1. 1
Erythrocyte Sedimentation Rate: Implementing a new method of
measurement in a hospital laboratory, and assessing its current
medical significance.
Abstract
The aim of this project was to validate the TEST 1 Alifax to replace the StaRRsed Compact (RR
Mechatronics) for measurement of the erythrocyte sedimentation rate (ESR) in a hospital
laboratory (Ysbyty Gwynedd, Bangor), and a review of the literature to determine the ESR’s
place in modern laboratory diagnostics. The Alifax output was first calibrated to be on a par with
the Westergren gold standard of ESR measurement used by the StaRRsed, then 137 samples
were analyzed to ensure sufficient correlation using the Bland-Altman method ‘for assessing
agreement between two methods of clinical measurement’ (Bland & Altman, 1986). Intra-Assay
Reproducibility was also assessed on the Alifax using 29 samples, each sample tested 5 times
on the instrument. The results showed acceptable correlation between the two instruments
when taking into consideration the number of variables affecting the Westergren method that
have no bearing on the Alifax measurement due to the advantage of environment control in the
newer instrument. The literature review concluded that the ESR only has place as a diagnostic
and monitoring tool and cannot itself be used alone for diagnosis or as a screening tool.
However, though the ESR has traditionally been used for the diagnosis of Rheumatoid Arthritis,
Polymyalgia Rheumatica and Temporal Arteritis, there are a number of new conditions with
potential for development. In summary, until other alternatives can match the user requirements,
(i.e. cheap, reliable and ease of use), the ESR will still have a place within the modern
Haematology laboratory, hence justifying the procurement of the new instrument.

2. 2
Introduction
The Erythrocyte Sedimentation Rate
The erythrocyte sedimentation rate (ESR) is the measure of how quickly red cells (erythrocytes)
fall through plasma over the period of one hour, and is expressed in millimetres per hour
(mm/hr). Recognition for the discovery of this phenomenon of red cell sedimentation is
somewhat disputed. Over the years a number of individuals have been attributed with its
discovery; from Robin Fåhraeus (1918), to Edmund Biernacki in 1894 who also detailed the
method of measurement, known as the ‘Biernacki Reaction’ (Kucharz, 1987) and more recently
evidence has been collated that suggests credit is due to John Hunter, a British surgeon and
anatomist in 1794 (Madrenas et al, 2005).
The rate at which the red cells sediment is predominantly defined by the presence of plasma
proteins, including fibrinogen, serum amyloid A protein (SAA), C-Reactive Protein (CRP) and
albumin, and their relative volumes. During an acute phase response, concentration of such
proteins change rapidly; there is an increase in fibrinogen, α1 proteins and immunoglobulins, but
a decrease in serum albumin (Hall & Malia, 1984).
The process of sedimentation occurs in three distinct stages:
 Stage 1: Aggregation; formation of red cell rouleaux which form spherical aggregates
 Stage 2: Aggregates sink through the plasma at approximately constant speed
 Stage 3: As the aggregates pack at the bottom of the tube, the rate of sedimentation
slows
(Lewis et al, 2001; Fabry, 1987)
Formation of rouleaux is mediated by the presence of plasma proteins; red cells have a net
negative charge, known as the zeta potential, due to surface sialic acid residues, which
prevents the cells from coming closer than 100nm (Fabry, 1987). The plasma proteins must be
of a sufficient length in order to overcome the electrical repulsion, and create a cross-link bridge
between erythrocytes (Chien & Jan, 1973). These rouleaux and aggregates sediment more
quickly, therefore in instances of increased prevalence of plasma proteins in an acute phase
response, the ESR will be higher. Immunoglobulins present in inflammatory reactions, such as
Rheumatoid Arthritis, may also result in the formation of aggregates through antibody-antigen
interactions (Hall & Malia, 1984; Lewis et al, 2001). Red cell aggregation is also aided by

