CHANGES IN PATTERN OF ERYTHROCYTE SEDIMENTATION RATE AND

C-REACTIVE PROTEIN FOLLOWING MAJOR ELECTIVE ORTHOPAEDIC

SURGERIES.

BY

EZEH, RICHARD CHUKWUNONYE

PG/M.Sc/O8/47768.

DEPARTMENT OF MEDICAL BIOCHEMISTY

UNIVERSITY OF NIGERIA, NSUKKA.

JANUARY, 2012

CHANGES IN PATTERN OF ERYTHROCYTE SEDIMENTATION RATE AND

C-REACTIVE PROTEIN FOLLOWING MAJOR ELECTIVE ORTHOPAEDIC

SURGERIES.

BY

EZEH, RICHARD CHUKWUNONYE

PG/M.Sc/O8/47768.

A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF

MEDICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA.

IN

PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

THE DEGREE OF MASTER OF SCIENCE (M.Sc)

IN

MEDICAL BIOCHEMISTRY

RESEARCH SUPERVISOR

PROFESSOR P.O. EGWIM

SPECIAL AREA OF STUDY

MEDICAL BIOCHEMISTRY

DATE: JANUARY 2012

CERTIFICATE

Mr. Richard Chukwunonye Ezeh, a Post-graduate student in the Department of Medical

Biochemistry majoring in Medical Biochemistry has satisfactorily carried out this

research work for the degree of Master of Science (M.Sc.) in Medical Biochemistry. The

work embodied in this thesis is original and has not been submitted in part or full for any

other diploma or degree of this or any other University.

SUPERVISOR…………………………………

P. O EGWIM, M.S, Ph.D

(PROFESSOR)

DEPARTMENT OF MEDICAL BIOCHEMISTRY

UNIVERSITY OF NIGERIA,

ENUGU CAMPUS.

DEDICATION

To God Almighty

To the memory of my late parents, Joseph and Catherine Eze-Nwaonyisi.

To my wife Pharm. (Mrs.) Ogechukwu Ezeh and our unborn children.

TABLE OF CONTENT

Acknowledgement ……………………………………………………………….i

Abstract…………………………………………………………………………..ii

CHAPTER ONE

INTRODUCTION…………………………………………………………………1

CHAPTER TWO

LITERATURE REVIEW…………………………………………………………8

2.1 Erythrocyte Sedimentation Rate (ESR)……………………………………..8

2.2 Normal values of ESR……………………………………………………..9

2.3 Factors affecting ESR……………………………………………………10

2.4 Methods of laboratory estimation of ESR………………………………..10

2.5 C-Reactive Protein (C-RP)…………………………………………………10

2.6 Methods of laboratory estimation of C-RP…………………………………12

2.7 Principle of high sensitivity (hs) ELISA Method………………………….12

2.8 Normal values………………………………………………………………..13

2.9 Pattern of changes of ESR and C-RP following major Elective

orthopaedic Surgeries…………………………………………………………..13

CHAPTER THREE

MATERIALS AND METHODS………………………………………………17

3.1 Sample collection……………………………………………………………18

3.2 Sample preparation…………………………………………………………..18

33 ESR estimation………………………………………………………………..19

3.4 C-RP estimation………………………………………………………………20

3.5 Stastical analysis………………………………………………………..21

CHAPTER FOUR

RESULTS 22

4.1 Sex distribution ………………………………………………………………23

4.2 Age distribution……………………………………………………………….24

4.3 Diagnosis/ Surgical procedure……………………………………………….25

4.4 Basal serum ESR levels……………………………………………………..26

4.5 Basal serum C-RP levels……………………………………………………..27

4.6 Pattern of changes of ESR following major elective

orthopaedic surgeries……………………………………….………… 28

4.7 Pattern of changes of C-RP following major

elective orthopaedic surgeries …………………………………………………….29

4.8 Comparative changes in serum ESR and C-RP levels following major elective

orthopaedic surgeries……………………………………………………………….30

CHAPTER FIVE 31

DISCUSSION 31

Conclusion …………………………………………………………………………37

Recommendation …………………………………………………………………..38

References …………………………………………………………………………39

APPENDIX 1A…………………………………………………………………….47

APPENDIX 1B……………………………………………………………………..49

APPENDIX 1C……………………………………………………………………..49

APPENDIX 1D…………………………………………………………………….50

APPENDIX 1E…………………………………………………………………51

APPENDIX 1F………………………………………………………………….51

APPENDIX 1G…………………………………………………………………52

APPENDIX 2A………………………………………………………………….63

APPENDIX 2B………………………………………………………………….64

APPENDIX 2C………………………………………………………………….65

LIST OF TABLES

Fig 4.4 Group statistics on ESR

Fig 4.5 Group statistics on C-RP

LIST OF FIGURES

Fig 4.1 Sex distribution

Fig 4.2 Age distribution

Fig 4.3 Diagnosis/ Surgical procedure

Fig 4.4 Basal serum ESR levels

Fig 4.5 Basal serum C-RP levels

Fig 4.6 Pattern of changes of ESR following major elective orthopaedic surgeries

Fig 4.7 Pattern of changes of C-RP following major elective orthopaedic surgeries

Fig 4.8 Comparative changes in serum ESR and CRP levels following major elective

orthopaedic surgeries.

ACKNOWLEDGEMENT

My very sincere gratitude and appreciation go to my supervisor Prof. P.O. Egwin for

making available to me his expert knowledge and experience. He was not just a

supervisor but a father in this project work.

My sincere thanks also go to Prof. P.O.J. Ogbunude, Prof. I. E. Ezeagu, Dr. F.E Ejezie,

Mrs. J.E Ikekpeazu, Dr. C.O. Ezeh and Mr. M. D. Ibegbu for their constant advice and

criticism at various stages of my work. The support of the entire non- academic staff and

post-graduate students of the Department of Medical Biochemistry U.N.E.C is also

warmly appreciated.

Furthermore, my sincere gratitude as follows-

To the management and entire staff of the National Orthopaedic Hospital, Enugu,

led by the Medical Director, Dr. C.B. Eze for their invaluable support in this

project.

To Mrs. Ngozi Sanni and entire staff of the Medical Laboratory Department of the

National Orthopaedic Hospital, Enugu for their support and assistance in the

various sample preparations and ESR estimation.

To Mr. E.J. A. Chukwukeje and Mrs. Chijioke Nnedimma who helped in typing

this work.

My acknowledgement cannot be complete without my dear wife Pharm. (Mrs.)

Ogechukwu Ezeh for providing me with the necessary conducive environment

needed for the completion of this work.

Mostly to the Almighty God, for the life, the good health, the strength and

capability to complete this work.

ABSTRACT

This study of pattern of changes of Erythrocyte Sedimentation Rate (ESR) and

C-Reactive Protein (C-RP) was a prospective observational study carried out on 23

selected Nigerian adults who underwent major elective orthopaedic surgeries at the

National Orthopaedic Hospital, Enugu, Nigeria.

Blood samples were collected from 23 patients (Males 10, Females 13) with a mean age

of 53.95 years, pre-operatively on Day 0 and post-operatively on Days 2,4,7 10, 14 and

42.

Blood samples were also collected from 21 adult volunteers (Males 9, Females 12) with

a mean age of 49.61years on one occasion as a negative control.

Erythrocyte Sedimentation Rate (ESR) estimation was by the Modified Westergreen

method while the C-RP assay was by the High Sensitivity (HS) ELISA technique.

The result showed a steep rise in both parameters (more than 300% constitutive levels)

within 2 days post-operatively.

The peak value for the mean serum C-RP level was attained on day 4 post operatively

with normalization to its pre-operative serum level on day 42 (P = 0.543), while the

mean serum ESR reached its peak on day 7 and remained significantly higher than its

pre-operative mean serum level even on day 42 post operatively (P = 0.003).

CHAPTER ONE

INTRODUCTION

A major surgery is defined as a procedure which given the locality, condition of patient,

level of difficulty or length of time to perform constitutes a hazard to life or function of

an organ or tissue (Mcgraw-Hill, 2002). Major surgeries are associated with various

changes in the neuro-endocrine and inflammatory cytokine system of the body.

Major orthopaedic surgical procedures are becoming more common in our society

because of

i. availability of the necessary expertise;

ii. increased awareness of the population;

iii. aging population with chronic joint diseases and osteoporotic fractures requiring

treatment;

iv. increasing number of traumatic fractures from road traffic accidents and other causes

of high energy injuries.

Most of these surgeries are performed with expensive implants and prostheses

made of metals and their alloys. These metals are foreign bodies which when infected can

lead to a disastrous outcome with attendant increased morbidity and mortality.

Early clinical diagnosis of deep surgical wound infection is made more difficult by the

normal adaptive response to trauma/surgery being mounted by the host in the immediate

post- operative period.

