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

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

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

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

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

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,

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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

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

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

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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 oC

Thermostat 37oC

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

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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

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

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)

Ali

fax E

SR

(m

m/h

r)

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)

Ali

fax E

SR

(m

m/h

r)

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)

Weste

rgre

n –

Alifa

x (

mm

/hr)

Mean difference

Mean + 2S.D.

Mean - 2S.D.

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.

Ali

fax E

SR

(m

m/h

r)

(Westergren ESR + Alifax ESR) /2 (mm/hr)

Weste

rgre

n –

Alifa

x (

mm

/hr)

Westergren ESR (mm/hr)

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.

Ind

ivid

ual

rep

eat

read

ing

s f

or

each

sam

ple

(m

m/h

r)

Mean of repeated readings (mm/hr)

Mean of repeated readings (mm/hr)

Mean of repeated readings (mm/hr)

Ind

ivid

ual

rep

eat

read

ing

s f

or

each

sam

ple

(m

m/h

r)

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

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

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

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

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