ABSTRACT INTRODUCTION:  Arthritis is the initial manifestation of many joint disorder. The most common causes of arthritis are crystal,trauma and infection.Synovial fluid[S.F] analysis provides an easy, noninvasive option in differentiating inflammatory from noninflammatory arthritis.OBJECTIVES:The aim of this study is to determine whether S.F Adenosine deaminase (ADA) and high sensitivity C-reactive protein (hs-CRP) can be useful in differentiating inflammatory from noninflammatory arthritis. METHODS:A total of 56 patients with knee arthritis with joint effusion who were admitted in Vinayaka Mission Kirupananda Variyar Medical College and Hospital, Salem and those who attended the orthopaedic outpatient clinic during the period of October 2011 to March 2012 have been included  in this  study.  There were 14 Rheumatoid Arthritis (RA), 7 Crystal induced arthritis, 3 Septic arthritis and 32 Osteoarthritis (OA).RESULTS:Statistically significant difference in mean synovial fluid ADA and hsCRP concentration between inflammatory 35.77±3.95 IU/L, 19.69±4.46 µg/ml and noninflammatory arthritis 12.93±2.06 IU/L, 2.77±0.91 µg/ml (p values<0.0001, 0.0001) was observed. Statistically significant difference in mean synovial fluid ADA and hsCRP concentration was observed when rheumatoid arthritis (22.98±3.21 IU/L,17.54±4.23 µg/ml), crystal induced (24.23±3.34 IU/L, 19.47±3.77 µg/ml), septic arthritis (60.1±5.31 IU/L,22.07±5.40 µg/ml) were compared with osteoarthritis (12.93±2.06 IU/L,2.77±0.91 µg/ml)CONCLUSION:Synovial fluid ADA and hsCRP helps to differentiate inflammatory from noninflammatory arthritis

KEYWORDS:

Adenosine deaminase, hsCRP, Synovial fluid, Rheumatoid arthritis, crystal induced arthritis, Septic arthritis

INTRODUCTION:

Arthritis is the bread and butter of orthopaedic practice. A comprehensive diagnostic approach is essential for early diagnosis. Synovial fluid analysis helps in this aspect. Analysis of synovial fluid has long been recommended as a routine procedure to assist in the diagnosis of arthritis. Available synovial fluid biochemical tests like glucose, protein have limited value and lack specificity in differentiating inflammatory from non inflammatory arthritis18.

Adenosine deaminase and high sensitive C reactive protein of the SF are among the important biomarkers in recent studies in differentiating inflammatory from non inflammatory arthritis.   Studies have reported that S.F ADA was higher in inflammatory arthritis (RA, Crystal induced and septic arthritis) when compared to non inflammatory osteoarthritis. ADA and CRP level are indicators18 of immune system activation.

The present study was undertaken to estimate  ADA, hsCRP, total protein, glucose concentration and total WBC count in synovial fluid obtained from patients with rheumatoid arthritis, osteoarthritis, crystal induced arthritis, septic arthritis  and to assess the value of the investigation in differentiating between inflammatory and noninflammatory  arthritis.

MATERIALS AND METHODS

A cross sectional study was performed in the SF of patients with clinical evidence of knee arthritis and joint effusion who consented for arthrocentesis . The subjects were patients who attended the outpatient clinic and those who were inpatients in the department of orthopaedics at Vinayaka Missions Kirupananda Variyar Medical College and Hospital, Salem. The study was approved by the ethics committee of the Institute.Written consent was obtained from all the patients in this study.