3. 3
increased levels of CRP and haptoglobin (Weng, 1996). The effect of albumin (the most
abundant plasma protein) on the ESR is somewhat contested; in a paper by Reinhart it is stated
that albumin generally retards the process (Reinhart, 1994), whereas in another paper the effect
of increased albumin levels on Immunoglobulin G-induced aggregation was found to be
inhibitory, whilst fibrinogen-induced aggregation is promoted (Maeda & Shiga, 1986).
The ESR is also affected by a number of physiological factors. The ESR of both sexes
increases with age, though females have a consistently higher ESR at each age range (Miller et
al, 1983) (Table 1), raised further through the occurrence of menopause, suggesting a hormonal
influence on the ESR (Bottiger & Svedberg, 1967). Race also affects the ESR; the mean ESR
for black individuals is approximately 2 to 13mm/hr higher than in white individuals for all age
groups and gender (Gillum, 1993).
Table 1: Normal ESR ranges, categorized by age and gender (Lewis et al, 2001).
Note ‘c’ denotes approximately.
Age
(years)
Men
(mm/hr)
Women
(mm/hr)
17-50 10 12
51-60 12 19
61-70 14 20
>70 c30 c35
Besides infection and inflammation, a raised ESR can be observed in pregnancy, anaemia, red
cell abnormalities such as macrocytosis, neoplasm, diabetes mellitus, hypothyroidism, collagen
vascular disease, and due to certain drugs such as heparin and oral contraceptives (Brigden,
1999; Olshaker & Jerrard, 1997). However, individuals suffering from liver disease, carcinoma
or other serious diseases may lack the ability to produce acute phase proteins and hence have
an unexpectedly normal ESR (Greer et al, 2008).
A lower than expected ESR, in relation to the normal range expected for the different age
groups, may occur in cases of extreme leukocytosis, polycythaemia, red cell abnormalities
(such as spherocytosis, acanthocytosis, microcytosis and sickle cells), haemolytic anaemia,

4. 4
pyruvate deficiency, disseminated intravascular coagulation (DIC), protein abnormalities
(hypofibrinogenemia, hypogammaglobulinaemia, dysproteinaemia with hyperviscosity state) and
due to anti-inflammatory agents and cortisone (Brigden, 1999; Greer et al, 2008; Olshaker &
Jerrard, 1997). The presence of a lower than expected ESR is of little diagnostic importance
with only a small percentage of individuals with a low ESR having an underlying condition
responsible (Zacharski & Kyle, 1965).
Other factors such as obesity, body temperature, time period since consuming previous meal,
aspirin and NSAIDS reportedly have no effect on the ESR (Brigden, 1999).
With the increasing pressure placed on hospital laboratories through an ever increasing work
load and the demand for reduced turnaround times, laboratories must aim to employ diagnostic
techniques that can process large number of samples quickly, with acceptable accuracy and
reliability. Instruments such as the Beckman Coulter LH750 full blood count analyzer presently
used at Ysbyty Gwynedd are able to process up to 110 samples per hour (Fernandez et al,
2001), and with the implementation of two of these machines, the laboratory can efficiently
process samples, even at peak times. In comparison, the current form of measurement of the
Erythrocyte Sedimentation Rate (ESR) employed at the hospital, using the StaRRsed Compact
can only process a maximum of 75 samples per hour, and requires the operator to load each
sample individually. With the hospital laboratory at Ysbyty Gwynedd receiving, on average, 400
to 500 samples requesting an ESR per day, with most of these samples being received during a
3 hour period in the afternoon, the slow rate of turnover means a sample backlog builds up and
thus the workload cannot be cleared as quickly. Although the ESR may not be of as great a
diagnostic significance as, for instance, a full blood count, quick turnover is still expected by the
laboratory service user.
Uses of the Erythrocyte Sedimentation Rate
The ESR is used as a diagnostic and monitoring tool, typically in Rheumatoid Arthritis and other
autoimmune conditions such as Temporal Arteritis and Polymyalgia Rheumatica.
Rheumatoid Arthritis (RA)
Approximately 350,000 people suffer from RA in the UK, predominantly women and individuals
over the age of 40[1]
. As described by Peakman & Vergani, RA is ‘a multisystem inflammatory
disease, principally affecting peripheral joints in a symmetric fashion and commonly leading to