The incidence of post- operative infection has been reduced to the barest minimum. This

is due to:

i. proper patient preparation;

ii. strict observance of the principle of asepsis;

iii. good surgical techniques;

iv. judicious use of antibiotics;

Still there are few incidents of breakthrough infection requiring early detection and

appropriate treatment. Consequently, concerted effort is being made through the use

of Erythrocyte Sedimentation Rate (ESR), C-Reactive Proteins (C-RP), and other

acute phase proteins (eg Haptoglobulin, Fibrinogen), as a screening tool for early

detection of deep surgical wound infections. The ESR is a less sensitive and specific

marker of deep wound infection than C-RP; however, it represents a cheaper, readily

available and less demanding screening tool in a developing country such as ours.

Efforts at determining the pattern of changes of ESR and C-RP following major

elective orthopaedic surgeries form the basis and rationale for this research work.

ACUTE PHASE PROTEINS

The term “Acute Phase” refers to local and systemic events that accompany

inflammation (Tarik and David 2002) and Acute Phase Proteins are those proteins

which reflect a measure of acute phase response.

Local responses include vasodilation, platelet aggregation, neutrophil chemotaxis and

release of lysosomal enzymes. Systemic responses include fever, leukocytosis, and a

change in synthesis of acute phase proteins. Stimuli for the increased synthesis of the

proteins include many different forms of injury such as trauma, surgery, infection,

immunological reaction, thermal injury, hypoxic injury and malignancy.

Acute phase proteins are used clinically as an aid to clinical diagnosis ( Van

Leeuwen and Van Rijswijk,1994 ). Because the response is relatively non specific,

the clinical value of measuring their concentration in the serum is to assess the extent

of inflammation reflecting momentary disease activity (Tarik and David, 2002).

Similar to tumour markers, acute phase proteins may be used to monitor the course of

disease in response to therapeutic intervention.

There are other acute phase proteins besides C-RP. They include the

Transport proteins (haptoglobulin, ceruloplasmin, and α- 1- antitrypsin), Coagulation

proteins (fibrinogen, prothrombin ) and Complement Components (C3,C4,C5 etc)

These proteins are believed to play a role in the body’s response to inflammation. For

example C-RP can stimulate the classical complement pathway, and α- 1-

antitrypsin can neutralize certain proteases released during acute inflammatory state(

Murray, 2006).

What makes ESR and C-RP the markers of choice in monitoring acute phase reaction

is the fact that they increase in concentration in the serum compared to basal values.

Additionally, they have a relatively short lag time from the moment of stimulus, and

are cost effective, (Van Leeuwen and Van Rijswijk, 1994).

BIOCHEMISTRY/PHYSIOLOGY OF ESR

Classical acute phase proteins are usually produced by the hepatic cells in response to

stimulation by interleukin I and VI. These cytokines are released by macrophages at

the site of injury or inflammation. The acute phase proteins are known to increase or

decrease in the serum concentration by at least 25% in response to stimuli.

The high post operative (post trauma) level of fibrinogen is doubled after 48hrs and

quadrupled at 96hrs( Badoe , 2000). This increase has a positive correlation with

ESR, though not absolutely.

ESR MEASUREMENT

When well mixed anticoagulated blood is placed in a vertical tube, erythrocytes tend

to fall toward the bottom. It is a common haematology test which is a non specific

measurement of inflammation. The ESR is governed by the balance between pro –

sedimentation factors mainly fibrinogen and those factors resisting sedimentation,

namely the negative charge of the erythrocytes (zeta potential). The length of fall of

the top of the erythrocytes in a given interval is called the Erythrocyte Sedimentation

Rate (ESR). Although the test does not correlate absolutely with any of the plasma

protein fraction, however, it is commonly used as a non specific and indirect

measurement of inflammation.

When an inflammatory process is present, the high proportion of fibrinogen in the

blood causes red blood cells to stick to each other. The red cells form stacks called

“Rouleaux” which settles faster. Rouleaux formation can also occur in association with

some lympho-proliferative disorders in which one or more immunoglobins are secreted in

high amounts.

BIOCHEMISTRY/PHYSIOLOGY OF C-RP

C-Reactive Protein (C-RP) is synthesized by hepatocytes and is classified as an acute

phase protein on the basis of its increase in plasma concentration during trauma, infection

and inflammation.

Cytokines, particularly interleukin 6(IL -6) induce C-RP synthesis in the liver (Kragsjerg

and Homberg, 1995). These cytokines appear to work at the level of gene transcription.

C-RP is an α globulin with a molecular mass of approximately 110,000- 140,000 daltons

and it is composed of five identical subunits, which are non-covalently assembled as a

cyclic pentamer (Dowling an Cook, 1972). The clearance rate of C-RP is constant;

therefore its level in the blood is regulated solely by synthesis. This acute phase protein

acts as opsonin for bacteria, parasites and immune complexes, activating the classical

complement pathway( Foglar and Lindsey, 1998).

AIMS AND OBJECTIVES

The aims and objectives of this study are as follows:

1. Determine the basal serum values for ESR and C-RP using the 23 selected

patients and the 21 recruited volunteer members of staff of the NOH, Enugu.

2. Develop a normal pattern of changes in ESR and C-RP following major elective

orthopaedic surgeries at NOH, Enugu.

3. Possibly use the normal pattern of ESR and C-RP changes as an effective tool to

predict early post- operative surgical wound infections.

EXCLUSION CRITERIA

The following categories of patients were excluded from the study:-

1. Patients without given consent for surgery or commitment to fully participate

in the research;

2. Patient with evidence of acute or chronic infections;

3. Poorly controlled diabetic patients and those on immunosuppressants;

4. Patient with known malignancies;

5. Pregnant women or women on contraceptive drugs

6. Patient with pre -operative (day 0) ESR value of more than 50mm per 1st

hour.

All subjects in this study (Patients and Controls) did not satisfy any of the six exclusion

criteria listed above,hence their inclusion/recruitment into the study.

CHAPTER TWO

LITERATURE REVIEW

2.1 Erythrocyte Sedimentation Rate (ESR)

The Erythrocyte Sedimentation Rate (ESR) also called Sedimentation Rate, is the rate at

which red blood cells sediment in a period of 1 hour. It is a common haematology test

that is a non specific measure of inflammation.

It was invented by the Polish doctor Edmund Biernarcki in 1897 (also referred to as the

Biernarcki test). In 1918 the Swedish pathologist Robert Sanno Fahraeus developed the

same along with Alf Vilhelm Alberston Westergreen. Both are eponymonsly remembered

for the Fahraus – Westergreen test). They used sodium citrate anti-coagulated specimens

(ICSH, 1993).

Although it is frequently ordered, ESR is of limited use as a screening test in

asymptomatic patients. It is useful for diagnosing disease such as multiple myeloma,

temporal arteritis, polymyalgia rheumatica, various auto-immune diseases, systemic

lupus erythematosus, Rheumatoid arthritis and chronic kidney diseases. In many of these

the ESR value may exceed 100mm/hour. It is commonly used for the differential

diagnosis of Kawasaki’s disease and is also usually increased in some chronic infective

conditions, like tuberculosis and infective endocarditis.

It can also be used as a crude measure of response in Hodgkins lymphoma. Additionally

its levels are used to define one of the several possible adverse prognostic factors in the

staging of Hodgkins lymphoma. It has been used for the prognosis of non- inflammatory

conditions such as prostate cancer, coronary artery disease and stroke. The use of ESR as

a screening test in asymptomatic persons is limited by its low sensitivity and specificity.

2.2 Normal Values of ESR

Westergreen original normal values in mm/1sthr (men, 3mm and women, 7mm) made

no allowance for a person’s age (Westergreen, 1957). It was later discovered that ESR

values tend to rise with age and to be generally higher in women (Bottiger and Svedberg,

1967). Values are increased in states of anaemia (Kanfer and Nicol 1997) and in black

populations (Gillum, 1993). A similar study by Obi (1984 ) in Nigeria, confirmed that

ESR value is higher in females. Ingram (1961) and Dappa,(2002) demonstrated a higher

value of serum fibrinogen in blacks compared to the Caucasians. The ESR reference

ranges from a large population study by Wetteland et al.(1996) revealed wide sex and

age variations confirming the earlier studies of Bottiger and Svedberg (1967).

2.3 Factors Affecting ESR

ESR is affected by temperature, pregnancy, drugs (decreases with adrenal steroids and

non steroidal anti-inflammatory drugs) and smoking. The value is known to be elevated

in anaemia, acute and chronic infections, malignancies especially the lympho-

proliferative disorders, rheumatoid arthritis and other causes of tissue inflammations.

The value of ESR is reduced in polycythermia, sickle cell disease, hereditary

spherocytosis and congestive cardiac failure.

