren, give rise to a large percentage of false negatives
and thus a risk of missed diagnoses.
This white paper provides an overview on the history and measurement of the
ESR and the different tests/instruments available on the market. In this paper
shows RR Mechatronics features its Starrsed Line of fully automated
Westergren ESR instruments. It shows several publications on analysis of these
tests in comparison with the gold standard of Westergren (and Starrsed). It
sheds light on the importance of the automated, accurate and safe use of the
Starrsed.
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Westergren ESR Measurement RR Mechatronics
Key Findings
The erythrocyte sedimentation rate (ESR) is a non-specific marker, used as a general
condition indicator. It is a classic test that measures how far erythrocytes settle in a test tube
over the course of time; 60 ± 1 minute. (page 4)
Erythrocyte sedimentation is governed by factors that stimulate or inhibit erythrocyte
aggregation and sedimentation. The clinically most relevant factors that influence ESR are
the erythrocytes themselves and plasma proteins associated with inflammation and tissue
damage. Erythrocytes usually aggregate into clumps that resemble a stack of coins and are
called rouleaux. (page 4,5)
The Westergren method is the gold standard and adopted as the reference method for ESR
measurement by the International Council for Standardization in Hæmatology (ICSH). (page
6,7)
In the original Westergren method, the ESR is read after 60 minutes. An ESR reading after 30
minutes can reliably be extrapolated to the corresponding ESR reading at 60 minutes
(correlation coefficient = 0.984). (page 10)
Test-1 is an ESR analyzer that produces ESR reading results within 20 seconds after sampling.
It takes however approximately 10 minutes before sedimentation starts at a constant rate.
This means that the Test-1 analyzer doesn’t actually measure sedimentation, but rather
calculates a mathematically derived ESR, based on aggregate measurements in the first,
rouleaux forming stage only. (page 11)
The Test-1, the iSED and the Ves-Matic Cube demonstrated clear flaws compared to the
original Westergren and the Starrsed, leading to an important number of false negatives.
(page 11)
The Starrsed ESR analyzers from Mechatronics are automated ESR analyzers that use the
reference Westergren method as recommended by the ICSH and CLSI. (page 11)
The Starrsed automated implementation of the Westergren ESR also takes care of the many
things that might influence the quality of the test result for example: temperature, stability,
dilution, washing and drying of the Westergren tubes and detecting problematic (hemolytic)
samples. (page 6,7,8,12)
ESR is a sensitive, non-specific marker of inflammation and is, in combination with clinical
history and physical examination, used as a “general physical condition” marker. (page 14)
ESR measurement is useful in the diagnosis of rheumatoid arteritis, temporal arthritis,
polymyalgia rheumatica, multiple myeloma and several autoimmune diseases. Clinical
studies have also suggested possible relevance of ESR levels in different other conditions.
(page 14,15)
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RR Mechatronics Westergren ESR Measurement
The erythrocyte sedimentation rate (ESR) is a non-specific marker, used as a general condition
indicator. ESR is a non-specific marker of inflammation. It can be used in combination with the
patient’s clinical history and physical examination and can serve as a guide to aid diagnosis,
management and follow-up of different auto-immune diseases, acute and chronic infections and
tumors (Bridgen, 1999).
ESR is a classic test that measures how far erythrocytes settle in a test tube over the course of time.
For this test, anti-coagulated whole blood is allowed to settle in an upright tube under standardized
conditions. The ESR is the distance in mm that the erythrocytes have fallen during that time. There
are many factors that affect the ESR, but the most clinically relevant factors that influence ESR are
the erythrocytes themselves and plasma proteins associated with inflammation and tissue damage.
The first one to notice and record changes in blood sedimentation during inflammation was John
Hunter, who mentioned this in “A treatise on the blood, inflammation and gunshot wounds”,
that was published posthumously in 1794. About a century later, in 1897, Edmund Biernacki, a
Polish physician, noticed that ESR was influenced by fibrinogen and developed his own ESR test,
which he published and presented to his peers. But since he published his findings in Polish and
German journals, his observations were hardly noticed in the English-speaking world.