5. 5
cartilage destruction, bone erosions and join deformities’ (Peakman & Vergani, 1997). ESR, in
conjunction with CRP, has been shown to be useful for assessing the severity of RA, correlating
with the prevalence of erosions visible on radiography. This allows for the effectiveness of drug
treatment to be assessed, i.e. is the drug only alleviating the symptoms or is it having a more
beneficial effect (Amos et al, 1977). Requests received in the laboratory typically require
monitoring of Methotrexate, a drug used in the treatment of RA patients.
Polymyalgia Rheumatica (PMR)
PMR generally affects women and individuals over 50 years of age, but is relatively uncommon
with approximately 11/100,000 new cases per year in England [2]
. A patient is most likely
suffering from PMR if 3 or more of the following criteria are met (Bird et al, 1979);
- bilateral shoulder pain or stiffness
- symptoms developed within the last 2 weeks
- initial ESR greater than 40mm/hr, usually elevated to >60mm/hr (Cush et al, 2005),
though in some cases a low ESR level of less than 40mm/hr has been reported (Proven
et al, 1999).
- duration of morning stiffness exceeding 1 hour
- aged over 65 years
- depression and/or weight loss
- bilateral tenderness in the upper arms
Though the ESR is an integrated part of the diagnostic and monitoring procedure, care should
be taken when relying on the ESR for monitoring of disease states. For instance, one study
described a patient diagnosed with PMR, including an elevated ESR, subsequently leading to
treatment with low-dose corticosteroids which in turn normalized the ESR. However, despite this
normal ESR, she developed symptoms of temporal arteritis which was subsequently confirmed
via biopsy (Papadakis & Schwartz, 1986). This case demonstrates that although the ESR is a

6. 6
useful tool in monitoring of disease states, it should never be completely relied upon as in some
cases it may not follow the predicted pattern.
Temporal arteritis (Giant Cell Arteritis, ‘GCA’)
Temporal arteritis (or Giant Cell Arteritis) is a vasculitis of vessels, predominantly the cranial
arteries (Fauchald et al 1972), which may develop following PMR though has a low prevalence
of approximately 7/100,000 per year in England [2]
. The 1990 Criteria for the Classification of
Temporal (Giant Cell) Arteritis includes five criteria of which three are required to diagnose the
condition, including an elevated ESR of ≥50mm/hr by the Westergren method (Hunder et al,
1990). However, as with PMR, there are reported cases of the ESR measurement falling within
the ‘normal’ range in patients suffering from temporal arteritis (Wong & Korn, 1986).
Other uses of the ESR
A number of studies have suggested the ESR may be included as a diagnostic tool for other
conditions, though its usefulness may be outweighed by other forms of measurement, such as
C - reactive protein (CRP).
The ESR is commonly used as a test in osteomyelitis, an inflammation of the bone marrow due
to infection, which may be complicated by septic arthritis. A study in 1994 observed its use in
acute haematogenous osteomyelitis in children, though showed that the CRP can provide a
more rapid diagnosis of the occurrence of septic arthritis, unlike the ESR measurement which
took 5 days to distinguish between cases presenting with septic arthritis and those without
(Unkila-Kallio et al, 1994). Speed of diagnosis or detection of progression is essential in many
medical conditions, therefore if an alternative method that delivers the same information in a
reduced time period is available, the original method becomes obsolete. However, in some
recent studies, the ESR has been shown to be a useful independent predictor for heart failure,
through monitoring of inflammation (Ingelsson et al, 2005) and could even be a useful
prognostic marker for indicating early relapse in Hodgkin’s disease patients, with the
persistence of a raised ESR after treatment suggesting a more resistant disease and likely
chance of relapse (Henry-Amar et al, 1991). The ESR has also been assessed for various other
usages, including: determining the risk of recurrence of bacterial otitis media (Del Beccaro et al,