2.4 Methods of Laboratory Estimation of ESR

Different methods for the measurement of ESR exist in the literature. They include the

Wintrobe method, Cutter method, Lindau – Adams method, the Smith method and the

Fahraeus- Westergreen method, which is by far the commonest method in current use.

However the Modified Westergreen method is the benchmark method for ESR analysis

(Bradford-Hunsly et al. 2010). ESR unit of measurement is mm/1st hour.

2.5 C-Reactive Protein (C-RP)

C-RP was first discovered in 1930 by Tilet and Francis in the serum of patients with

pneumonia, but it was not actually isolated until 1941. It was named for its ability to bind

and precipitate the C-polysacharide of pneumococcal cell wall( Schultz and Arnold,

1990).

Early laboratory methods were only qualitative in nature until the late 1970s, when

significant advances in isolating C-RP and measuring to the picogram range were made

(Tarik and David ,2002).

C-RP is synthesized in the liver and is normally present as a trace constituent of serum or

plasma at levels less than 0.3mgldl ( Macy et al.,1997).

C-RP is one of the acute phase proteins, the serum or plasma levels of which rise during

general, non specific response to a wide variety of diseases. This includes bacterial

infections, acute phase of rheumatoid arthritis, abdominal abscesses and inflammation of

the bile ducts (Dowling and Cook, 1972).

C-RP may also be elevated in patients with Guillain- Bare syndrome and multiple

sclerosis, certain viral infections, tuberculosis, burned patients and surgical trauma (

Hedlund, 1947).

Although the detected elevated level of C-RP in the serum is not specific for any

particular disease, it is a useful indicator of inflammatory process ( Morley and

Kushner,1982). Its level rises in serum within 24hours to 48hours following acute tissue

damage, reaching a peak during the acute stage (approximately 1000 times the

constitutive level) and decreases with the resolution of inflammation or trauma (Kushner,

1991).

The assay of plasma/serum C-RP is a more reliable and sensitive indicator of the

inflammatory process than the ESR, which may be affected by physiological changes not

associated with the inflammatory process (Hind and Pepys, 1984) . The C-RP is a direct

and quantitative measure of Acute Phase Reaction.

2.6 Methods of Laboratory Estimation of C-RP

Different methods for the estimation of C-RP exist in the Literature. Current assay

methods including latex agglutuation, Nephelometry and Radio- Immuno Diffusion

(RID) have the general disadvantage of low sensitivity. However Enzyme Linked

Immunosorbent Assay (ELISA) method provide the highest sensitivity and specificity

(Roberts, et al. 2000); it is therefore the current method of choice. Additionally,

measurement of C-RP by high-sensitivity C-RP assays may add to the predictive values

of other cardiac markers (myoglobin, Creatine-kinase-MB, troponin I and T), which is

used to assess the risk of cardiovascular and peripheral vascular disease ( Ridker, 1998).

2.7 Principle of High Sensitivity (HS) ELISA Method

The C-RP hs ELISA is based on the principle of a solid phase enzyme-linked

immunosorbent assay (Votila et al., 1981). The assay system utilizes a unique

monoclonal antibody directed against a distinct antigenic determinant on the C-RP

molecule. This mouse monoclonal anti-C-RP antibody is used for solid phase

immobilization (on the microtiter wells). A goat anti-C-RP antibody is in the antibody –

enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react

simultaneously with the two antibodies, resulting in the C-RP molecules being

sandwiched between the solid phase and enzyme-linked antibodies. After 45 minutes

incubation at room temperature, the wells are washed with water to remove unbound

labeled antibodies. A tetramethylbenzidine (TMB) reagent is added and incubated for

20minutes, resulting in the development of blue colour. The colour development is

stopped with the addition of 1N HCI changing the colour to yellow. The concentration of

C-RP is directly proportional to the colour intensity of the test sample. Absorbance is

measured spectrophotometrically at 450nm.

2.8 Normal Values

The plasma level of C-RP in most healthy subjects are usually less than 1mg/L, with

normal value defined as less than 10mg/L.

2.9 Pattern of Changes of ESR and C-RP Following Major Elective Orthopaedic

Surgery

The normal pattern of changes of ESR and C-RP following major elective orthopaedic

surgeries has been studied by many workers. Also studied are deviation from the normal

pattern following some post- operative complications like wound infections.

In a serial ESR measurement done in 110 patients who underwent lumber spine surgery,

Johnson et al.(1991) demonstrated a rapid increase of ESR with a peak value on the

fourth post operative day. The values returned to normal for the majority of the patents

after 2weeks. The ESR values at the time of diagnosis for five patients with post-

operative deep wound infection were used for comparison. All patients with deep wound

infection had an ESR value exceeding the corresponding mean value (+2SD). They

concluded that ESR values showed a homogenous pattern with rapid increase and decline

after lumbar spinal surgery in patients without post-operative infection. In another

independent study by the same authors, serial serum C-RP and ESR serum estimations

were done in 45 patients who underwent uncomplicated anterior cruciate ligament

reconstruction. Analysis of the blood sample collected both pre-operatively and post-

operatively revealed a marked increase after surgery and peaking between the third and

the seventh post-operative days, with the latter showing a faster return to normal. They

concluded that C-RP could be used as a more accurate predictor than ESR for post-

operative septic complications if the blood level remained elevated (or unexpectedly

rises).

Mustard et al.(1987) conducted a study on 108 patients undergoing different

orthopaedic procedures.. Blood samples were collected every day from immediate pre-

operative period to post-operative day 14. The result showed that C-RP testing was very

predictive. They concluded that a normal C-RP response to surgery without secondary

rise may exclude the possibility of post-operative septic complications and that C-RP was

a better marker for post operative infection than fever, white blood cells(WBC) or ESR,

which are more easily affected by the surgical procedure itself.

In their study of 66 Patients who underwent various types of hip implant operations,

Okafor and MacLellan (1998) demonstrated a significant difference in the post-operative

ESR and C-RP values between infected and the non infected patients. In the normal

group the variations, for ESR, were significantly different for day 0 versus day 2 and day

0 versus day 7, but not for day 0 versus day 21. This reflected the process of

normalization of ESR by day 21 in the normal population. In the infected group the post-

operative values of ESR were significantly different from the day 0 value reflecting the

persistent inflammatory state. C-RP rose and peaked at day 2 in the normal group and by

the day 21, it had normalized. With respect to the infected sub-set, C-RP also peaked at

day two and then showed a persistent elevation on day 7 and 21.

Larsson et al. (1995) performed a prospective study focused on C-RP levels in 193

patients undergoing 4 types of uncomplicated elective orthopaedic surgery procedures.

All four procedures had a peak C-RP response 2 to 3 days after surgery, followed by a

biphasic rapid decline. In the first phase there was a rapid decline 3 to 5 days after

surgery. In the second phase there was a more gradual decrease until day 14 to day 21

after surgery. ESR values tended to be more variable, remaining elevated after 42 days.

The conclusion from the study was that the normalized C-RP response that followed the

typical biphasic response indicated an uneventful recovery.

Waleczek et al. (1991) had studied orthopaedic procedures in 108 patients in which

post-operative C-RP levels were compared to ESR, white blood cells, body temperature

and clinical symptoms. Normal patterns of C-RP levels were seen in 101 patients that had

uneventful outcome. Seven (7) patients that had atypical C-RP pattern all had wound

infections. The results for the other parameters studied were more variable and less

predictive.

In a retrospective study involving 373 patients who had undergone various joint

replacement arthroplasties of the hip and knee between 1989 and 1994, Niskanem et al.

(1996), demonstrated a maximum (peak) serum level of C-RP on day 3. They also

showed that the C-RP curves were identical for different ages and genders. Furthermore

blood transfusions, the use of antibiotics and the amount of bleeding during and after the

operations, had no effect on the serum C-RP levels.

Kalhor and Najd (2005) characterized the pattern of the post-operative changes with

respect to serum C-RP, ESR and WBC, using ninety-four (94) patients with normal

values for these parameters, pre-operatively. They found that extensive (major) surgeries

resulted in higher values for C-RP and ESR only; changes in WBC values were irregular,

and the factors of age and sex of the patients had no effect whatsoever.

Finally, it should be noted that quite a number of similar/related studies has been

carried out by other workers,( Song et al.(1997); Meyer et al. (1995); Schmidt and

Oramek (1990); Mok et al.; (2008)) . Taken together, the results obtained have clearly

shown that C-RP and ESR levels could serve as rapid indicators (markers) of post-

operative early infection. Furthermore, C-RP values were by far more sensitive,

predictive, reliable and applicable for a wide range of major, elective orthopaedic

surgeries in the early post-operative period.