In the 1920’s, Swedish pathologist Robert Fåhræus and physician Alf Westergren made similar
observations of the ESR in pregnant and tuberculosis patients. Together, they developed the
Fåhræus-Westergren method of measuring ESR, which was quickly and widely adopted in clinical
laboratories over the world and became known as the Westergren method.
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Westergren ESR Measurement RR Mechatronics
Rouleaux formation
Fig. 2: At low shear stress, RBC’s adhere side to side and form stacks called rouleaux, followed by
end to end connections creating 3D aggregates (the rouleaux formation)
(Fabry, 1987)
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RR Mechatronics Westergren ESR Measurement
Of these methods, only the Westergren method and Wintrobe method are still in use today. The
Wintrobe method uses tubes of only 100 mm long with a smaller diameter than standard
Westergren tubes. EDTA blood without extra diluent is added to the tube and allowed to
sediment for 60 minutes. After 60 minutes the distance that the blood cells have fallen is
registered in mm.
Because the Wintrobe tubes are shorter than the Westergren tubes, the method is less sensitive
than the Westergren method.
Procedure
The Westergren method as referenced by the ICSH consists of the following steps:
Blood collection
It is recommended that the EDTA sample is tested within 4 hours after collection, but it has been
reported that storage for up to 24 hours at 4°C still results in a stable ESR value. When ready to test,
the blood sample is thoroughly mixed and diluted 4:1 using a sodium citrate solution.
Tube handling
The Westergren method uses standardized colorless, circular glass or plastic tubes, with an inner
diameter of at least 2.55 mm and sufficient length to include a 200 mm sedimentation scale. The
inner diameter should be constant (± 5%) over the whole length; a so called Westergren tube.
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Westergren ESR Measurement RR Mechatronics
The diluted sample is aspirated and transferred to the Westergren tube. The Westergren tube is then
placed in a stable, vertical position at a constant temperature (± 1°C) between 18°C and 25°C in an
area free from vibrations, drafts and direct sunlight.
After 60 ± 1 minute, the distance from the bottom of the plasma meniscus to the top of the
descended erythrocytes is read and recorded in mm. The buffy coat that is made up of leukocytes
should not be included in the erythrocyte column.
(Jou, Lewis, Briggs, Lee, De La Salle, & McFadden, 2011) (CLSI, 2011)
a. b. c.
Fig 3: a. The diluted sample is aspirated and transferred to the Westergren tube.
b. A normal ESR after 60 minutes; <20 mm plasma.
c. An elevated ESR after 60 minutes.
Because of contamination risks, disposable, tubes are sometimes used. These need to be replaced
after use and generate a lot of waste. Some plastic tubes can have a strong attractive force on
erythrocytes or release plasticizers that can affect sedimentation rate. Mold-release agents used in
the manufacturing process may sometimes alter sedimentation characteristics. Reusable tubes need
to be cleaned thoroughly after use.
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RR Mechatronics Westergren ESR Measurement
Sample storage
The storage conditions of the sample are permitted, conform the CSLI standard (CLSI, 2011), to be up
to 4 hours at room temperature or up to 24 hours at 4 degrees Celsius in cold storage. These storage
conditions are in line with the sample storage requirement for the hematology analyzers.
Changes in plasma viscosity and hematocrit can cause variable plugging of the long Westergren tube
by rapidly falling erythrocyte aggregates. Correct dilution (4:1) of the blood sample in sodium citrate
prevents this and makes the measurement independent of viscosity and hematocrit differences.
Temperature variation
The sedimentation process is substantially influenced by temperature variations. One example would
be when sunlight would shine on some tubes but not others. The ICSH recommends a constant
temperature (± 1°C) between 18°C and 25°C.
Vibrations
Vibrations can artificially increase sedimentation rate and should therefore be avoided.
Problematic samples
In some cases, erythrocyte abnormalities can result in hazy, cloudy samples that are difficult to read.
Also hemolytic or lipemic samples can cause difficulties in the accurate reading of ESR.
