ERYTHROCYTE SEDIMENTATION RATE

Technology, correlation and reference range

considerations

Introduction
The erythrocyte sedimentation rate (ESR) is widely used as a
screening test that indirectly and non-specifically measures
the presence of inflammation. Elevated ESR results may be
seen in a variety of conditions including infections,
malignancies, renal disease, inflammatory states and Figure 1: Depiction of RBC Aggregation using Capillary Photometry Technology
autoimmune diseases.
The Alifax® Erythrocyte Sedimentation Rate Analyzer Series
Traditionally, the ESR has been measured using EDTA
uses capillary photometry technology which eliminates the
anticoagulated whole blood which is diluted and placed into a
disadvantages of the manual ESR method; by providing ESR
specialized glass tube and allowed to sediment over a
results in 20 seconds, laboratory efficiency and
one-hour period. After the one-hour sedimentation period, the
turn-around-times are improved. Capillary photometry
height of the red blood cell (RBC) column is measured to
technology is listed in the Clinical and Laboratory Standards
determine the distance the RBCs have settled. While simple to
Institute guideline for ESR testing (HO2-A5) as an alternative
perform, manual ESR methods are time consuming, labor
to traditional ESR methods.².
intensive and increase the potential for operator exposure to
Proficiency testing samples for the Alifax systems are available
biohazardous materials. Additionally, manual ESR methods are
from proficiency testing providers such as the College of
impacted by numerous other factors including: environment
American Pathologists.
(vibration, temperature fluctuation and drafts in the test area),
sample specific conditions (anemia and abnormal RBC
Specimen requirements
morphology) and operator technique (setting up the test and Specimens must be collected in either K₂. or K₃. EDTA
reading the result).¹. anticoagulant. Optimal time to analyze a specimen stored at
Normal RBCs have a net negative charge which causes them to
room temperature is within 4 hours of collection. Refrigerated
repel each other. An increase in proteins like fibrinogen, alpha
specimens are acceptable for testing for up to 24 hours after
and beta globulins and immunoglobulins reduces the net
collection. The sample must be allowed to come to room
negative charge of the RBCs. This promotes formation of
temperature for approximately 15 minutes before testing.
rouleaux and agglutination which causes the RBCs to sediment
Refer to the analyzer instructions for use for more information
more quickly and increases the ESR. Erythrocyte
on specimen collection and handling.
sedimentation occurs in three stages: rouleaux formation in Alifax systems do not require the use of special collection
which RBCs aggregate for the first 10 minutes; sedimentation, tubes for determination of ESR results. One EDTA tube can be
which occurs over the following 40 min utes; and RBC packing, used for all routine hematology testing, decreasing collection
which occurs in the last 10 minutes. Despite the test name and tube costs and increasing pre-analytic efficiencies. Unlike
reporting units of millimeters/hour, the ESR is not timing the systems which read the ESR result through the tube wall, the
rate of sedimentation. Rather, it is a measurement of the Alifax systems perform closed tube sample aspiration,
amount of sedimentation as determined by the height of the eliminating interference from excessive tube labels.
RBC column at one hour. Various ESR methods may not
consider all phases of sedimentation and can be susceptible to Correlations
different interferences and proteins generated in diverse A correlation study was performed by Sysmex using 201 fresh,
disease states. whole blood samples collected in K₂-EDTA from an acute care
hospital comparing the Alifax® Roller 20PN™ ESR Analyzer
and the DISPETTE® 2 . Saline Modified Westergren method. • History of autoimmune, • Pregnancy
The samples were processed first on the Alifax Roller 20PN inflammatory disorders or • Menstruating
with no pre-mixing before analysis. The internal analysis mode diabetes • Recent injury or surgery
with analyzer sample mixing was used for all samples tested on • Taking any medicines or • Elevated biochemical
the Alifax system. After processing on the Alifax system, the supplements markers of inflammation
samples were tested using the DISPETTE method according to • Tobacco use like CRP or fibrinogen
manufacturer’s instructions.³ All test setup and resulting was • Ethanol use
completed by the same laboratory scientist to eliminate
Since ESR results are age and gender dependent, sufficient
operator-to-operator variability associated with the manual
‘normal’ subjects for males and females in different age ranges
ESR.
Results on the Alifax Roller 20PN ranged from 2 to 120 mm. would also be required. Such a reference range study to
Using regular linear regression to analyze the data, the slope identify ‘normal’ subjects is not possible or practical. This is
was 1.064 (95% Confidence Interval (CI) 0.973 to 1.156), the similar to other analytes like tumor markers or therapeutic
intercept was 12.5 (9% CI 9.5 to 15.5) and the correlation drugs which require extensive clinical or other evaluation to
coefficient (R) was 0.8526. (See Figure 2) determine a reference range.
An extensive reference range study, excluding subjects
meeting the above criteria, was performed by Piva et al.⁴. to
determine the reference ranges for the Alifax ESR method
(Table 1).

