New approach for Laboratory diagnosis of Rheumatoid
Arthritis
Rheumatic diseases
 Group of diseases of different etiology
 Common clinical features:
o Temporary or chronic pain
o Degeneration changes
o Inflammation related mainly to the motor system
 Changes are related mainly to the:
o Joints and muscles
o Tendons
o Ligaments
o Peripheral nerves
o Subcutaneous tissue
 10 different classes
o Rheumatoid arthritis (Systemic connective tissue diseases)
o Juvenile arthritis (Systemic connective tissue diseases)
o Systemic lupus Erythematosus,
o Systemic sclerosis
o Polymyositis and dermatomyositis
o Sjögren's syndrome
In rheumatic diseases – a specific organ or tissue is the main target:
Joint structure elements (RA)
Immunologic disturbances lead to inflammatory process  organ/ tissue
damage.
Frequency of Rheumatic disease:
• Around 25% of population in well developed countries
• Frequently leads to disability
Etiopathogenesis of Rheumatic disease:
 Complex, multifactorial still not fully understood
 Immunologic mechanisms (synthesis of auto-Ab as a result of
activation of B lymphocytes and disturbances of lymphocytes T
function)
 Genetic factors
 Environmental factors
 Hormonal factors
 Recognition of presented autoantigens by specific lymphocytes T
appears to be the most important in the pathophysiology of
rheumatoid inflammation of synovial tissue
 Immunologic mechanisms (autoimmune response)
o Induction of immunologic response leading to arthritis,
specially synovial membrane, by proinflammatory cytokines
(IL-15, IL-17, TNF-α, IL-1, IL-6)
Autoimmune response in RA:
↑ Effect of citrullinated peptides (posttranslational deimination of Arg
into citrulline); genetically determined.
Some environmental factors as:
 Smoking and bacterial infections
 Stimulate increased citrullination of proteins.
Citrullinated vimentin (a protein of cellular skeleton)
Has been found to activate T lymphocytes in RA patients.
T lymphocytes activated by citrullinated vimentin are present in RA
patients HLA-DRSE+/anti-CPAb+.
In >60% of RA patients peripheral blood T lymphocytes recognize other
citrullinated peptides, such as aggrecan, and release IL-17.
Lymphocytes T, specific for citrullinated peptides (autoantigens such as
vimentin, enolase α) stimulate generation of anti-CPAb
whereas activated lymphocytes B produce RF (rheumatoid factor).
Role of autoantibodies in RA
Most anti-CPAb (IgG class) act as proinflammatory factors:
 Activate complement cascade
 Stimulate release of proinflammatory cytokines from
macrophages.
Anti-CPAb activate mast cells  histamine release
More About RA
 Chronic, immune-dependent systemic connective tissue disease
characterized by:
o Symmetric or non-symmetric arthritis
o Perijointal changes
o Organ complications leading to joint destruction and disability
 One of the most frequent systemic CTD
 Frequency of RA worldwide 0,5-2%; in Polish population approx. 1%
Diagnosis of RA
 Primarily based upon 7 classification criteria of ACR (ARA)
o Clinical criteria (5)
o Radiological criterion (1)
o Immunologic criterion (1)
 Inflammation of at least one joint plus score 6 out of 10 points in 4
categories
o (0-5 points) Joint damage (number and site)
o (0-3 points) Blood serology - anti-CCP (ACPAb), RF
o (0-1 points) Inflammatory markers - ESR, CRP
o (0-1 points) Duration of clinical symptoms
New criteria for RA diagnosis allow the recognition of RA in the early
stage when compared to the older ones.
Laboratory markers for RA
(Score) Serology markers:
 (0 points) RF (-) and anti-CCP/ACPAb (-)
 (2 points) Low (+) RF or anti-CCP/ACPAb
 (3 points) High (+) RF or anti-CCP/ACPAb
(0-1 points) Proinflammatory markers:
 (0 points) CRP or ESR within the reference range
 (1 points) CRP or ESR >reference range
Laboratory diagnosis in RA
 Routine laboratory diagnosis:
o Blood count
 Anemia,
 Leukocytosis
 Increased PLT number
 Low Hb
 ↓ sFe
o ESR ↑, CRP ↑ (good prognostic marker)
 SAA (new markers):
o Urinalysis (proteinuria, leukocytosis)
 Determination of antibodies:
o RF (IgM +seropositive from; IgM - seronegative)
o Anti-CCP Ab
 New antibodies
 Additional tests – COMP, YKL-40, MMP-3
Markers of RA
 Genetic: HLA-D4, HLA DRB-1
 Autoantibodies:
o Rheumatoid factor (IgM, IgG, IgA)
o Anti-CCP/ACPAb
 Novel markers
o Anti-RA33 (anti-nuclear protein A2)
o Anti-Sa (anti-citrullinated vimentin filaments = anti-enolase α)
o Anti-Annexin V
Rheumatoid factor (IgM, IgG, IgA, IgE)




RF IgM main class – occurs in 60 – 80% patients,
RF IgG in 36 – 66% RA patients
RF IgA in 19 – 88% patients (marker of erosive changes in early RA)
RF IgE in 68% RA patients
Specificity (%) of RF IgM:
 RA: 60-80
 Sjøgren: 70-95
 Lupus: 15-35
 Scleroderma: 20-30
 Tuberculosis 8-15
 Healthy: < 5
Other:
 Dermato-myostisis and polymystosis
 Bacterial Endocardits
 Viral Hepatits
ACPAb/Anti-CCP
Detected very early in the disease course with the sensitivity of 40-60%,
which has a great diagnostic utility for early diagnosis of RA.
