This document summarizes various laboratory investigations used in rheumatology. It discusses tests such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), uric acid, rheumatoid factor, anti-CCP antibodies, HLA-B27, antinuclear antibodies (ANA), and antineutrophil cytoplasmic antibodies (ANCA). It provides the clinical utility, interpretation, and limitations of these tests for evaluating rheumatic conditions like rheumatoid arthritis, ankylosing spondylitis, and vasculitis. The document emphasizes that laboratory tests should be used in conjunction with clinical history and examination for accurate diagnosis.

1. Laboratory Investigations
In Rheumatology
Dr.Amol S. Raut
MBBS.MD.Medicine
Fellowship in Rheumatology
Consultant Rheumatologist
Relief Arthritis, Rheumatology
and Dental Clinic
Aurangabad

2. Content
• Introduction
• Acute phase Reactants- ESR and CRP
• Uric Acid
• RA and Anti-CCP Antibody
• HLA-B27
• Antinuclear Antibody (ANA)
• ANA Blot
• ANCA
• Miscellaneous Investigations

3. Three Things To Remember
1. What you are expecting from the particular
investigation?
2. What action you are going to take once the reports
are available?
3. Can’t replace through history taking (80%)and
clinical examination (15%)

4. Systematic is always better….
1. Basic Investigations
2. Specific Investigations to reach the diagnosis
3. To know the disease extent/complications/damage

5. In Mathematics
2+2= 4
In Rheumatology…..
???

6. ESR
• Erythrocyte Sedimentation rate is simply the speed at
which RBCs fall when the blood is taken in a tube
• An indirect screen for elevated concentrations of acute
phase proteins
• Westergreen Method is the gold standard

7. • The usual accepted upper limits
Men-15 mm/hr
Women- 20 mm/hr
• A simple formula for calculating the upper limit of
normal ESR at any age has been used regularly:
Men - Age in years divided by 2
Women- Age in years plus 10 divided by 2

8. Noninflammatory Conditions of Raised ESR
• Aging
• Female sex
• Obesity
• Pregnancy
• Anaemia
• Diabetes
• End-stage renal disease
• Heart disease
• Alterations in number (polycythemia), size
(microcytosis), or shape (spherocytosis, sickle cell)

9. • Markedly elevated ESR (>100 mm/hour)
• Infection, bacterial
• Connective tissue disease: giant cell arteritis,
polymyalgia rheumatica, SLE, other vasculitides
• Malignancy: lymphomas, myeloma
• Markedly low ESR (0 mm/hour)
• Afibrinogenemia/dysfibrinogenemia
• Agammaglobulinemia
• Extreme polycythemia (hematocrit > 65%)
• Increased plasma viscosity

10. C-Reactive Protein
• Plasma C-reactive protein is synthesized by hepatocytes.
• After an acute inflammatory stimulus, CRP concentration
increases rapidly and peaks at 2 to 3 days at levels that
reflect the extent of tissue injury.
• If the stimulus is removed, serum CRP levels drop rapidly,
with a half-life of roughly 19 hours.
• Persistent elevations in CRP - chronic inflammatory
states
eg. active RA, pulmonary tuberculosis, or extensive
malignant disease

11. • Normal value : less than 0.5 mg/dL
• Greater than 1 mg/dL reflect clinically significant
inflammatory disease.
• 1 to 10 mg/dL - moderate increase
• Greater than 10 mg/dL - marked increase

12. • No uniformity in reporting concentrations has been
noted between laboratories, and values can be
conveyed in mg/L, μg/mL, or mg/dL.
• Similar to ESR, population studies show a skewed,
rather than Gaussian distribution, leaving parametric
statistical tests inappropriate for interpretation of
CRP data.
Limitations of CRP

13. Acute Phase Reactants In The
Management Of Rheumatic
Diseases
(1) In evaluating the extent or severity of inflammation
(2) In monitoring changes in disease activity over time
(3) In assessing prognosis

14. Rheumatoid Arthritis
• ESR & CRP - 45% of patients may have normal serum
levels at presentation
• ESR values have been found to remain stable over
the years and CRP levels correlate better with
disease activity.
• Effective disease-modifying antirheumatic drug
therapy decreases CRP by about 40%.

15. Prognostic value in RA
• Elevated acute phase reactant levels are associated
with early synovitis and erosions
• Predict radiographic progression
• Correlate with work disability on long-term follow-up
and predict progression to major joint replacement.
• Elevated CRP levels : death from cardiovascular
diseases

16. Systemic Lupus Erythematosus
• Many (such as patients with nephritis) show mild or
no elevation in CRP during periods of activity
• Subsets of patients, such as those with serositis or
chronic synovitis may have high CRP.
• In contrast, ESR correlates with disease activity and
accrued tissue damage in SLE.