The arthritis were diagnosed as RA, crystal induced arthritis, septic and OA based on physical examination, laboratory results and radiological findings. Synovial fluid was aspirated from the arthritic knee collected in sterile plain and heparinized tubes and analyzed for the following

SF was centrifuged with the speed of 1500 RPM supernatant was used for biochemical analysi

  1. SF hsCRP  was quantitated by ELISA method
  2. SF ADA quantitated based on the principle adenosine deaminase hydrolyses adenosine to ammonia and inosine, finally under alkaline medium blue indophenols complex is formed which is measured by  using  semiautoanalyser[microlab 300 – Merck]
  3. SF Total protein estimated by Biuret method and SF Glucose estimated by GODPOD method  were measured   using semiautoanalyser[microlab 300 -Merck]

II      a.   Gram staining done to see the presence of microorganism under light microscope,

b.   Presence of crystals using polarised microscope

c.   Microbiological SF culture were also done

III         SF  collected in sterile heparinised vial, centrifuged at 1500rpm and the smear was prepared      from the sedimentation part of centrifuged SF and  analysed  to determine the number of white blood cells.

STATISTICAL ANALYSIS:

Results are expressed as the mean ±SD. Statistical comparison between the two groups were carried out using student’s t test. P value less than 0.05 was considered significant.

DISCUSSION:

Arthrocentesis 10 is therapeutic in knee joint effusion as it provides pain relief, prevents stretching of the ligaments and eliminates a potent source of infection. The synovial fluid or pus aspirated, in addition to being therapeutic provides us with a diagnostic tool. The fluid analysis should include a cell count, gram staining, culture, examination of crystals, 8,14 ADA 3 and hsCRP  which helps in differentiating inflammatory from noninflammatory condition 18.

Adenosine deaminase is one of the key enzymes of the purine2 metabolic pathway17, catalyzing the irreversible deamination of adenosine to inosine22 and deoxyadenosine to deoxyinosine.22,17 During inflammatory reactions the enzyme is released into extracellular fluid7 and in serous effusion of different pathology the levels of ADA activity increases considerably. ADA exists in all nucleated cells and its concentration varies in different tissues. Thymus1 that contains T lymphocytes has major role in immune system contain highest level of  ADA enzyme activity 4, therefore determination of ADA in synovial fluid  also indicates lymphoreticular activities.

Adenosine is a purine nucleoside with tissue protective property against injuries. It has an anti-inflammatory function by decreasing proinflammatory cytokines6, increasing anti-inflammatory cytokines, cytokine modulation of macrophage and monocytes , endothelial cell inflammatory functions  and neutrophil adherance to fibroblast and adherence of neutrophil to endothelial cell.

Adenosine acting through A2 receptors, exerts a potent inhibitory effect on the synthesis of leukotriene B4 and thus contribute to its anti-inflammatory properties6. Adenosine, called a ‘retaliatory metabolite’because it is a regulatory autocoid 18 that is generated as a result of cellular injury or stress, interacts with specific G protein-coupled receptors on inflammatory and immune cells to regulate their function.

The leukocyte count in the S.F is helpful in distinguishing inflammatory from noninflammatory arthritis.

CRP a member of the pentraxin family of proteins.11 CRP  activates the classical complement cascade and mediates phagocytosis, but it is also capable of regulating inflammation12.   CRP is secreted by the liver 21 in response to a variety of inflammatory cytokines. Levels of CRP increase very rapidly in response to trauma, inflammation and infection and decrease rapidly with resolution of the condition. Thus, the measurement of CRP is widely used to monitor various inflammatory states. In our study statistically  significant increase  was seen in ADA and hsCRP between inflammatory and in noninflammatory arthritis.

The results of Yuksel et al.,4 also showed significant increase of ADA in RA (21.5±8.4 IU/L) than in OA (9.7±4.9 IU/L).

The study of Nakamachi et al., showed that mean ADA concentration in  the synovial fluid of patient with RA  was higher than in OA17, Similar results  were found in the study of Seung young Kim et al.,5

Batool Zamani et al., 22. showed significant raise in  SF ADA  and  hsCRP in inflammatory arthritis[26.06±8.96 IU/L and 12.72±9.25µg/ml] when compared to noninflammatory arthritis[14.8±2.79IU/L and 2.36±2.7µg/ml].