7. 7
1992), assessing the severity of pelvic inflammatory disease (Miettinen et al, 1993), evaluation
of febrile IV drug users (Gallagher et al, 1993), prostate cancer prognosis (Imai et al, 1990),
renal cell carcinoma survival (Ljungberg et al, 1995) coronary heart disease prognosis (Erikssen
et al, 2000), and in early prediction of stroke severity (Chamorro et al, 1995). Studies have
shown conditions in which the ESR is not affected by the condition itself, thus meaning it is still
viable for use in the detection of inflammatory disorders in the patient. One such study
concluded that in patients with chronic renal failure the condition itself did not effect the ESR
measurement, therefore it can still be used to evaluate the patient as normal (Brouillard et al
1996).
Screening Use of the ESR
Papers, such as that by Sox & Liang in 1986, state than in current day medicine there is no
place for the ESR as a screening method in asymptomatic individuals. However, the number of
ESRs requested suggests that the diagnosing physician still finds the measurement a useful
tool for indicating a possible subclinical change.
Other methods of assessing the acute phase response, and their comparison to the ESR.
Though it is cheap and historically used as an inflammatory marker, the ESR is not the only
method for monitoring the acute phase response, other tests include: C-Reactive Protein (CRP),
Plasma Viscosity (PV) and, less commonly, cytokines (Kokkonen, 2010).
CRP levels rise more quickly than the ESR, and has a shorter half life making is useful for
assessment of treatment (Buess & Ludwug, 1995), though this can result in contradiction of the
ESR and CRP measurements, i.e. a high ESR/low CRP or low ESR/high CRP, despite both
being inflammatory markers (Costenbader et al, 2007).
Plasma viscosity has been shown to be more sensitive in detecting changes in plasma proteins
than the ESR, with a greater reliability and usefulness (Hutchinson & Eastham, 1977).

8. 8
The attributes and downfalls of each of the three most common methods; ESR, CRP and PV
are summarised below.
Table 2: Comparison of the ESR, C-Reactive Protein and Plasma Viscosity Tests (adapted from
Brigden, 1999 with additional information from Lewis, Bain & Bates, 2001)
Several other less known alternatives to the ESR have also been suggested, including blood
echogenicity; a computerized method that subjects the flowing blood to ultrasonic echoes
(Kallio, 1991), and the Zeta Sedimentation Ratio; a method similar to the Westergen method of
ESR measurement yet claims to be unaffected by anaemia (Bull & Brailsford, 1972).
Test Advantages Disadvantages
ESR Low cost
Simple procedure
Affected by a variety of factors, as previously
documented; not sensitive enough for
screening, slow to respond to acute disease
CRP Rapid response to inflammation, and hence
can often be detected before clinical features
become apparent, serum level rapidly
decreases as cause
is resolved
Expensive necessitating batch processing which
may delay individual results.
Wide reference range.
PV Unaffected by anaemia or red blood cell size
Less dependent on age and sex variable
Expensive
Not widely available
Technically awkward to perform

9. 9
Measurement of the Erythrocyte Sedimentation Rate
Currently used in the Haematology laboratory is the StaRRsed Compact by RR Mechatronics
(Figure 1) though the new TEST1 Alifax (Figure 2) is being validated to replace this instrument.
Numerous papers provide evidence that the Alifax instrument provides improved quality control
measures (Piva et al, 2007), reliable and precise results (Ozdem et al, 2006) and the
measurements obtained better reflect inflammation than by the Westergren method (Cha et al,
2009). Technical factors that can influence the ESR when performed using the Westergren
method, including; dilution, temperature (sedimentation is normally accelerated as temp
increases,), tilted ESR tube, inadequate mixing, clotting of sample, vibration during testing, short
ESR tube, drafts and sunlight (Hall & Malia, 1984; Lewis et al, 2001), claim to be effectively
counteracted through the action of environment control and technique of measurement in the
Alifax.
The following table (Table 3, pg.10) compares the two instruments for a variety of properties,
compiled from the instrument brochures and through use of the instruments.
Figure 1: StaRRsed Compact Figure 2: TEST 1 Alifax