Number of 2.5 Percentile 97.5 Percentile

Age (Years) Gender
Subjects (95% CI) (95% CI)
Figure 2: Alifax® Roller 20PN™ ESR Analyzer versus DISPETTE® 2 Saline Modified
Westergren Method
0-14 80 M and F 2 (2-2) 34 (26-41)
The ESR is measuring the phenomenon of RBC sedimentation 15-50 190 F 2 (2-2) 37 (36-39)
which is impacted by the presence of various plasma proteins 15-50 150 M 2 (2-2) 28 (20-30)
and sample specific factors. It is not measuring an actual
51-70 120 F 2 (2-2) 39 (38-45)
analyte such as hemoglobin or cholesterol. Tests for analytes
51-70 130 M 2 (2-2) 37 (31-44)
like hemoglobin and cholesterol are highly standardized
between manufacturers and calibrated with calibrators >70 170 M and F 3 (3-3) 46 (45-55)

traceable to a reference method. There is not an

Table 1: Alifax ESR reference ranges
internationally recognized calibration standard for the ESR. It
is important to note that, because of how the ESR is measuring A concordance study was performed by Sysmex using 100
the phenomenon of RBC sedimentation rather than an analyte fresh K2.-EDTA whole blood samples. Samples were residual
(such as hemoglobin or cholesterol in an end-point samples from male and female patients ranging in age from 18
photometric reaction) there can be more imprecision with ESR to 94.
results. Due to these factors, comparison studies between Samples were tested using the Alifax Roller 20PN and the
different ESR methods may not show the high degree of DISPETTE 2 Saline Modified Westergren method. Considering
correlation that one would expect with a highly standardized the age and gender of the patient, results were classified as
analyte. The phenomenon of erythrocyte sedimentation is normal or abnormal using the reference ranges shown in Table
impacted by the effect of all the various plasma proteins as . for the Alifax method. For the DISPETTE method, reference
well as other sample related factors. Because of this, ESR ranges from Clinical hematology: Principles, procedures,
results may not correlate to C-reactive protein (CRP) and correlations⁵. (Lotspeich-Steininger, et al) were used and are
brinogen results. shown in Table 2.
Age (Years) Gender
Reference ranges
Due to the many factors that influence the ESR, laboratories <50 Male 0-15
have historically used reference ranges obtained from >50 Male 0-20
literature when reporting ESR results. Intensive screening to <50 Female 0-20
identify truly ‘normal’ subjects would be necessary when >50 Female 0-30
performing an ESR reference range study. Table 2: DISPETTE method reference ranges.
This would involve excluding subjects from the study if they
Of the 100 samples, 84 had the same interpretation (normal
had any of following conditions:
or abnormal) considering the reference ranges for each
method. Of the 16 samples with discordant interpretations, 9
samples had results that were borderline and near the upper
limit of the reference range for one or both methods.
Some discordant result interpretations are expected when
comparing ESR results from differing methodologies. This
may possibly be due to the presence of plasma proteins
associated with certain disease states or other sample
specific factors.⁶
To monitor disease progression in certain clinical states (i.e.,
rheumatic disease or multiple myeloma), some clinicians
monitor ESR results over time. When changing ESR
methodologies, laboratories should consider notifying
ordering clinicians of the method change. Another option is
Range
to report all ESR results with a message about the method
change for a time after going live with the new methodology.
This will help raise awareness with clinicians that a change in
the result is likely due to the method change rather than a
change in the patient condition.

Summary
Automated systems such as the Alifax family of ESR analyzers
provide results more quickly and efficiently than manual ESR
methods. They also eliminate the issues associated with the
manual ESR method including influences from environmental
factors and operator-to-operator technique variability.
Different ESR methods can have varying susceptibilities to
interferences and proteins generated in different disease
states. Method specific reference ranges should be adopted
when implementing new ESR methodologies.

Bibliography
1. Jacobs, David S., Dwight K. Oxley, and Wayne R. DeMott.
Jacobs & DeMott, Laboratory Test Handbook, Hudson
(Cleveland), OH: Lexi-Comp, 2001.

2. “H02A5, Procedures for the Erythrocyte Sedimentation

Rate Test, 5th Edition”, Clinical and Laboratory Standards
Institute, Wayne, PA, May 2011.

3.DISPETTE® 2. Saline Package Insert (FH1600), March 2016.

4.Piva, E., Sanzari, M., Servidio, G., et al. (2001). Length of

Sedimentation Reaction in Undiluted Blood (Erythrocyte
Sedimentation Rate): Variations with Sex and Age and
Reference Limits. Clinical Chemistry and Laboratory
Medicine, 39(5), pp. 451-454. Tomado el 4 Dic. 2018, de
doi:10.1515/CCLM.2001.071.

5. Lotspeich-Steininger, C. A., Stiene-Martin, E. A., & Koepke,

J. A. (1992). Clinical hematology: Principles, procedures,
correlations. Philadelphia:vLippincott.

6. Kim M, Ju Y-S, Lee EJ, et al. Erythrocyte sedimentation rate

measured using microhemagglutination is not elevated in
monoclonal gammopathy compared with other diseases. Int
J Lab Hem2018;40:540-578. https://doi.org/10.1111/ijlh.12859.
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