CHAPTER THREE

MATERIALS AND METHODS

A prospective observational study in which samples from twenty-three(23) adult

Nigerian patients who underwent major elective orthopaedic surgeries at the National

Orthopaedic Hospital, Enugu were used.

Such major surgeries includes:

i. total joint replacement arthroplasty

ii. open reduction with internal fixation using plate and screws.

iii. spinal decompression

Twenty-one(21) study subjects made up of adult Nigerian subjects working with the

NOHE were also recruited as negative control.

The study was approved by the Hospital Ethical Committee.

All the 23 patients and 21 control adult subjects met the inclusion criteria.(i.e, they did

not meet the exclusion criteria listed earlier)

The patients (N = 23) underwent a series of ESR and C-RP level estimations pre-

operatively on day 0, and post operatively, on day 2, day 4, day 7, day 10, day 14 and day

42.

Samples from the 21 healthy subjects ( control group) were collected once and analysed

fresh together with the 161 frozen samples from the 23 patients.

3.1 Sample Collection

Five (5) ml of venous whole blood was collected on each occasion (from the antecubital

vein) for both ESR and C-RP using EDTA bottles and plain bottles respectively.

Samples were usually collected in the morning period.

Two (2)ml of whole blood was put in the EDTA bottle while three (3) ml was put into the

clean plain bottle

Each bottle was properly labeled with Names, Hospital number, Sex, Age and the Day

of collection.

3.2 Sample Preparation

ESR: The EDTA bottles containing 2ml of whole blood were placed on a roller mixer

(Denley Mixer 15).

C-RP: The whole blood sample in the plain tubes were centrifuged at a speed of 3000

rev/min(550g) for 5 minutes– using the Gallenkomp tube centrifuge.

The supernatant (serum) was then taken and transferred to another clean plain bottle

using a pasteur pipette. The plain tubes were then covered and frozen at a temperature of

-20 degrees Centigrade until the time of analysis.

3.3 ESR Estimation

The ESR estimation was done by the modified Westergreen method using (Dispette).

These estimations were usually done within 2hours after the collection of the samples.

Values were read in millimeter per 1st one hour.

Materials required for Modified Westergreen Method

1. Caped Dispette filling reservoirs containing sodium Citrate

2.Pasteur pipettes

3. Dispette tubes

4. Metal stand (Rack)

Procedure

The caps of the Dipette filling reservoir and the EDTA bottle from the Denley mixer

were removed. With the aid of Pasteur pipette about 1ml of mixed whole blood was

added into the reservoir to the fill line level as indicated before the cap is securely

replaced. The reservoir was gavity mixed by inversion ensuring that all the blood has

returned to the bottom section of the reservoir. The Dispette tube was inserted gently

through the reservoir membrane into the bottom of the reservoir, ensuring that the whole

blood rises up to the “O” level of the Dipette tube. The Dispette assembly (both reservoir

and tube) were placed upright (900) in a metal stand (Rack), away from direct sunlight.

Readings are done manually and recorded in milimeters at exactly one hour after setting

upright.

3.4 C-RP Estimation

All the frozen samples were taken to the reference laboratory (Lifecare Specialist

Medical Laboratory, Coal Camp, Enugu) where they were thawed and C-RP estimation

done using the DRG Diagnostic C-RP High Sensitivity (HS) ELISA method (DRG

instruments GmbH D-35039 Marburg, Germany). The C-RP estimation on the 21 fresh

samples from the volunteer healthy adults were done simultaneously with the thawed

samples of the patients.

Materials required for HS-C-RP assay

Antibody-Coated wells (microtitre wells coated with mouse monoclonal anti- C-

RP)

Reference standard set

HsC-RP Sample Diluent

C-RP Enzyme Conjugate Reagent

Tetramethylbenzidine (TMB) Reagent

Stop Solution (diluted hydrochloric acid (1N HCL) )

Distilled water

Micropipettes

Disposable pipette tips

Microtitre well reader

Absorbent paper

Graph paper

Test tubes

Assay procedure for HS-ELISA method

All the patients and control serum samples were diluted 100 fold in a set of test tubes

prior to use (5µl of serum was mixed with 495µl (0.495ml) sample diluent using a

micropipette. 10µl of C-RP standards, Diluted specimens and Diluted controls were

dispensed into the microtitre wells. A 100µl of C-RP Enzyme conjugate reagent was

added into each well. The solution was thoroughly and completely mixed for 30seconds

and incubated at room temperature for 45 minutes. The incubation mixture was removed

by flicking plate contents into a waste container. The microtitre wells were additionally

rinsed and flicked 5 times with distilled water. 100µl of TMB solution was dispensed into

each well and gently mixed for 5 seconds. It was further incubated at room temperature

for 20 minutes. The ensuring reaction was stopped with a 1N HCL. All the solution were

gently mixed until its colour changed from blue to yellow. The absorbance was measured

spectrophotometrically at 450nm using a microtitre well reader.

Values were read in milligram per litre (mg/L)

3.5 Statistical analysis

Patients data and records were collected manually

Data statistical analysis was done using the Statistical Package for Social Sciences

(SPSS) software for windows, version 15 (SPSS Inc, Chicago, Illinois, USA).

Result presentation was made in prose format, based on pie charts, bar charts/bar graphs,

regular graphs-all as figures. Additionally,Tables depicting raw data details and/or

statistical data analysis are presented as Appendices 1A, 1B, 1C, 1D, 1E, 1F- for

reference purposes.

CHAPTER FOUR

RESULTS

A total of thirty five patients who met the inclusion criteria as outlined above was

recruited into the study. Out of this number only twenty- three(23) patients constituting

65.71% of the study group completed the research at forty- two days post operative

period. Other patients either refused to continue with the investigation because of the

multiple venipunctures involved or were lost to follow up.

Blood samples were also collected from the twenty one (21) adult members of staff from

the National Orthopaedic Hospital Enugu who volunteered to participate in the research.

This constitute the negative control samples.

The major results of the various aspects of the study are presented in the following

Figures (Figures 4.1-4.8),pictorially and graphically. Additionally, relevant raw

data,embracing some Group Statistics, Mean ESR and C-RP values + SD,(with Standard

Error of Means) for the various sub-sets and parameters studied, are presented in

Appendices (`1A,1B,1C,1D,1E,1F on Pages 37-48).

4.1 SEX DISTRIBUTION

Figure 4.1: Sex distribution of patients/control

Of the twenty three patients who completed the research, thirteen (56.52%) were females

while ten (43.48%) were males. For the control group out of the twenty one members

recruited twelve (57.14%) were females, while nine (42.86%) were males.

4.2 Age Distribution

0

1

2

3

4

5

6

7

8

9

10

21-30 31-40 41-50 51-60 61-70 71-80

Figure 4.2: Age Distribution of Patients.

The age range of the patients varied from 24 to 80years with a mean age of 53. 95years.

For the Control group, the age range varied from 27 to 62 years with a mean age of

49.61years. Only 4 out of the 23 patients were below 30years of age while 2 patients

(8.7%) were above 71years of age.

Age Range in Years

No of Pts

4.3 Diagnosis/Surgical procedure

13.04, 13%8.79%

78.26

TJA ORIF LAM

Figure 4. 3: Profile of Diagnosis/Surgical Procedure

Out of the 23 patients admitted for surgery 18 patients (78.26%) had severe osteoarthritis

of the Hip/knee joints requiring total joint arthroplasty (TJA) of the affected joints, 3

patients (13.04%) had old fracture non union requiring open reduction and internal

fixation with plate and screws (ORIF), while the remaining 2 patients (8.79%) with spinal

pathology had laminectomy

4.4 Basal Serum ESR Levels:

Figure 4.4: Mean basal ESR values of both patients and control with reference to

sex.

Figure 4.4 shows the mean basal ESR values for different sexes and control.

The breakdown according to individual sex reveals a mean basal ESR values of

10.8mm/1st hr and 11.8mm/1

st hr for male patient and control respectively and 20mm/1

st

hr and 21.25mm/1st hr for female patients and control respectively.

4.5 Basal serum C-RP levels

Figure 4. 5: Mean basal C-RP values for both patients and control with reference to sex.

The mean basal C-RP values of both patients (test group) and control, with reference to

sex, are shown graphically in Figure4. 5.

The breakdown according to sex shows mean basal CRP levels of 11.65 and 7.887 mg/L

for male patient and control respectively and 7.63 and 3.05 mg/L for female patient and

control respectively.

4.6 Pattern of Changes of ESR following Major Elective Orthopaedic Surgeries

Surgical Procedures

Figure 4. 6: Pattern of Changes of ESR following Surgery

Figure 4. 6 shows the bar graph presentation of the pattern of changes of ESR following

surgery.