10. 10
Table 3 – Comparison of Instruments for performing the ESR
StaRRsed Compact [3] Alifax Test 1 [4]
Method Westergren Red cell agglutination kinetics
Turnaround time/sample 60 minutes (or 30 mins programme available) 20 seconds
Capacity 84 Westergren pipettes 60 samples (4 racks of 15)
Max. output in 1 hour 75 samples 180 samples
Volume of blood used 1.3ml (1300μl) 0.15ml (150μl) (min. dead vol. of 1ml)
Dilution Requires citrate solution Not required
Solutions required Detergent, water, saline, disinfectant None
Internal barcode reader Yes Yes
Temperature control Temperature corrected to the value of 18 o
C Thermostat 37o
C
Tube required Accepts open and closed sample tubes of
virtually any brand and type
Works with primary collection tube
Cost Approx. 10p/test £140 per 10,000 tests
(<1.5p/test)
Maintenance
- cleaning
- waste
Automatic at the end of each cycle
Automatic waste control, disposal straight into
drain
3 tube cleaning system with a photometer
check, when required
Enclosed waste container indicated when
full
Quality controls One normal and one high control 3 controls; normal, slightly raised and high
Disadvantages Affected by;
- low levels of haematocrit
- environmental conditions
Each sample has to be loaded manually
Advantages Possible hazy conditions, results interpreted via
standard algorithm
Microbiological filters
Not affected by low levels of haematocrit.
Enclosed environment allows for tighter
control of variables

11. 11
Method of Verification
The experimental portion of the verification was relatively simple. The officially recognized
reference procedure is the Westergren method (Westergren, 1921) recommended by the
International Committee for Standards in Haematology (1993), therefore all calibration of new
methods or instruments should be done against this method. The StaRRsed Compact uses the
Westergen method for analysis of ESR, however, as this verification was taking place in a busy
hospital laboratory, it was unrealistic to be able to run all the test samples on the StaRRsed
instrument as, with the long process time, both the patient and test samples could not be run
and hence patient samples took priority.
Initial Comparison of the Alifax and StaRRsed Compact
Initially, 79 samples were run on both the Alifax and StaRRsed Compact to determine a
preliminary evaluation of the agreement, and if the Alifax presented with a bias, i.e. consistently
producing higher values than the StaRRsed Compact. The output of the Alifax was then
adjusted to calibrate the instrument to produce results on a par with the StaRRsed instrument.
Verification of the Alifax Post Adjustment
For the remainder of the verification, the comparison took place between the Alifax, and the
manual Westergren method, the same method used by the StaRRsed, though obviously human
error in performance of the method was now introduced as a possible source of error. 137
samples were picked at random and the ESR analysed using both the Westergen method of
measurement and the newer method on the Alifax.
Westergen Manual Method
This method employs a 30cm straight glass, graduated plugged dispette with a uniform
diameter, which must be clean, dry and free from dust, and the method performed at room
temperature (Lewis et al, 2001). EDTA blood samples are inverted multiple times to ensure
even spread of the constituents, then 1.6ml is mixed in a fresh, labelled tube with 0.4ml of
trisodium citrate diluent (32g/l stock solution). The tube is inverted to ensure adequate mixing of

12. 12
the contents, and the open end of the glass tube placed in the mixture, held in a vertical position
by a stand. Using a suction device, the diluted blood is drawn up the glass tube, until it reaches
the stopper. The mixture is then left to stand for 1 hour to allow settling of the red blood cells.
After exactly one hour, the ESR is read to the nearest millimeter, by observing the upper limit of
the red cell sediment. Whilst performing manually, the result recorded relied upon the
individual’s assessment of this upper limit, which is complicated when the red cells do not
separate evenly, resulting in a hazy appearance, which may occur when there is a high
reticulocyte count (Lewis et al, 2001).
Alifax Method
The measurement of the ESR via the Alifax requires minimal input from the operator, therefore
virtually eliminating human error of measurement. The racks are loaded into the instrument,
then rotated slowly for 2 minutes to provide adequate disaggregation of the sample which may
have settled between collection and processing. The blood is then drawn from the tube, via an
aspiration needle, into a capillary with samples separated by an air bubble of approximately
530nm. The sample is then subjected to centrifugation at 20g, and a photometer of wavelength
950nm measures the rate of sedimentation, taking one thousand readings during the 20 second
measurement period. The information, as electrical impulses, is collected by a photodiode
detector, and a mathematical algorithm is applied to convert the analysis of optical density into
an ESR result, equivalent to Westergren values (de Jonge et al, 2000; Cha et al, 2009). During
the verification, the Alifax is configured to print the results, however, once implemented in the
laboratory, the results can be sent directly to the laboratory information management system
‘TelePath’. Though the method of measurement employed is not officially recognized by the
International Committee for Standards in Haematology, calibrating the Alifax to produce results
on a par with the Westergren method allows for integration of the instrument within the
laboratory with confidence in the measurements produced.
Statistical Method of Assessing Agreement
To compare the variation in results between the old and new instrument, the Bland - Altman
method for assessing agreement between two methods of clinical measurement (Bland &
Altman, 1986) was used. This method allows for: a clear interpretation of any bias present (i.e.
is the Alifax consistently producing higher or lower results), observation of the differences