Finding of this study correlates well with that of Batool zamani et al.,22 .,where mean of SF ADA and hs-CRP activity were increased in inflammatory arthritis (35.77 ± 3.95 IU /L, 19.69 ± 4.46 µg/ml) when compared to noninflammatory arthritis (12.93±2.06 IU/L, 2.77±0.91 µg/ml). SF ADA and hsCRP concentrations were higher in septic arthritis9,16 when compared to other arthritis. SF ADA was  significantly raised in septic arthritis when compared to RA and crystal induced arthritis, but SF hsCRP concentration did not differ significantly when septic arthritis were compared with crystal induced and RA. SF ADA concentration helps in differentiating various inflammatory arthritis but hsCRP does  not differentiate the inflammatory arthritis from each other.

SF protein19 and glucose did not show significance in differentiating inflammatory from noninflammatory arthritis.

 

 

RESULTS

Totally 56 patients were included in this study. Noninflammatory OA group comprised of 32 patients. Inflammatory arthritis comprised of 24 patients which includes 14 RA, 7 crystal induced arthritis  and 3 septic arthritis.

Table.1 shows ADA ,hsCRP, total  protein  and glucose in the synovial fluid of different types of arthritis. Patients with inflammatory arthritis shows statistically significant increase in mean SF ADA and hsCRP [35.77±3.95 IU/L, 19.69±4.46 µg/ml  ] when   compared  with patients of  noninflammatory arthritis[12.93±2.06 IU/L, 2.77± 0.91 µg/ml  ][P values<0.0001,0.0001]

Figure 1  and  figure2  shows the concentration of ADA and hsCRP in synovial fluid of different arthritis respectively.

Statistically significant difference was observed in mean SF ADA  and  hsCRP  concentration   of  RA, crystal induced and septic arthritis when compared with OA [P<0.0001,0.0001,0.0001,0.0001,0.0001,0.0001]. Statistical significant difference was observed for SF  ADA concentration between septic and crystal induced arthritis, also between septic arthritis and RA[P<0.0001,0.0001].There was  no statistical significant difference between SF hsCRP concentration between septic and crystal induced arthritis, between RA and crystal induced arthritis and  also between septic arthritis and RA [P=0.40,0.31,0.12] . Total   WBC count of more than 2000 cells per cumm of  SF were observed in inflammatory arthritis and total WBC were less than 2000 cells per cumm  of SF in noninflammatory arthritis,which correlates well with the findings of H.Amer et al., and chokkalingam et al. 13,15

There was no statistical significant difference in mean SF total protein and glucose concentration between inflammatory (4.06±1.8g%, 66.33±8mg%) and noninflammatory arthritis (3.6±0.5g%,74±19mg%) [p values= 0.16,0.07]

This study showed positive correlation between ADA, hsCRP and WBC count. This documents that ADA and hsCRP concentration in tissues rises when immune system is activated  in response to infection or inflammatory reactions.

CONCLUSION:

Synovial fluid ADA is a biomarker in differentiating inflammatory from non inflammatory arthritis and it also helps to differentiate septic from rheumatoid and crystal induced arthritis. Further studies on comparison of synovial fluid ADA and hsCRP with serum  ADA and hsCRP of different arthritis with a large sample size will throw more light on differentiating arthritis.

 

 

 

 

 

 

 

 

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  4. H Yuskel and T F Akoglu. Serum and synovial fluid adenosine deaminase activity in patients with rheumatoid arthritis, Osteoarthritis and reactive arthritis. Annals of the Rheumatic Disease.1988; 47:492-495
  5. Seung Young Kim,MD., et al., Synovial fluid adenosine deaminase activity in the patients of Rheumatoid Arthritis, Osteoarthritis, Ankylosing Spondylitis and Gouty Arthritis.1995; 2(1)46-51
  6. E. Krump, G. LemayetalAdenosine A2 receptor-induced inhibition of leukotriene B4

synthesis in whole blood ex vivo, British Journal of Pharmacology.1996; 117: 1639-1644