(CLSI, 2011) (Hardeman, Levitus, Pelliccia, & Bouman, 2010)
In some cases sampling errors, e.g. a low sample volume, can lead to foam or bubbles in the sample.
In these cases, the reduced quality of the sample can lead to incorrect results and false clinical
interpretation, if the analyzing system does not perform internal quality controls and flags low
quality samples and unreliable readings.
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Westergren ESR Measurement RR Mechatronics
DM Dissanayake of the University of Peradenya in Sri Lanka has tested whether it was possible to
use an inclined tube to get a faster reading of the ESR.
Dissanayake tilted tubes at an angle of 45 degrees and registered sedimentation distances every
30 seconds from 4 to 13 minutes by reading the lowest level of the meniscus. These results were
compared with a traditional Westergren reading of the same sample in another tube that was
kept vertically. The experiment contained a wide range of ESR readings, from 0 mm/hr to well
over 150 mm/hr. The correlation between the traditional Westergren reading and the tilted tube
was maximal between 10 and 11.5 minutes (correlation coefficient=0.985-0.986) for both low
and high ESR readings.
(Dissanayake, 2006)
The accuracy of the results was considered acceptable. It demonstrates however that a tilted
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RR Mechatronics Westergren ESR Measurement
Correlation coefficient:
- The value of a correlation coefficient ranges between -1 and 1.
- The strongest linear relationship is indicated by a correlation coefficient of -1 or 1.
- The weakest linear relationship is indicated by a correlation coefficient equal to 0.
NB: The correlation coefficient 0.984 is considered a very strong linear relationshipThe accuracy
of the results was considered acceptable. It demonstrates however that a tilted tube has a strong
influence on the optimal testing time.
Fig. 5: The 30 minute ESR readings correlate highly with the corresponding 60 minute ESR readings
over a wide range of blood samples (correlation coefficient = 0.984).
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Westergren ESR Measurement RR Mechatronics
Fig. 6: Results reported into the upper left quadrant are considered normal according to the
Westergren gold standard, but high according to method X. These so called “false positives” will lead
to additional costs for supplementary testing or unnecessary treatment. Results reported into the
lower right quadrant are considered high according to the Westergren gold standard, but low
according to method X. These so called “false negatives” may lead to missed diagnosis.
In addition, sedimentation characteristics of the second and third stage can be relevant for some
diseases, e.g. multiple myeloma. Test-1 was not as sensitive to the presence of paraproteins as the
Westergren method (Raijmakers, Kuijper, Bakkeren, & Vader, 2008) and could produce significantly
different results, especially in the higher ESR readings (Hardeman, Levitus, Pelliccia, & Bouman,
2010). In one comparison it was found that in 11.5% of the samples, the differences in results could
lead to either a missed diagnosis (false negative) or additional testing costs (false positive)
(Hardeman, Levitus, Pelliccia, & Bouman, 2010).
The figures below are Passing Bablok regression plots taken for three independent publications. By
evaluating and connecting the dots of three publications it is possible to compare the Test1 and iSED
with the Starrsed and the gold standard Westergren. It again articulates the quality of the original
Westergren method and the Westergren related methods in determining ESR.
The Test1, the iSED and the Ves-Matic Cube demonstrated clear flaws compared to the original
Westergren and the Starrsed leading to an important number of false negatives.
Fig. 7: Higher than 40 on the Westergren scale and lower than 40 on the compared instrument scale
are potentially missed diagnoses.
(Hardeman, Levitus, Pelliccia, & Bouman, 2010) (Raijmakers, Kuijper, Bakkeren, & Vader, 2008)
(Bogdaycioglu, Yilmaz, Sezer, & Oguz, 2014) (Curvers, et al., 2010)
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RR Mechatronics Westergren ESR Measurement
Pre-mixing, sampling and dilution of standard whole-blood EDTA samples in sodium citrate is fully
automated, which ensures accuracy and frees up time for the operator, who only needs to load the
samples into the analyzer. The analyzers contain a built-in barcode reader that automatically
identifies and registers the correct blood samples. Starrsed analyzers use a specifically designed
needle for sampling that minimizes damage to the rubber stopper and ensures that blood vials can
be sampled reliably multiple times.