13. 13
between the measurement on the old instrument and new instrument and if there is any trend in
this difference (i.e. is the agreement better when measuring lower values?) and subsequent
statistical analysis to assess if the sample size was sufficient to truly represent the agreement.
Assessing the Precision of the Alifax (Intra-Assay Reproducibility)
The precision was tested by performing a reproducibility test, whereby 29 samples were
randomly selected and analysed 5 times by the Alifax in one session.
Results
Assessing agreement between methods
Figure 3 - Initial comparison of 79 ESR results from Alifax and StaRRsed Compact – Prior
to adjustment of the Alifax output (Trend line indicates perfect correlation between the two
instruments)
The data shows a consistent
bias, with the Alifax producing
higher results than the StaRRsed
Compact. From this initial
comparison, it was surmised that
the Alifax required an adjustment
factor in order to produce results
on a par with those produced by
the StaRRsed. Effectively this
moved all the data points down
or up by a percentage, affecting
higher values to a greater degree, with the aim of gaining a more even spread of the data points
around the line of agreement. After experimenting with this adjustment factor on the Alifax to
gain a tighter correlation around the line of agreement, the Alifax was compared to the
Westergren method for 137 samples. For all subsequent tests on the Alifax, and indeed when
the instrument was integrated into routine use in the laboratory, checking of this adjustment
StaRRsed Compact ESR (mm/hr)
AlifaxESR(mm/hr)

14. 14
factor was integrated into the Standard Operating Procedure (SOP). The adjustment factor is
included in the detailed print-out when the control samples are analysed at the beginning of
each day, and is described as the ‘Y factor = 0.8656’.
Figure 4 - Comparison of Manual Method (Westergren) vs Alifax ESR for 137 samples
This is a simple scatter graph to display the raw data collected. A clear interpretation of the data
cannot be made from this scatter plot alone, therefore the data was analysed using the Bland-
Altman method.
Westergren ESR (mm/hr)
AlifaxESR(mm/hr)

15. 15
Figure 5 - Evaluation of Manual (Westergren) vs Alifax ESRs using the Bland-Altman
Method for Assessing Agreement between Two Methods of Clinical Measurement for 137
samples
The Bland-Altman analysis of the 137 samples allows for an observation of the spread and bias
of the data, in addition to the degree of correlation.
The mean difference between the two instruments is 1.47mm/hr, with a standard error of
± 1.37mm/hr. The 95% confidence interval for the mean difference was calculated as
-1.22mm/hr to 4.16mm/hr. For the original 137 samples, 94% of the results lie within the limits of
agreement (mean ± 2S.D.), which are approximately -31mm/hr to 33mm/hr.
The data is then subjected to further analysis by removing the data points for all the samples
with a haematocrit level of less than 35.0, and removing samples for which the haematocrit data
was unavailable (i.e. no Full Blood Count had been performed). This reduced the sample size
down to ninety-eight.
(Westergren ESR + Alifax ESR) /2 (mm/hr)
Westergren–Alifax(mm/hr)
Mean difference
Mean + 2S.D.
Mean - 2S.D.

16. 16
Figure 6 – Comparison of Westergren vs Alifax ESR with Samples of Low Haematocrit
(<35.0) or Unknown Haematocrit Status Removed (98 samples).
Again, analysis of the data
cannot be made with
observation of the simple plot
of the raw data, therefore the
information is again
subjected to the Bland
Altman analysis.
Figure 7 - Evaluation of Manual (Westergren) vs Alifax ESRs using the Bland-Altman
Method for Assessing Agreement between Two Methods of Clinical Measurement, with
Samples of Low Haematocrit (<35.0) or Unknown Haematocrit Status Removed (98
samples).
The mean difference is now
0.45mm/hr with a standard
error or ±1.21mm/hr and a
confidence interval of 1.92 to
-2.82mm/hr.
The limits of agreement are
narrower than previous at
-24.4mm/hr to 23.5mm/hr,
with 90% of the data falling
within this 95% interval.
AlifaxESR(mm/hr)
(Westergren ESR + Alifax ESR) /2 (mm/hr)
Westergren–Alifax(mm/hr)
Westergren ESR (mm/hr)