17. Ankylosing Spondylitis
• AS does not lead to a substantial increase in ESR or CRP.
• The ESR and CRP values are lower in patients with AxSpA
than in those with RA, are elevated in only 50% to 60% of
patients
• Patients with elevated CRP levels are likely to respond
better to TNFi treatment with average decrease of 75% in
CRP concentration after 12 weeks.

18. Case
• 67/F
• Polyarticular pains with swelling
• B/L symmetrical, gradual onset
• Small joints of hands, knees, shoulders, low back
• RF- 28U (>20IU)
• MTx, HCQ
• No response

19. • Mechanical pains
• DIP
• ‘H’ and ‘B’ nodes
• Diagnosis : Osteoarthritis
• Advise : stop DMARDs, Physiotherapy SOS
analgesics

21. RHEUMATOID FACTOR
• RFs are a family of autoantibodies that recognize antigenic
determinants on the Fc portion of IgG.
• Major RF species - IgM isotype, IgG-RF and IgA-RF less
frequent
• A transient increase in IgM-RF is part of the normal immune
response that occurs during infections, triggered by immune
complexes containing microbial antigens
• The physiologic role of RF during a normal immune response is
to enhance the avidity and size of immune complexes, thereby
improving clearance of the immune complexes

22. Methods Used
• Latex agglutination – Interobserver Variability
• Nephalometry or Turbidimetry – reproducible
• ELISA – More sensitive than the above two and can
detect other isotypes

23. Causes Of Positive Test For RF
• Rheumatoid arthritis
• Sjogren’s syndrome
• Vasculitis such as polyarteritis nodosa
• Sarcoidosis
• Systemic lupus erythematosus
• Cryoglobulinemia
• Chronic liver disease
• Infections- tuberculosis , bacterial endocarditis, infectious
mononucleosis, leprosy, syphilis, leishmaniasis.
• Malignancies
• Old age(5% women aged above 60)

24. • Low-titre IgM-RF can be found in 10% to 15% of healthy individuals,
whereas chronic persistence of high-affinity IgM-RF at elevated
titre, as well as the presence of IgG and IgA subtypes, is a
characteristic feature of RA
• At the commonly used cut off value of 15 or 20 IU/mL, IgM-RF
shows only moderate specificity
• Specificity increases at higher titre, and RF above 40 or 50 IU/mL
(high-titre RF) has been found to be quite specific for RA.

25. Role As Diagnostic And Prognostic Marker
• Sensitivity - 60% to 90% in patients with established disease,
and in early RA it ranges between 50% and 60%
• Specificity – 48-92% (IgM isotype Specific for RA, igG and IgA
in the absence of IgM isotype- Other CTDs)
• High Titre Positivity suggests aggressive articular disease,
increased radiographic damage, increased extra-articular
manifestations
• Patients with RF positivity show better response with
rituximab treatment whereas IgA subtype shows decreased
response to TNFi

26. Bursting
The Myths
• Myth 1 : All RF positive patients have Rheumatoid arthritis
• Fact : Low titre positivity in normal individuals, in other CTDs
and many other conditions
• Myth 2 : All the first line relatives with RF will develop RA
• Fact : May remain positive lifelong without getting joint
symptoms
• Myth 3 : Doing serial RF levels for disease activity monitoring
• Fact: RF levels fluctuate independent of disease activity, serial
titres measurements has no role

27. ANTI–CITRULLINATED PROTEIN/
PEPTIDE ANTIBODIES(ACCP/ACPA)
• IgG Antibody directed against citrullinated proteins
• With high sensitivity (approximately 60% to 70%) and high
specificity (approximately 95%), the CCP2 assay (method of
detection is now considered a “gold standard” diagnostic test for
RA
• Presence of ACPA pertains more erosive disease and Extraarticular
manifestations such as scleritis, sicca sympoms, Interstitial lung
disease
• Monitoring of ACPA ( and RA) titres is of no use as the titres
doesn’t correlate with disease activity

29. Antistreptolysin O- ASO
• Antistreptolysin O produced in response to infection with
group A Streptococci (GAS)
• A rise in ASO titres suggests preceding GAS infection
• Limitations
1. As per revised Jones criteria detection of GAS
infection is helpful but does not confirm ARF
2. Healthy School going children have 200-300 todd
units of ASO titres as GAS tonsillopharyngitis is
common in this age group
3. False positives
Hence not routinely recommended