Starrsed analyzers use standardized, reusable glass tubes that are especially made and tested. The
tubes are cleaned using detergent and protease enzymes, rinsed and dried after each cycle, ensuring
that the tubes are clean before use. This reduces waste and minimizes biohazard risks and the cost of
operation.
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Westergren ESR Measurement RR Mechatronics
Fig. 9: The tubes are cleaned using detergent and protease enzymes. The inside of the tube is dried
and disinfected by air that has passed through a heating element.
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RR Mechatronics Westergren ESR Measurement
During acute phase reactions, macromolecular plasma proteins, particularly fibrinogen, are produced
that decrease the negative charges between erythrocytes and thereby encourage rouleaux
formation.
Paraproteins are positively charged molecules that are abundantly present in multiple myeloma and
Waldenström’s macroglobulinemia patients. Like fibrinogen, paraproteins decrease the negative
charges between erythrocytes and increase rouleaux formation. As described earlier the aggregation
based ESR tests (Test-1 and iSED) typically miss detecting these disorders, see paragraph above on
“Aggregation versus Sedimentation”. (Raijmakers, Kuijper, Bakkeren, & Vader, 2008)
High protein concentrations increase plasma viscosity, which slows down the fall rate and thus ESR.
However, the effects of fibrinogen and paraproteins on the negative charges between erythrocytes
and rouleaux formation far outweigh the effect of increased plasma viscosity, resulting in a strong
net increase of ESR.
In anemia, erythrocyte numbers are reduced, which increases rouleaux formation. In addition, the
reduced hematocrit affects the velocity of the upward plasma current so that erythrocyte aggregates
fall faster.
In macrocytosis, erythrocytes are changed into a shape with a small surface-to-volume ratio, which
leads to a higher sedimentation rate.
(Saadeh, 1998) (Bridgen, 1999)
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Westergren ESR Measurement RR Mechatronics
Erythrocyte abnormalities can affect aggregation, rouleaux formation and fall rate. Erythrocytes with
irregular or small shapes tend to settle slower and decrease ESR.
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RR Mechatronics Westergren ESR Measurement
ESR measurement is useful in the diagnosis of Rheumatoid Arthritis and the follow-up of RA-patients
when combined with other parameters as outlined in the ACR guidelines. However, ESR can be
elevated when RA is clinically quiescent and vice versa. ESR is also useful in the follow-up of SLE, but
not for inflammatory myopathy or spondyloarthropathy.
An elevated ESR is one of the diagnostic criteria for temporal arthritis and polymyalgia rheumatica.
The ESR is almost always elevated in these conditions, in some cases exceeding 100 mm/hr. However
a normal ESR in suspected patients does not rule out diagnosis. If clinical features are present, a
temporal artery biopsy is highly recommended, even when ESR is not elevated.
Multiple myeloma
An increased ESR is helpful in multiple myeloma, but the diagnosis depends on other criteria
(monoclonal spike or serum electrophoresis, marrow plasmacytosis and lytic bone lesions). ESR in
benign monoclonal gammopathy is not well studied. ESR measurements should only serve as a guide
to disease progression or response to therapy in symptomatic patients.
Other uses
Clinical studies, often small studies, have suggested possible relevance of ESR levels in different
conditions, e.g. bacterial otitis media, acute hematogenous osteomyelitis in children, sickle cell
disease, pelvic inflammatory disease, febrile IV drug users, prostate cancer, coronary artery disease
and stroke.
An extreme elevation of ESR, defined as >100 mm/hr is indicative for a serious underlying disease,
most notably infection, collagen vascular disease, metastatic malignant tumors or renal disease. In
most cases, the underlying condition is clinically apparent. In < 2% of patients with an extremely
elevated ESR, no obvious cause can be found, but the underlying cause can usually be found in
combination with the clinical history, physical examination and other standard laboratory tests.