  1. Gyorgy Hasko et al., Adenosine Receptor Agonists Differentially Regulate IL-10, TNF- , and nitric oxide production in RAW 264.7 Macrophages and Endotoxemic Mice. Journal of immunologyr.1996;157:4634-39
  2. Ma DT, Carroll GJ. Monoarthropathy. Could this be infection? Aust Fam Physician.1998 Jan-Feb; 27(1-2):28-31
  3. Soderquist B et al., Bacterial or crystal-associated arthritis? Discriminating ability of serum inflammatory markers. Scand J Infect Dis.1998;30(6):591-6

10.  Simon H Till, M L Snaith. Assessment, investigation, and management of acute monoarthritis, JAccid Emerg Med.1999;16: 355-361.

11.  Du Clos TW. Function of C – reactive Protein. Ann Med.2000;32(4):274-8

12.  Mortensen RF. C reactive protein, inflammation and innate immunity. Immunol Res. 2001, 24(2):163-76

13.  H.Amer et al., The utilization of synovial fluid analysis in the UK. Rheumatology.2001;40:1060-1063

14.  A Swan et al., The value of Synovial fluid assays in the diagnosis of joint disease: a literature survey. Ann Rheum Dis.2002; 61:493-498

15.  Chokkalingam siva,M.D., et al., Diagnosing Acute Monoarthritis in adults: A practical Approach for the Family Physician. American Family Physician.2003;68(1):83-90

16.  Levine MJ et al., Assessment of the test characteristics of C-reactive protein for septic arthritis in children. J pediatr Orthop. 2003 May-Jun 23(3); 373-7.

17.  Yuji Nakamachi et al., Specific increase in enzymatic activity  adenosine deaminase 1 in rheumatoid synovial fibroblasts Arthritis & Rheumatism. March 2003; 48: 668-674

18.  Gyorgy Hasko 1,2 and Bruce N. Cronstein3 adenosine; an endogenous regulator of innate immunity.Trends in immunology.2004;25:33-39

19.  Sangeeta B Kulkarni et al., Role of synovial fluid analysis and synovial biopsy in joint diseases. Bombay Hospital Journal.Oct 2004; 46: No.04

20.  Khalid Hassanein et al., Role of Adenosine Deaminase (ADA) in the diagnosis of pulmonary tuberculosis. Egyptian Journal of Bronchology. 2010;4(1):11-18

21.  Praveen Hariharan et al., Sensitivity of Erythrocyte Sedimentation Rate and C reactive protein or the exclusion of septic arthritis in emergency department patients. The Journal of Emergency Medicine.2011;40(4):428-431

22.  Batool Zamani et al., Synovial fluid adenosine deaminase and high sensitivity C reactive Protein activity in differentiating monoarthritis. Rheumatol Int. 2012; 32:183-188

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1. Synovial fluid ADA, hsCRP, Glucose and Total Protein  concentration in

arthritis patients

PARAMETERS

NON INFLAMMATORY ARTHRITIS

INFLAMMATORY ARTHRITIS

OA

RA

CRYSTAL INDUCED ARTHRITIS

SEPTIC ARTHRITIS

ADA (IU/ L)

12.93±2.06

22.98±3.21

24.23±3.34

60.1±5.31

hs-CRP (µg/ml)

2.77±0.91

17.54±4.23

19.47±3.77

22.07±5.40

Glucose(mg%)

74.0±19.0

65.0±9.0

68.0±7.0

66.0±8.0

Protein (g%)

3.6±0.5

4.1±1.3

4.5±3.3

3.6±0.8

 

 

 

 

 

 

 

 

Fig 1.Synovial fluid ADA Concentration in different types of arthritis.

 

 

Fig 2. The Synovial fluid hs-CRP Concentration in different types of arthritis.

 

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