The serum ESR in the immediate post operative period shows a remarkable change in

pattern. It rises

from its mean basal serum level el of 16.00mm/1st hr to 51.84mm/1

st hr on Day 2, peaks

on Day 7 at 83.70mm/1st hr and gradually falls to 24.13mm/1

st hr on Day 42.

C

-

R

P

(

m

e

a

n

)

4.7 Pattern of Changes of C-RP following Major Elective Orthopaedic Surgeries

Figure 4.7: Pattern of Changes of C-RP following Surgery

The pattern of changes of C-RP following surgery is presented in bar graph form in

Figure 4.7.

The serum C-RP changes after major elective orthopaedic surgical procedure shows a

steep increase from its mean basal level of 9.38mg/L to 30.15mg/L on Day 2 and

31.37mg/L on Day 4 when it peaks.

This is followed by a steady decline in the serum CRP levels until Day 42 when it has

almost normalized to its pre-surgery serum level,(10.10mg/L).

4.8 Comparative Changes in serum ESR and C-RP levels following Major Elective

Orthopaedic Surgeries

0

10

20

30

40

50

60

70

80

90

Day 0 Day 2 Day 4 Day 7 Day10 Day 14 Day 42

ESR CRP

Figure 4.8: Comparative Plots of Changes in Serum ESR and C-RP

Mean ESR

and C-RP

The comparative changes in the pattern of serum ESR and C-RP following major elective

orthopaedic surgical procedures are presented in figure 8

The graph shows that the C-RP peaks faster on Day 4 and has almost completely

normalized by Day 42, (p value 0.543). The ESR on the other hand continues to rise

from its pre-injury level (Day O) to Day 7 when it peaks. It then starts a gradual fall

which is still significantly higher than its pre-injury levels at Day 42,(p value = 0.003)

CHAPTER FIVE

DISCUSSION

Out of the 35 patients who consented to the study, 23 patients (65.71 %) completed the

study. Five( 5)

patients got tired of the multiple venepuncture and opted out of the study while the

remaining 7 patients were lost to follow up.

Regarding sex distribution, 13 patients (56.52%) out of the 23 patients that

completed the study were females while 10 patients (43.48%) were males. This

distribution was just a chance occurrence when they were being recruited into the study.

Overall, it is known from the hospital statistics that more males present to our regional

centre for treatment than their females counterparts.

The age distribution of the patients range from 24 years to 80 years with a mean

age of 53.95 years. The highest concentration of patients; N=10(43.48%) was seen in the

7th

decade (61-70 years age group). Incidentally, all the patients in this group had total

joint replacement arthroplasty for degenerative osteoarthritis. Also, of note is that four (4)

patients (17.39%) were below 30 years of age. Three (3) out of the 4 patients below 30

years have sickle cell arthropathy of the hip joints requiring early total joint replacement

surgery, while the fourth patient had open reduction and internal fixation with plate and

screws for an old fracture non union. Interestingly, the majority of the patients (69.56%)

who participated in the study were above 50 years of age. This is not surprising

considering the fact that most of the patients who were recruited into the study presented

with degenerative osteoarthritis which is an age related disease.

Of the 23 patients who completed the study, 18 (78.26%) had severe osteoarthritis

of the hip and knee joints. The predominance of the patients with degenerative

osteoarthritis was because it was convenient for the research since the hospital was

operating on them in batches at the time of the study.

Of the 18 patients with severe osteoarthritis, 12 were of the hip joints while 6

affected the knee joints. The distribution was also a purely chance occurrence as more

hip surgeries were done in the Hospital, at the commencement of the programme than

knee replacement procedures.

The basal serum ESR level varies from 3mm/1st hr to 40mm/1

st hr with a mean

level of 16.00mm/1st hr and 17.71mm/1

st hr for the patients and control respectively. The

result showed a higher mean basal ESR values in females in both patients and control

groups. It is 10.80mm/1st hr and 20.00mm/1

st hr for the males and female patients

respectively, and 11.88mm/1st hr and 21.25mm/1

st hr for the male and female control,

respectively.

The significantly higher mean basal ESR levels in females compared to their male

counterparts is in keeping with earlier published works of Bottiger and Svedberg, (1967)

, Obi,(1984) and Wetteland et al.(1996).

From this result it could be clearly seen that there is no significant difference in

the mean basal serum ESR levels between the patients and Control(p=0.666)

The basal serum C-RP levels of the patients varied from 1.40 to 27.90mg/L with a mean

of 9.38 mg/L , and 1.2 to 28.8mg/L with a mean of 5.12mg/L for the control.

The result clearly shows that the mean serum C-RP level is significantly higher in

males, 11.65mg/L and 7.63mg/L for the male and female patients respectively and

7.89mg/L and 3.05mg/L for the male and female controls, respectively. The higher mean

serum C-RP level in males is in keeping with the works of Milcan et al. (2004) and

Yamada et al. (2001),both of which

are consistent with other cross sectional Japanese studies.

However, this finding contrasts with most published articles from the West. For

instance Shaffer (2001) and Harlan (2005) in a population based Dallas study, showed

that the median C-RP values were higher in females . One explanation for this higher C-

RP level in females is that the dysfunctional adipose tissue, as indicated by elevated

Triacylglycerol (TAG) levels, appears to accelerate inflammation in women compared to

men.

Especially noteworthy from this study is the higher mean serum C-RP levels in our

population( both patients and control irrespective of sex) compared to Literature Western

values. Again this finding is supported by the result of Harlan in the same population-

based Dallas heart study to determine whether C-RP distributions varied by sex and self

reported race. They showed that the median C-RP levels were significantly higher in

blacks than in whites (3.0mg/L vs 2.3 mg/L and in women than in men (3.3mg/L vs

1.8mg/L)

Put together both the Dallas and Japanese studies have shown that both sex and

race may affect the C-RP significantly. In addition, the Japanese study also demonstrated

the influence of environmental factors on serum C-RP levels

Also noted in our study was a significant difference in the mean serum C-RP

levels (unlike for the ESR) between the patients and control,( 9.35mg/L) and (5.12mg/L)

respectively( p=0.050) This observation may be due to some undiagnosed inflammatory

arthritis in some of the patients or some unknown consequences of the prolonged frozen

storage of the samples before analysis.

The changes in serum ESR following major elective orthopaedic surgeries present

an interesting pattern. The mean serum ESR level increased from its pre-surgery (Day 0)

level of 16.00mm/1st hr to 51.84mm/1

st hr on day 2. The mean serum ESR level peaked

on day 7 at 83.70mm/1st hr and by Day 42 has decreased to 24.13mm/1

st hr, still

significantly higher than its basal mean level (p=0.003). This significant rise and fall of

ESR following major surgeries is in keeping with the earlier works of Johnson et

al.(1991), Okafor and Maclellan,(1998), Larson et al.(1992). The acute rise and fall of

ESR following elective surgeries can be explained by the high post -operative( post

traumatic) level of serum fibrinogen which has a direct correlation with serum ESR level.

Also the reduction in the serum fibrinogen level with the resolution of the inflammatory

process within seven days explains the subsequent ESR peaking within 4-7 days and the

gradual normalisation within 42 days, in the absence of infection.

Similar to the profile seen with the ESR, the pattern of changes of serum C-RP

level following major elective orthopaedic surgeries is even more interesting. The mean

serum C-RP level also rises from its pre-surgery basal (Day 0) level of 9.38mg/L to

30.15mg/L on day 2. It continues to rise until day 4 when the peak level of 31.37mg/L is

reached. Its serum level subsequently falls to 10.10mg/L on day 42 almost back to its

basal level of 9.38mg/L (p=0.543). This acute rise and subsequent fall of serum C-RP

level is in keeping with works of Waleczek et al. (1991) and Niskanen et al. (1996)

The acute rise and subsequent peaking of C-RP on day 4, with gradual decline in

the serum level up to day 42, can be explained as follows. Following major

surgeries/trauma the serum mononuclear cells especially the macrophages are recruited

and activated to release increased levels of interleukin I, VI and even some amount of

tumour necrosis factor α.. These factors stimulate the liver to produce increased level of

Acute Phase Proteins including the C-RP. Subsequent resolution of inflammation

(absence of wound complications like, infections) leads to the normalization of serum C-

RP level.

The C-RP and ESR values rise from their mean basal levels, peak at day 4 and

day 7 respectively, and decline gradually with the C-RP values almost falling to their

pre-surgery level on day 42.

The initial rise and peaking of C-RP on Day 4 is faster than for ESR, and so

could be a better indicator of early post operative complications such as infections than

ESR (whose initial rise is also rapid, but with a slower and irregular fall).

CONCLUSION

1. Measurement of blood Erythrocyte Sedimentation Rate(ESR),and assay of serum

C-Reative Protein, C-RP (a typical acute phase protein),are both useful for

monitoring early post- operative complications such as surgical wound infections in

patients.