17. 17
Intra-Assay Reproducibility
Figure 8 – Assessing the Precision of the Alifax: The mean of 5 repeat ESR readings
plotted against the individual 5 readings for the 29 samples.
The vertical spread for each mean reading describes the variation in results; the greater the
vertical spread, the greater the variation in results. Up to a mean reading of approximately
50mm/hr, the readings do not differ by more than 5mm/hr and in 4 cases the Alifax produced
the exact same result for all 5 runs. In only 3 of the samples did the standard deviation exceed
10mm/hr, these were all for samples which has a raised ESR of greater than 50mm/hr.
Mean of repeated readings (mm/hr)
Mean of repeated readings (mm/hr)
Individualrepeatreadingsforeachsample(mm/hr)

18. 18
Discussion
When the raw data for the comparison of 137 samples was plotted as a simple scatter graph,
some correlation at the lower end of the scale is observed, indicated by clustering of the data
points close to the line of agreement, though as the ESR values increase, this correlation
becomes less pronounced (Figure 4, pg.14). In this comparison, the data points are spread
relatively equally around the line of agreement, indicating the final adjustment value decided
upon was sufficient.
When the data was analysed using the Bland Altman analysis (Figure 5, pg.15), the mean
difference is 1.47mm/hr, the proximity of this value to zero indicates that the Alifax does not
display any significant bias; neither consistently producing higher or lower results than the
Westergren method. On observation of the actual spread of results, it is shown that as the ESR
mean value increases from 20mm/hr upwards, the difference between the measurements made
by the two instruments increases noticeably. If another set of samples were to be run, the
sample mean difference would undoubtedly vary by some degree. By determining the standard
error of the mean difference, an estimate can be made by how much this value may vary, to
indicate the proximity of the mean difference gained here to the true mean difference. The
standard error is calculated to be ± 1.37mm/hr, attributing to the beneficial large sample size,
and thus allows for an assurance in the validity of data obtained. The 95% confidence interval
was calculated at -1.22mm/hr to 4.16mm/hr, showing that even if the sample size were
increased further, the true mean difference between the two instruments would fall between
these values, which roughly centre on a difference of zero.
The limits of agreement indicate that it is possible for the Alifax to produce results approximately
30mm/hr higher or lower than the Westergren method, though this is a generalised observation
of all the data over the entire range of the ESR measurements. In reality, for ESRs below
20mm/hr this value, the limits of agreement would be significantly lower, showing a better
agreement between the two instruments as presented on the graph.
When the samples with low haematocrit or no haematocrit data were removed from the
analysis, the basic plot comparing the two methods displayed a distribution pattern similar to the
original comparison of 137 samples (Figure 6, pg.16). Bland-Altman analysis shows the majority
of data is more concentrated around the mean difference of 0.45mm/hr with a reduced standard

19. 19
error of ±1.21mm/hr and confidence interval of 1.92 to -2.82mm/hr, and narrower limits of
agreement (-24.4mm/hr to 23.5mm/hr), within which 90% of the data lies (Figure 7, pg.16).
Though, again, the difference in the measurements doesn’t really come into effect until about
20mm/hr, below which the measurements only differ by a few millimetres per hour maximum.
To expect perfect correlation between the two instruments would be unrealistic; as previously
detailed, the Westergren method of analysis is affected by a number of environmental variables
in addition to the haematocrit level, whilst the Alifax process claims to be unaffected by these
variables. However, despite this possible source of variation, the results of the comparison via
Bland Altman analysis has shown that, for the most part, the results produced by the newer
machine are on a par with the older machine, i.e. a normal result remains normal with only a few
millimetres difference at the most, whereas an elevated result indeed remains elevated to a
level of diagnostic or clinical importance. The statistical analysis of the data allows for
confidence that the mean difference gained is close to the true difference between the two
instruments, reflecting the strength of the verification process due to its large sample number.
The Alifax presented extremely accurate intra-assay repeatability, especially at the lower end of
the scale as shown by the reduced vertical spread of results for each sample (Figure 8, pg.17).
In realistic terms, it is unusual to have to repeat an ESR reading on the same patient sample,
though gaining concurrent results in up to 5 readings improves the confidence that the readings
being gained are precise. On the StaRRsed Compact, it was in fact previously impossible to run
a single sample more than once as the test used too great a volume of the sample to allow for a
second run.
Throughout the verification of the Alifax, it became apparent that daily checks must be made to
ensure the instrument remained properly calibrated and maintained. This allowed for
development of a ‘Daily Check Record’ as part of the Standard Operating Procedure (SOP), to
record the quality control results, adjustment ‘Y’ factor and T100 value which is used to ensure
the instruments analytical components were sufficiently clean.
The outcome of this assessment of accuracy and precision of the Alifax allows for installation of
the new instrument in place of the old StaRRsed Compact, with confidence that the new
instrument is calibrated effectively to reflect the Westergren values. However, despite this
conclusion, the results could not be released without informing the recipient physician of the