Highly specific for RA. Detected in approx. 35-40% of (RF-) RA patients.
May be used as prognostic factor of disease activity.
In patients with anti-CCP (+) greater degree of radiologic changes are
observed and increased joint destruction.
Anti-CCP concentration correlates with the development of bone
erosions.
Sensitivity and specificity of anti-CCP
SLE
Sjögren’s Syndrome
Scleroderma
Polymyositis
Other types of arthritis
Chronic inflammatory bowel
disease
Autoimmune thyroiditis
Boreliosis
Viral diseases
Blood donors
Specificity for RA
Number
357
121
125
61
370
80
Anti-CCP (+)
8%
3%
5%
0%
6%
1%
50
45
126
376
1711
0%
2%
1%
0%
97%
Anti-RA33
(anti-nuclear protein)
Marker of early stage of RA detected also in RF (-) patients.
Assay: ELISA
Anti-Sa
(anti-citrullinated vimentin filaments)
Ag Sa- its activity is similar to human enolase α.
Early marker of rapid and progressive joint destruction.
Prognostic markers of disease progression.
Assay: ELISA
Sensitivity and specificity of markers in RA
Markers
Sensitivity [%]
Specificity [%]
Anti - CCP
60 – 80
98
APF
29 - 79
89 - 98
AKA
27 - 47
84 - 94
AFA
33 - 76
93 - 98
Anti-Sa
22 - 40
85 - 95
anti - RA33
29 - 36
40 - 87
Markers of RA
 Markers of inflammatory state
o ESR,
o CRP hs,
o SAA
o Cytokines: IL-6, TnF-α, IL-1,
 Markers of angiogenesis:
o VEGF
o Angiopoetin-1 and Ang-2 (indices of angiogenesis activity and
prognostic markers of joint destruction)
SAA in rheumatoid arthritis
Frequency of amyloidosis in Finnish RA patients is approx. 30% (AA in
cardiac muscle or kidneys).
Amyloidosis was found in patients with more pronounced
proinflammatory state and longer disease duration.
It is suggested that patients with active and long disease duration should
be screened for SAA concentration, which enables early antiinflammatory treatment.
Markers of RA
 Biochemical bone markers:
o CTxI,
o Pyridoline (PYD),
o Deoxypyridoline (DPD)
o Osteocalcin,
o PINP
o Bone sialoprotein
 Markers of cartilage turnover and degradation:
o Hyaluronic acid
o COMP
o YKL-40
o CTx II, MMP-3
COMP – in the diagnosis of early stage of
 Cartilage oligomeric matrix protein - marker of cartilage turnover,
prognostic marker of joint damage in RA
 Increased serum COMP – not dependent on DAS-CRP, allows
selection of patients with early RA stage with bone erosion (MRI)
related to inflammation of synovial membrane.
MMP-3 (matrix metalloproteinase 3)
Secreted by proliferating synoviocytes from inflamed synovium into SF.
Marker of disease activity correlates with CRP concentration and degree
of joint destruction.
MMP-3 – a predictor of the degree of joint destruction in patients with
early RA.
Allows selection of patients for more aggressive therapy.
Systemic Lupus Erythematous (SLE)
 Chronic multi-systemic autoimmune disease affecting:
o Joints,
o Skin
o Heart
o Lungs
o CNS
o Kidneys
o Hematopoietic system
 Occurs at every age but mostly 20-40 years
 9-fold more often occurs in women.
 Characteristic feature – immune complexes deposition and presence
of Ab.
 Immune complexes are deposited in glomeruli (glomerulonephritis).
 Antibodies against erythrocytes, leukocytes, platelets are present.