2. Both parameters rise steeply (more than 300% of their constitutive levels)

48hours after surgery.

3. ESR is an indirect qualitative test with a direct correlation with another acute

phase protein

( fibrinogen); C-RP assay, on the other hand, is a direct quantitative

measurement.

4. C-RP attains its peak faster ( day 4) compared to day 7, for ESR. Furthermore,

its fall is also more rapid and regular. Therefore C-RP could be a better

indicator/predictor of early post-operative complications, than ESR.

5. However, ESR is a cheaper and more readily available test in a developing

country such as ours.

6.Results of this study indicate as follows:

i. higher ESR values in females than in males (both patients and controls);

ii. higher C-RP values in males than in females (both patients and controls);

iii. higher C-RP values in our population compared to Western Literature C-RP

values.

RECOMMENDATION

1. Being a direct quantitative assay for Acute Phase Protein, the use of C-RP test

should be encouraged in our Hospital Laboratories.

2. There is however, a possibility of alterations of serum C-RP values if there was a

prolonged frozen storage of sample prior to assay.

3 . A large multi-center population based study to determine the mean/ median serum

levels of C-RP among Nigerians seems necessary.

4. Further research into the role of interleukins (particularly interleukin 1 and VI)

and tumour necrosis factor α, following trauma/surgery, should be vigorously pursued

in our sub- region.

REFERENCES

Badoe, E. A. (2000). – Metabolic response to trauma. In Principles and Practice of

Surgery, 3rd Edition: Ghana Publishing Company: pp 98.

Bottiger, L.E. and Svedberg, C. A, (1967). Normal ESR and age. Br. Med. J.. (2) : 85 -87

(ICSH): International Council for Standisation in Haematology(Expert Panel on Blood

Rheology) (1993). J.Clin.Pathol.46(3): 198-203

Bradford-Hunsley, B.S., Rebecca-Reiss, B.S.and Amanda-Miller L (2010)

Streck ESR-Vacuum tubes yield stable erythrocyte sedimentation rates with variable

temperatures.

Wikipaedia

Dappa, D. V. (2002). Haemorrheological parameters in some hypertensive Nigerians:

M.Sc Thesis, UNIBEN. Nigeria.

Dowling, P. and Cook, S. (1972). Immune events in demyelinating diseases : In

Wolfgang, F. Ellison, G W. Stevens, J G. and Andrew, J M. eds ; Multiple sclerosis.

Academic Press Inc. New York, pp 269-277

Foglar, C. and Lindsey, R.W. (1998). C-reactive protein in Orthopaedics: Orthopaedics.

21(6):687-691

Gillum, R. F. (1993). A racial difference in ESR : Journal of the National Medical

Association 85(i) 47 – 50, pmid – 8426384.

Harlan, M.K. (2005). C-RP levels vary by Sex and Race : Journal Watch Cardiology,

September 16.

Hedlund P. (1947). The appearance of acute phase proteins in various diseases: Acta

Med. Scand:128, (Suppl, 196): 579-601.

Hind, C.R.H. and Pepys, M.B. (1984). The role of serum C-reactive (CRP) measurement

in clinical practice: Int. Med.( 5):112-151.

Ingram, J. K. (1961). A suggested schedule for the rapid investigation of acute

haemostatic failure: J. Clin. Path. 41:521 – 534.

Jensen, C. K., Brandslurd, I., Sogaard, I. (1988). Lumbar disc surgery and variations in

C-reactive protein, erythrocyte sedimentation rate and the compliment split product C3d:

Acta Neurochir . 90: 42 – 44

Jönsson, B.O., Söderholm, R., Strömqvýst, B. (1991). Erythrocyte

Sedimentation rate after lumbar spine surgery. Spine 16: 1049-1050.

Kanfer, E. J. and Nicol, B. A. (1997). Haemoglobin concentration and erythrocyte

sedimentation rate in primary care patient: J Roy Soc Med : 90 (1): 16-8

Kalhor, M. and Najd, M.F.(2005). The cause of inflammatory factors after elective

orthopeadic surgeries . Journal of Iran University of Medical Science and Health

Services : 11(44 Special Issue 2) 1029-1034

Kragsbjerg, P. and Holmberg, H. (1995). Serum concentrations of interleukin 6, tumour

necrosis factor – a, and C-reactive protein in patients undergoing major operations: Eur.

J. Surg. 161:17-22

Kushner, I. (1991). “C-reactive protein in rheumatology:

“Arthritis Rheum. 34:1065-1068,

Larsson, S. Thelander, U. Friberg, S. (1992).

C –Reactive protein (C-RP) levels after elective orthopedic surgery:

Clin.Orthop. 275: 237-242.

Macy, E.M., Hayes, T.E., and Tracy, R.P. (1997)

Variability in the measurement of C-reactive protein in healthy subjects; Implications for

reference interval and epidemiological applications: Clin. Chem. 43;(1):52-58,.

Mcgraw-Hill Concise Dictionary of Modern Medicine @2002 by the Mcgraw-Hill

companies inc., 2002.

Meyer, B., Schaller, K., Rohde, V. and Hassler, W. (1995).

The C-reactive protein for detection of early infections after lumbar microdiscectomy:

Acta Neurochir: 136(3-4):145-150.

MILCAN, A. KANDEMIR, O. POLAT, G. BAGDATOGLU,C. COLAK,M.

KUYURTAL, F. (2004). Acute phase proteins after orthopaedic surgery. Journal of

Arthroplasty & Arthroscopic Surgery: 15 : (2) :85 - 89.

Mok, J.M., Pekmezci, M., Piper, S. L., Boyd, E., Berven, S.H., Burch, S., Deviren, V., Tay, B.

and Hu, S. S.(2008).

Use of C-Reactive protein after spinal surgery: Comparism with Erythrocyte

sedimentation rate as predictor of early postoperative infections complications: Spine 33

:(4): 415-421

Morley, J. J. and Kushner, I.. (1982). Serum C-reactive protein levels. In Kushner, I.,

Volanakis, J.E., and Gerwutz. Annals of N.Y. Acad. Sci. 389: 406-417.

Murray, R.K. (2006).Plasma proteins & Immunoglobulins. In Harpers Illustrated

Biochemistry : 27th Edition,:McGraw-Hill and Lange Publishing Company: pp591.

Mustard, R.A., Bohnen, J.M.A., Haseeb, S. (1987)

C-reactive protein levels to predict post-operative septic complications:

Arch. Surg ;122:69-73

Niskanen, R.O., Korkala, O. and Pammo, H. (1996).

Serum C-reactive protein levels after total hip and knee arthroplasty: J. Bone

Joint Surg. 78-B: 431-33.

Obi, G. O. (1984). Packed cell volume and Erythrocyte Sedimentation Rate in healthy

Nigerian adults:

Af.r J. Med. & Med. Sci. March – June 13 (1-2) ;1 – 6.

Okafor, B. and Maclellan, G.(1998). Acta Orthoppaedica Belgica, 64-1.

Ridker, P.M.(1998). Plasma concentration of C-reactive protein and the risk of

developing peripheral vascular disease: Circulation 97:425-428,

Roberts, W. L., Moulton, L and Law, T.C. ( 2001). Evaluation of nine automated High-

Sensitivity C-Reactive Protein Methods; Implications for Clinical and Epidemiological

Applications: Clin. Chem. 47: 461-468.

Schmidt, M.A.and Oremek, G. (1990). C-reactive Protein zur Erkennung postoperative

infektioser Komplikationen. Chirurg . 61:895 -899.

Schultz, .R. and Arnold, P.I. (1990).

“Properties of four acute phase proteins: C-reactive protein, serum amyloid A protein,

glycoprotein, and fibrinogen” Seminar in Arthritis an Rheumatism ; 20: 129-147,.

Shaffer, L E.T. (2009).

Analysis of factors affecting the sex differences in C-RP levels

Ph.D thesis. Ohio State University.

Song, K.S., Hang, C.H., Mni, P.W. and Cho, Y.L(1997).

The Changes in the rate of C-RP in orthopaedic surgery: J. Korean Orthopeadic

Association Jun 32(3) ;697-703.

Tarik, M.H.and David, H. K.(2002). C-Reactive Proteins and Erythrocyte

Sedimentation Rate in orthopaedics: The University of Pennsylvania Orthopaedic

Journal: (15): 13- 16.

Van Leeuwen, M., and vain Rijswijk, M.H. (1994). Acute phase proteins in

monitoring inflammatory disorders. Bailliere’s Clin Rheumatol 8:531-552.

Votila, M., Ruslanti, E. and Engull, E.(1981). Two site sandwich enzyme:

Immunol. Methods 42: 11-15.