30. Case
• 30/M
• Left 3rd toe pain and Swelling since 2 months
• No other complaints
• Uric acid - 8.1
• Treated as Gout
• No relief

31. • On through history and examination
• H/o excess dandruff and scaly plauqes behind the
ears s/o Psoriasis
• Diagnosis : Psoriatic arthritis
• Treated with Mtx-Responded

32. Uric Acid
• Uric acid is the final breakdown product of purine metabolism, it
circulates n the plasma as sodium urate and excreted through
kidneys
• Elevated uric acid levels is called as hyperuricemia (> 7mg), can be
due to increased urate formation or decreased excretion
• Deposition of uric acid crystals in the joints leads to Gout, cause
renal stones leading to urate nephropathy
• Varies with Gender, Weight, Diet, Drugs
• Reference Values
Males- 3.4-7.2mg/dl
Females- 2.4-6.5mg/dl
Treatment target level- Below 5 mg/dl

33. • Gout- M:F is 9:1 (most common Arthritis in Men)
• Can be normal during the acute episode
• Extremely rare in the woman of child bearing age due to
uricosuric action of estrogen
• Can have predominance of upper extremities with polyarticular
onset when present in postmenopausal woman
• Hyperuricemia in an otherwise asymptomatic individual does
not need treatment

34. When to Send???
• Typical presentation
• History s/o acute episodes of Gout in the past
• Tophi in the hands and feet (Chronic Gout)
• H/o renal calculi
• Chronic Kidney Disease

35. Case
• 27/M
• C/o Low back pain since 3 months
• Early Morning Stiffness for 5-10 mins
• HLA-B27 was Positive
• Treated as Spondyloarthropathy

36. • On detailed history
• Back pain used to increase after activity and
exertion and better with rest
• MRI sacroiliac joint was normal
• Diagnosis : Mechanical Back Pain
• Managed with Muscle relaxant and
physiotherapy

37. HLA-B27
• The strong association of HLA-B27 with
Spondyloarthropathy has been known for more than 40
years
• HLA-B27 is found in 80% to 95% of patients with AS
compared with 6% to 9% of the general populations
• Associated with a younger age at onset and predisposes
to axial involvement

38. HLA-B27 positivity in other
conditions
Percentage
Ankylosing Spondylitis 80-95%
Reactive Arthritis 60-80%
Psoriatic 60%
Enteropathic Arthritis 60%
Uveitis 40-70%

39. HLA-B27 Associated Conditions

40. • Although the HLA-B27 genetic test can be helpful,
limitations need to be kept in mind:
(1) Fewer than 1 in 10 individuals possessing HLA-B27
develops disease
(2) Only 6% of HLA-B27–positive individuals get AS, and
even in HLA-B27–positive individuals with back pain
only 22.5% have AS
(3) A significant proportion of AS patients, especially
African Americans, do not have HLA-B27
(4) Although the gene is autosomal codominant, it has
variable penetrance and thus can appear to skip
generations.

41. Bursting The Myths
• Myth 1: All HLA-B27 positive patients have AS
• Fact: HLA-B27 is positive in roughly 6-9% of general
population. Less than 5% of HLA-B27 carriers develop
disease
• Myth 2: All patients with back pain and HLA-B27
positivity can be deemed to have AS
• Fact: Only inflammatory back pain in presence of HLA-
B27 positivity has significance
• Myth 3 : All AS patients are HLA-B27 positive
• Fact: Only 80-95% of AS patients are positive for HLA-
B27

43. Antinuclear Antibodies-ANA
• Antinuclear antibodies (ANAs) are a diverse group of
autoantibodies that recognize nuclear macromolecules and
their complexes . Considered as gold standard screening test
for Connective Tissue diseases
• ANAs target macromolecular components in the cell nucleus
and can bind to DNA, RNA and proteins, as well as complexes
of nucleic acids with proteins
• It can also bind to certain cytoplasmic antigens such as
SSA,SSB

44. When is an ANA indicated?
• Inflammatory polyarthritis
• Signs/symptoms that suggest SLE/
• Sjogren’s/Myositis/Scleroderma:
• Raynaud’s
• Rashes
• Oral ulcers
• Eye inflammation
• Sicca symptoms
• Proximal muscle weakness
• PUO with Cytopenias
• Other organ involvement(nephritis)

45. ANA Assays
• IMMUNOFLUORESCENCE (IFA)- Gold standard
• Highly sensitive
• Semiquantitative –Expressed in Titres and pattern
• Cost effective and easy to perform
• Different antigens can be tested at the same time
• Requires trained person to report , varies with observer
• The initial dilution for routine testing is usually 1:40 or
1:80,

46. • ELISA
• Automated
• Reported as Numeric value- Quantitative
• Manufacturers will provide recommended cut off
value for the kit used
• High sensitivity

47. How to Interpret ANA by IF?
• ANA is positive, Speckled pattern,3+ intensity at
1:320 or at 1:1000
• The Pattern of Fluorescence
• The Titre of Positivity
• Clinically significant titers are usually ≥1:160.