(Saadeh, 1998) (Bridgen, 1999)
Conclusion
Erythrocyte Sedimentation Rate following the gold standard of Westergren is a useful general
condition indicator and marker for inflammation. Modern and fully automated instruments have
made the ESR test even more accurate and safe in comparison with the manual Westergren version.
The Starrsed automated implementation of the Westergren ESR in addition takes care of the many
things that might influence the quality of the test result for example: temperature, stability, dilution,
washing and drying of the Westergren tubes and detecting problematic (hemolytic) samples. All
these possible quality influencers need to be under control to perform an optimally accurate test.
Several published studies highlight that alternatives to the ICSH and CLSI declared gold standard ESR
method that use a test principle that is very different from Westergren, give rise to a large
percentage of false negatives and thus a risk of missed diagnoses.
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Westergren ESR Measurement RR Mechatronics
References
Bogdaycioglu, N., Yilmaz, F., Sezer, S., & Oguz, E. (2014). Comparison of iSED and Ves-Matic Cube 200
Erythrocyte Sedimentation Rate Measurements With Westergren Method. Journal of clinical
laboratory analysis.
Bridgen, M. L. (1999). Clinical Utility of the Erythrocyte Sedimentation Rate. Am Fam Physician.
CLSI. (2011). Procedures for the Erythrocyte Sedimentation Rate Test; Approved Standard—Fifth
Edition. CLSI document H02-A5. Wayne, PA: Clinical and Laboratory Standards Institute.
Curvers, J., Kooren, J., Laan, M., van Lierop, E., van de Kerkhof, D., S. V., et al. (2010). Evaluation of
the Ves-Matic Cube 200 erythrocyte sedimentation method: comparison with Westergren-
based methods. Am J Clin Pathol.
Dissanayake, D. M. (2006). A rapid method for testing the erythrocyte sedimentation rate. Journal of
Diagnostic Pathology, 47-51.
Fabry, T. L. (1987). Mechanism of Erythrocyte Aggregation and Sedimentation. Blood, 1987 (Blood,
Vol 70, No5), 1572-1576.
Frankel, S., Reitman, S., & Sonnenwirth, A. (1963). A Text Book on Laboratory Procedures and Their
Interpretations. In Gradwohl Clinical Laboratory Methods and Diagnosis. The C.V. Mosby
Company London.
Grybowski, A., & Sak, J. J. (2011 ;38). Who Discovered the Erythrocyte Sedimentation Rate? The
Journal of Rheumatology, 1521-1522.
Hardeman, M., Levitus, M., Pelliccia, A., & Bouman, A. (2010). Test 1 analyser for determination of
ESR. 1. Practical evaluation and comparison with the Westergren technique. Scand J Clin Lab
Invest.
Jou, J., Lewis, S., Briggs, C., Lee, S., De La Salle, B., & McFadden, S. (2011). ICSH review of the
measurement of the erythocyte sedimentation rate. Int J Lab Hematol.
Madrenas J, P. P. (2005). Giving credit where credit is due: John Hunter and the discovery of
erythrocyte sedimentation rate. . The Lancet, 366.
Penchas, S., Stern, Z., & Bar-Or, D. (1978). Heparin and the ESR. Arch Intern Med.
Raijmakers, M., Kuijper, P., Bakkeren, D., & Vader, H. (2008). The effect of M-proteins on the
erythrocyte sedimentation rate; a comparison between the StarrSed and TEST 1 analyzers.
Ned Tijdschr Klin Chem Labgeneesk.
Rogers, R. (1994). The development of 30 minute ESR's on the 'StaRRsed' ESR Analyser. Medical
Laboratory World.
Saadeh, C. (1998). The erythrocyte sedimentation rate: old and new clinical applications. South Med
Journal.
www.rrmechatronics.com
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RR Mechatronics Westergren ESR Measurement
Notes
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Westergren ESR Measurement RR Mechatronics
Notes
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RR Mechatronics
De Corantijn 13, 1689 AN Zwaag, The Netherlands
P.O. Box 225, 1620 AE Hoorn, The Netherlands
T + 31 229 291 129
sales@rrmechatronics.com
www.rrmechatronics.com