Westergreen A, (1957). Diagnostic tests the erythrocyte sedimentation rate range

and limitation of the technique: Triangle 3(1); 20–5 pmo13455726.

Waleczek, H., Kozianka, J. and Everts, H. D. (1991). C-reactive protein zur

Fruherkennung postoperative Infektionen nach Knochenoperationen

Chirung;62:866-870.

Wetteland, P., Roger, M., Solberg, H.E and Wersen, O.H.(1996). Population

based ESR study in 3910 subjectively healthy Norwegean adults, a statistical

study based on men and women from the Oslo area: J. Intern. Med. 240 (3): 125 –

31

Yamada, S., Gotola, T., Natashima, Y., Kayaba, K., Ishikawa, S., Wayo, N.,

Nakamura, Y., Hoh, Y. and Kajii, E. (2001). Distributions of serum C-RP and

association with artherosclerotic risk factors in a Japanese population: Am. J.

Epidemiol. 153 :1183 -90.

APPENDIX 1A

Sex

Code Age Age group

ESR Day

0 CRP Day 0 ESR Day 2 CRP Day 2 ESR Day 4 CRP Day 4 ESR Day 7 CRP Day 7 ESR Day 10

CRP Day

10

ESR Day

14

CRP Day

14 ESR Day 42

CRP Day

42

1 25 1 4 1.4 5 32.6 9 22.2 15 5.6 9 1.7 8 1.6 5 1.5

1 36 2 19 27.9 43 30.1 50 31.2 56 31.2 48 30.4 50 32.1 16 29.8

1 45 3 4 9.8 20 28.1 70 30.4 65 27.2 62 25.8 54 23.9 28 7.8

1 60 4 3 18 5 31.2 9 32.5 15 32.4 9 32.8 8 33.4 4 22.1

1 61 5 7 5.5 60 32.5 100 32.4 121 32.1 78 19.5 50 27.4 18 12.1

1 62 5 15 25.2 42 32.6 39 32.4 72 32.7 65 30.2 45 14.2 22 12.1

1 64 5 13 6.5 58 31.3 72 32.1 95 29.4 65 24.1 64 23.3 36 10.2

1 65 5 22 11.6 91 32.1 130 32.6 88 32.7 68 22.2 45 15.4 25 12.2

1 70 5 5 3.1 32 32.3 74 32.5 89 32 75 32.1 50 20.2 12 8.4

1 72 6 16 7.5 41 30.4 61 30.5 70 27.8 70 26.2 50 22.4 30 10.1

2 24 1 12 3.5 28 7.3 50 32.2 55 31.5 47 32.2 45 30.2 20 4

2 24 1 28 13.2 73 32 92 32.9 110 31.4 110 20.3 118 18.1 45 14.2

2 26 1 15 6.2 54 30.2 74 34.8 85 32.7 80 26.1 65 20.1 25 6.5

2 46 3 24 1.9 43 30.1 70 32.6 75 28.1 75 27.8 76 28.9 36 4.6

2 51 4 11 1.5 75 32.6 115 32 120 30.3 107 27.7 103 27.5 20 5.5

2 54 4 20 8.4 60 31.1 80 31.9 90 29.5 76 23.2 60 21.2 12 8.5

2 56 4 5 4.5 25 31 64 31.8 45 30 50 28.8 30 18.2 8 1.7

2 61 5 20 12.1 57 30.2 75 30.6 90 28.1 72 27.2 62 25.6 16 11.2

2 62 5 25 3.5 80 31.1 95 31.2 94 30.2 68 24.3 71 23.4 20 12.4

2 65 5 32 18.1 70 29.7 105 30.2 108 31.7 98 31.9 83 31.7 40 26.5

2 65 5 28 18.1 100 31.4 122 32.4 150 30.8 125 30.4 97 20.1 27 2.6

2 67 5 26 3.4 90 32.4 110 32.4 125 29.4 103 28.5 129 31.6 30 4.1

2 80 6 14 4.8 40 31.1 55 27.7 92 28.2 70 17.4 53 17.7 60 4.1

APPENDIX 1B

Group Statistics

Sex Code 2222 N Mean Std.Deviation

Std. Error

Mean

ESR Day 0 and

control

male subject 10 10.8000 7.02060 2.22011

male control 9 11.8889 7.55719 2.51906

ESR Day 0 and

control

female subject 13 20.0000 9.13567 2.23607

male control 12 21.2500 10.18131 2.93909

APPENDIX 1C

Group Statistics

Sex Code 2222 N Mean Std.Deviation

Std. Error

Mean

CRP Day 0 and

control

male subject 10 11.6500 9.13567 2.88895

male control 9 7.8778 9.97607 3.32536

CRP Day 0 and

control

female subject 13 7.6308 5.86734 1.62731

female control 12 3.0500 1.11966 .32322

APPENDIX 1D

Variables Mean +2SD Std. error of

Mean

Mean

difference

t P value

ESR Day 0 16.00 + 8.8 1.83 - - -

ESR Day 2 51.83+ 26.30 5.48 35.83 -8.47 .000

ESR Day 4 74.83+ 31.86 6.64 58.83 10.21 .000

ESR Day 7 83.70+ 32.89 6.86 67.70 11.38 .000

ESRDay 10 70.87+ 27.86 5.81 54.87 11.40 .000

ESR Day

14

61.57 + 29.92 6.24 45.57 -8.90 .000

ESR Day

42

24.13 + 13.32 2.78 -8.13 -3.33 .003

APPENDIX 1E

Variables Mean +2SD Std. error of Mean T P value

Mean difference

CRP Day 0 9.38 +7.55 1.58 - - -

CRP Day 2 30.15+ 5.11 1.07 20.77 11.61 .000

CRP Day 4 31.37+ 2.41 .502 21.99 14.01 .000

CRP Day 7 29.35+ 5.46 1.14 19.97 12.39 .000

CRP Day

10

25.69 + 6.74 1.41 16.31 -9.43 .000

CRP Day

14

22.97 + 7.30 1.52 13.59 -6.78 .000

CRP Day

42

10.10 + 7.45 1.55 - 717 -.61 .543

APPENDIX 1F

Group Statistics

Group N Mean Std.Deviation

Std. Error

Mean

ESR Day 0 and

control

Day 0 23 16.0000 8.80 1.83

Control 21 17.24 10.12 2.21

ESR Day 0 and

control

Day 0 23 9.38 7.55 1.58

Control 21 5.12 6.82 1.49

APPENDIX 1G

Descriptives

N Mean

Std.

Deviatio

n

Std.