49. Homogeneous
pattern
Speckled pattern

50. Centromere pattern

51. Can a positive ANA occur in a normal
healthy individual?
YES
• The frequency depends on ANA titre and patient
characteristics:
• ANA 1:40: 20%–30% of healthy individuals can be
positive
• ANA 1:80: 10%–15% positive
• ANA 1:160: 5% positive
• ANA 1:320: 3% positive
• Healthy relative of an SLE patient: 5%–25% positive
(usually low titres)
• Elderly (age >70 years): up to 70% positive at ANA
titre 1:40

53. Antineutrophil Cytoplasmic
Antibodies
• ANCAs represent a subgroup of neutrophil-specific
autoantibodies. They are commonly directed to the
azurophil granule proteins myeloperoxidase (MPO)
and proteinase 3 (PR3)
• Two types of assays :
- Immunofluorescence (IF)
- Enzyme immunoassay (EIA)

54. • With IF, three principal patterns of fluorescence are recognized:
- cytoplasmic (c-ANCA)
- perinuclear (p-ANCA)
- atypical (x-ANCA)
• In patients with vasculitis,
- c-ANCA pattern: anti-PR3 antibodies (PR3- ANCA)
- p-ANCA pattern: anti-MPO antibodies (MPO-ANCA)
• These antibodies can be detected by EIA.

55. • Atypical ANCA patterns : seen in- inflammatory bowel
disease, systemic immune-mediated diseases, infections,
Malignancy
• Thus, regardless of the pattern revealed by IF, positive
results on IF assays should be confirmed by EIAs, which
also determine the specific antigen targeted by the ANCA
(e.g., PR3, MPO)
• If C-ANCA is not against PR3 or P-ANCA is not against
MPO, look for causes other than vasculitis for the positive
ANCA.

56. Disease associated with ANCA
• C-ANCA (PR3-positive):
• Granulomatosis with polyangiitis,
• Microscopic polyangiitis (usually P-ANCA),
• Eosinophilic granulomatosis with polyangiitis (rare)
• P-ANCA (MPO-positive):
• Microscopic polyangiitis,
• Eosinophilic granulomatosis with polyangiitis,
• Pauciimmune glomerulonephritis (renal-limited
vasculitis),
• Goodpasture’s disease
• Drug-induced syndromes (hydralazine, propylthiouracil,
minocycline, others)

57. • Patterns of disease are well recognized, with typical dominance of
kidney, lung, and upper airway involvement in patients with
granulomatosis with polyangiitis (GPA), previously termed
Wegener granulomatosis.
• Lack of upper airway involvement but significant renal and lung
involvement occurs in microscopic polyangiitis (MPA).
• There is a predominance of upper and lower airway involvement
together with neurologic (peripheral nerve) features in EGPA
(Churg Strauss Syndrome)

58. Role In Diagnosis
• When IF and EIA results are considered together, ANCAs seen in :
- 90% to 95% of generalized GPA cases during active disease
- 70% to 80% of cases of the limited forms
• The detection of a c-ANCA pattern on IF in combination with PR3-
ANCA detection by EIA strongly suggests GPA
• Negative ANCA test result does not preclude the diagnosis of GPA
• Despite advances in ANCA testing techniques, the cornerstone of
diagnosis in AAV remains the combination of typical clinical features
and histopathologic findings

59. Miscellaneous
• APLA profile-Lupus Anticoagulant, Beta2-
Glycoprotein-1, Anticardiolipin Antibodies
• Serum ferritin- acute phase reactant, diagnosis
and monitoring of Adult Onset Still’s Disease and
Systemic onset JIA (SoJIA)
• CPK Total- Diagnosis and monitoring of myositis
• ACE levels-Sarcoid
• Complement Levels- SLE disease activity,
Hypocomplentemic urticarial vasculitis

60. Conclusion
• Laboratory tests are useful tools for diagnosis of systemic
rheumatic diseases and for monitoring of adverse events related
to medication use in some rheumatic conditions
• Laboratory results are considered in combination with clinical
signs and symptoms and should not replace a thorough history
and physical examination in the diagnostic evaluation

61. Thank you……