20. 20
instrument changeover, as the recorded ESR measurement will differ to previous values due to
influencing factors such as the haematocrit level. Release of results generated by the new
machine was instigated in conjunction with a memo that informed the recipient of the change in
measurement procedure, therefore implying that if an unexpected value was obtained, i.e. in the
case of using the ESR to monitor treatment for a condition, say Rheumatoid Arthritis, the doctor
be aware of this change and wait for a second result before instigating any regime change in
treatment. In terms of non-monitoring use of the ESR, it is again enforced that the ESR is not a
diagnostic tool in itself, and must be teamed with other laboratory results, physical exam and
assessment of presenting symptoms in the diagnosis of a medical condition.
Conclusion
The TEST 1 Alifax not only has better control of the environmental variables and improved
quality control measures, but also has greatly reduced manual input and processing times. The
older instrument required manual input of each sample and a processing time of one hour,
whereas the Alifax has drastically reduced the turnaround time; samples are loaded in racks of
up to 12 (or 15 if new racks are purchased) with a capacity for 4 racks, requiring a fraction of the
manual operator input time, and a processing time that means over double the number of
samples can be processed within a given time frame. Subsequently, the sample backlog is
prevented to a degree, and the laboratory is better able to deal with the pressure from an
increasing workload.
As with every facet of the working Haematology laboratory, a great emphasis is placed on the
economical impact of sample processing and instrument maintenance. Though purchase of the
new instrument itself required expenditure, the long term cost of the instrument more than
balances its expense. Unlike the predecessor ESR instrument, the Alifax has no solutions
required therefore there is no need for preparation of samples or reliance on stock availability,
the quality controls are cheaper, and the cost per sample is almost one tenth of the previous
price.

21. 21
In light of the data collected and the subsequent analysis, it was therefore found to be
acceptable to replace the StaRRsed Compact with the TEST1 Alifax in the Haematology
laboratory, with confidence that the new instrument provides accurate and reliable results.
The literature review concluded that, though the ESR only has a place as a diagnostic and
monitoring tool and cannot itself be used alone for diagnosis or as a screening tool, until
alternatives match the user requirements, (i.e. cheap, reliable and ease of use), the ESR
remains integral to the evaluation of inflammation. In addition to this, conditions for which the
ESR has potential use, apart from the traditional Rheumatoid Arthritis, Polymyalgia Rheumatica
and Temporal Arteritis, are constantly being considered and developed suggesting that it will be
many years before the ESR is phased out of routine laboratory use.
Acknowledgements
I would like to thank the Haematology Department at Ysbyty Gwynedd for giving me the
opportunity to do this project and providing an enjoyable work atmosphere. In particular, thanks
go to my project supervisor Robert Walters for his guidance throughout my project and time in
Haematology during my year at the hospital, and to Enid Lloyd Jones for her help whilst
completing the practical portion of this project and figuring out the programming instructions!
Thanks also to the Phlebotomy Department at Ysbyty Gwynedd for providing me with a surplus
of samples, even at short notice.
In addition I would like to thank Dr Pat Gadsdon and Merfyn Williams of Bangor University for
their help and support throughout my degree.

22. 22
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