Error

95% Confidence

Interval for

Mean

Minimu

m

Maximu

m

Lowe

r

Boun

d

Upper

Bound

Lower

Bound

Upper

Bound

Lower

Bound

Upper

Bound

Lower

Bound

Upper

Bound

ES

R

Day

21-

30

year

4 14.7500 9.97914 4.98957 -1.1290 30.6290 4.00 28.00

0 s

31-

40

year

s

1 19.0000 . . . . 19.00 19.00

41-

50

year

s

2 14.0000

14.1421

4

10.0000

0

-

113.062

0

141.062

0

4.00 24.00

51-

60

year

s

4 9.7500 7.63217 3.81608 -2.3945 21.8945 3.00 20.00

61-

70

year

s

10 19.3000 9.04372 2.85988 12.8305 25.7695 5.00 32.00

71-

80

year

s

2 15.0000 1.41421 1.00000 2.2938 27.7062 14.00 16.00

Tota

l

23 16.0000 8.79566 1.83402 12.1965 19.8035 3.00 32.00

CR

P

Day

0

21-

30

year

s

4 6.0750 5.14028 2.57014 -2.1043 14.2543 1.40 13.20

31-

40

year

s

1 27.9000 . . . . 27.90 27.90

41-

50

year

s

2 5.8500 5.58614 3.95000

-

44.3395

56.0395 1.90 9.80

51-

60

year

s

4 8.1000 7.17914 3.58957 -3.3236 19.5236 1.50 18.00

61-

70

year

s

10 10.7100 7.67470 2.42695 5.2199 16.2001 3.10 25.20

71-

80

year

s

2 6.1500 1.90919 1.35000

-

11.0034

23.3034 4.80 7.50

Tota

l

23 9.3783 7.55453 1.57523 6.1114 12.6451 1.40 27.90

ES

R

Day

2

21-

30

year

s

4 40.0000

29.7433

5

14.8716

7

-7.3283 87.3283 5.00 73.00

31-

40

year

s

1 43.0000 . . . . 43.00 43.00

41-

50

year

s

2 31.5000

16.2634

6

11.5000

0

-

114.621

4

177.621

4

20.00 43.00

51-

60

year

s

4 41.2500

31.9830

7

15.9915

3

-9.6422 92.1422 5.00 75.00

61-

70

year

s

10 68.0000

22.2161

1

7.02535 52.1076 83.8924 32.00 100.00

71-

80

year

s

2 40.5000 .70711 .50000 34.1469 46.8531 40.00 41.00

Tota

l

23 51.8261

26.2966

5

5.48323 40.4546 63.1976 5.00 100.00

CR

P

Day

2

21-

30

year

s

4 25.5250

12.1927

2

6.09636 6.1237 44.9263 7.30 32.60

31-

40

year

s

1 30.1000 . . . . 30.10 30.10

41-

50

year

s

2 29.1000 1.41421 1.00000 16.3938 41.8062 28.10 30.10

51-

60

year

s

4 31.4750 .75443 .37722 30.2745 32.6755 31.00 32.60

61-

70

year

s

10 31.5600 1.00687 .31840 30.8397 32.2803 29.70 32.60

71-

80

year

s

2 30.7500 .49497 .35000 26.3028 35.1972 30.40 31.10

Tota

l

23 30.1478 5.10996 1.06550 27.9381 32.3575 7.30 32.60

ES

R

Day

4

21-

30

year

s

4 56.2500

35.8922

0

17.9461

0

-.8625

113.362

5

9.00 92.00

31-

40

year

s

1 50.0000 . . . . 50.00 50.00

41-

50

year

s

2 70.0000 .00000 .00000 70.0000 70.0000 70.00 70.00

51-

60

year

s

4 67.0000

44.1437

0

22.0718

5

-3.2425

137.242

5

9.00 115.00

61-

70

year

s

10 92.2000

27.3487

8

8.64844 72.6359

111.764

1

39.00 130.00

71-

80

year

s

2 58.0000 4.24264 3.00000 19.8814 96.1186 55.00 61.00

Tota

l

23 74.8261

31.8614

2

6.64357 61.0482 88.6040 9.00 130.00

CR

P

Day

4

21-

30

year

s

4 30.5250 5.65766 2.82883 21.5224 39.5276 22.20 34.80

31-

40

year

s

1 31.2000 . . . . 31.20 31.20

41-

50

year

s

2 31.5000 1.55563 1.10000 17.5232 45.4768 30.40 32.60

51-

60

year

s

4 32.0500 .31091 .15546 31.5553 32.5447 31.80 32.50

61-

70

year

s

10 31.8800 .87914 .27801 31.2511 32.5089 30.20 32.60

71-

80

year

s

2 29.1000 1.97990 1.40000 11.3113 46.8887 27.70 30.50

Tota

l

23 31.3696 2.40538 .50156 30.3294 32.4097 22.20 34.80

ES

R

Day

7

21-

30

year

s

4 66.2500

40.9013

0

20.4506

5

1.1669

131.333

1

15.00 110.00

31-

40

year

s

1 56.0000 . . . . 56.00 56.00

41-

50

year

s

2 70.0000 7.07107 5.00000 6.4690

133.531

0

65.00 75.00

51-

60

year

s

4 67.5000

46.6369

0

23.3184

5

-6.7097

141.709

7

15.00 120.00

61-

70

year

s

10

103.200

0

22.9434

1

7.25534 86.7873

119.612

7

72.00 150.00

71-

80

2 81.0000

15.5563

5

11.0000

0

-

58.7683

220.768

3

70.00 92.00

year

s

Tota

l

23 83.6957

32.8916

1

6.85837 69.4723 97.9190 15.00 150.00

CR

P

Day

7

21-

30

year

s

4 25.3000

13.1466

1

6.57330 4.3808 46.2192 5.60 32.70

31-

40

year

s

1 31.2000 . . . . 31.20 31.20

41-

50

year

s

2 27.6500 .63640 .45000 21.9322 33.3678 27.20 28.10

51-

60

year

s

4 30.5500 1.27671 .63836 28.5185 32.5815 29.50 32.40

61- 10 30.9100 1.58496 .50121 29.7762 32.0438 28.10 32.70

70

year

s

71-

80

year

s

2 28.0000 .28284 .20000 25.4588 30.5412 27.80 28.20

Tota

l

23 29.3478 5.45659 1.13778 26.9882 31.7074 5.60 32.70

ES

R

Day

10

21-

30

year

s

4 61.5000

43.4396

1

21.7198

1

-7.6221

130.622

1

9.00 110.00

31-

40

year

s

1 48.0000 . . . . 48.00 48.00

41-

50

year

s

2 68.5000 9.19239 6.50000

-

14.0903

151.090

3

62.00 75.00

51-

60

year

s

4 60.5000

41.4929

7

20.7464

9

-5.5246

126.524

6

9.00 107.00

61-

70

year

s

10 81.7000

20.2212

8

6.39453 67.2346 96.1654 65.00 125.00

71-

80

year

s

2 70.0000 .00000 .00000 70.0000 70.0000 70.00 70.00

Tota

l

23 70.8696

27.8638

0

5.81000 58.8204 82.9188 9.00 125.00

CR

P

Day

10

21-

30

year

s

4 20.0750

13.1783

6

6.58918 -.8947 41.0447 1.70 32.20

31-

40

year

s

1 30.4000 . . . . 30.40 30.40

41-

50

year

s

2 26.8000 1.41421 1.00000 14.0938 39.5062 25.80 27.80

51-

60

year

s

4 28.1250 3.94747 1.97373 21.8437 34.4063 23.20 32.80

61-

70

year

s

10 27.0400 4.33441 1.37066 23.9393 30.1407 19.50 32.10

71-

80

year

s

2 21.8000 6.22254 4.40000

-

34.1073

77.7073 17.40 26.20

Tota

l

23 25.6870 6.74377 1.40617 22.7707 28.6032 1.70 32.80

ES

R

Day

14

21-

30

year

s

4 59.0000

45.8766

5

22.9383

2

-

14.0000

132.000

0

8.00 118.00

31-

40

year

s

1 50.0000 . . . . 50.00 50.00

41-

50

year

s

2 65.0000

15.5563

5

11.0000

0

-

74.7683

204.768

3

54.00 76.00

51-

60

year

s

4 50.2500

41.1207

6

20.5603

8

-

15.1823

115.682

3

8.00 103.00

61-

70

year

s

10 69.6000

26.9328

4

8.51691 50.3334 88.8666 45.00 129.00

71-

80

year

s

2 51.5000 2.12132 1.50000 32.4407 70.5593 50.00 53.00

Tota

l

23 61.5652

29.9284

4

6.24051 48.6232 74.5072 8.00 129.00

CR

P

Day

14

21-

30

year

s

4 17.5000

11.8493

3

5.92467 -1.3549 36.3549 1.60 30.20

31-

40

year

s

1 32.1000 . . . . 32.10 32.10

41-

50

year

s

2 26.4000 3.53553 2.50000 -5.3655 58.1655 23.90 28.90

51-

60

year

s

4 25.0750 6.76923 3.38462 14.3036 35.8464 18.20 33.40

61-

70

year

s

10 23.2900 6.02429 1.90505 18.9805 27.5995 14.20 31.70

71-

80

2 20.0500 3.32340 2.35000 -9.8096 49.9096 17.70 22.40

year

s

Tota

l

23 22.9652 7.29643 1.52141 19.8100 26.1204 1.60 33.40

ES

R

Day

42

21-

30

year

s

4 23.7500

16.5201

9

8.26009 -2.5373 50.0373 5.00 45.00

31-

40

year

s

1 16.0000 . . . . 16.00 16.00

41-

50

year

s

2 32.0000 5.65685 4.00000

-

18.8248

82.8248 28.00 36.00

51-

60

year

s

4 11.0000 6.83130 3.41565 .1299 21.8701 4.00 20.00

61- 10 24.6000 8.85940 2.80159 18.2624 30.9376 12.00 40.00

70

year

s

71-

80

year

s

2 45.0000

21.2132

0

15.0000

0

-

145.593

1

235.593

1

30.00 60.00

Tota

l

23 24.1304

13.3154

2

2.77646 18.3724 29.8885 4.00 60.00

CR

P

Day

42

21-

30

year

s

4 6.5500 5.49333 2.74666 -2.1911 15.2911 1.50 14.20

31-

40

year

s

1 29.8000 . . . . 29.80 29.80

41-

50

year

s

2 6.2000 2.26274 1.60000

-

14.1299

26.5299 4.60 7.80

51-

60

year

s

4 9.4500 8.88050 4.44025 -4.6809 23.5809 1.70 22.10

61-

70

year

s

10 11.1800 6.42353 2.03130 6.5849 15.7751 2.60 26.50

71-

80

year

s

2 7.1000 4.24264 3.00000

-

31.0186

45.2186 4.10 10.10

Tota

l

23 10.0957 7.45248 1.55395 6.8730 13.3183 1.50 29.80

APPENDIX 2 A

APPENDIX 2B

APPENDIX 2C