SLE Diagnostic criteria
 Disturbances of kidney function (proteinuria, casts in the urine)
 Hematologic disorders:
o Hemolytic anemia,
o Retikulocytosis
o Leukopenia
o Thrombocytopenia
 Immunologic disorders
o LE cells
o Anti-DNA Ab
o Anti-Sm Ab
o Anti-Ro
o Anti-La
 ANA Ab
New Ab in SLE:
 Anti-CCP in approx. 12,5%
 Anti-MCV (mutated citrullinated vimentin)
 In around 10,5%
SLE – Laboratory findings
 Hematology:
o Hemolytic anemia
o Normocytosis/ normochromatosis (decreased
erythropoesis),
o Leukopenia (<4ths./mm3)
o lymphocytopenia
o Granulocytopenia
o Thrombocytopenia (<100 ths./mm3)
 ↑ ESR
 Coagulation: prolonged clotting time (aPTT) caused by antiphospholipid Ab (lupus anticoagulant)
 Kidney dysfunction- persistent proteinuria > 0,5 g/24h and/or casts in
the urine
 Serology: positive syphilitic reaction (effect of aPL-Ab)
 Immunology:
o ↓ concentration of complement C3, C4,
o Hypergammaglobulinemia,
Characteristic antibodies
 Anti-dsDNA in active disease process
 Anti-histone Ab
 Ab anti small nuclear ribonucleoproteins:
o Anti- Sm (Smith): typical for SLE
o Anti- U1RNP
o Anti- Ro (a component of snRNP) : typical for SLE with skin
lesions and overreaction for day-light; also occur in Siögren
Syndrome
Occurrence of antibodies in SLE
(Occurrence) Antibodies:
• (70 - 95 %) ss-DNA
• (30 - 90 %) ds-DNA
• (40 - 80 %) Histones
• (5 - 30 %) Sm
• (20- 60 %) SS-A
• (10 - 20 %) SS-B
• (5 - 15 %)RNP
Scleroderma (SCL)
Chronic systemic connective tissue disease with excessive collagen
deposition in the skin and viscera along with vascular abnormalities and
disorders in immune system and inflammatory state.
Pathogenesis:
 Autoimune response/ factors
 Genetic predisposition
 Functional and morphopathological vascular disorders
 Disorders of collagen synthesis
Laboratory diagnosis:
 ESR
 CRP
 Proteinogram
 Blood cell count
 Urinalysis
 RF, ANA, anti- Scl-70 determination
Ab characteristics for SCL:
(Frequency) Ab anti:
• (80 - 95 %) Centromers
• (25 - 75 %) Scl - 70
• (30 - 60 %) ss DNA
• (4 %) RNA polymerase
• (3 %) PM - Scl (overlapping syndrome)
• (1 - 7 %) Ku (overlapping syndrome with polymyositis)
Psoriatic Arthritis
• Chronic inflammatory disease: arthritis with concomitant psoriasis of
skin and nails
• Seronegative (RF-; anti-CCP +) arthritis with vertebral lesions
Laboratory Diagnosis:
• Seronegative form RF IgM(-)
• Anti-CCP (+) in 5-13% of patients
• In active disease:
o ↑ ESR, leukocytosis,
o ↑ of acute phase proteins
o Normochromic anemia
o Presence of CIC (circ. immune complexes)
Juvenile chronic arthritis (JCA/JIA)
 The most prevalent inflammatory systemic connective tissue disease
in children/ adolescents
 Different clinical features: polyarthritis (>5 joints involved) or
oligoarthritis (<4 joints involved); totally different from RA
 In children – mostly systemic disorders, changes in big joints or single
articular joint. RF IgM occurs rarely
Laboratory Diagnosis:
 RF IgM, RF IgA
 Anti-CCP in polyarthritis (frequency 2-40%)
 CRP
 Proteinuria
 Cartilage degradation markers – COMP
 Combined measurement of COMP, anti-CCP, RF IgM/ IgA for
evaluation of disease stage
Sjøgren Syndrome
Primary Sjögren’s Syndrome:
Chronic inflammatory autoimmune disease affecting exocrine glands
(lacrimal and salivary) by infiltration of lymphocytes
Secondary Sjögren’s Syndrome
Combination of sicca syndrome (dry eyes and dry mouth) with other
autoimmune CTD.
Laboratory diagnosis:
 Anemia
 Leukopenia in 30% of patients
 Rarely thrombocytopenia
 ↑ immunoglobulins
 In 70% of patients ANA (+)
Antibodies in Sjørgren syndrome:
(Prevalence) Antibodies anti-:
 (40 - 95 %)SS-A
 (40 - 95 %)SS-B
 (40 - 95 %)SS-DNA
SS-A:
Antigen:
 RNA type Y1, Y3, Y4 or Y5 associated with 2 proteins (52 and 60 kDa)
Function:
 Regulation of active translational mRNA
Associated diseases:
 (40 - 95 %) Sjögren’s Syndrome
 (20 - 60 %) SLE
SS-B
Antigen:
 Phosphoprotein (48 kDa) associated with low molecular RNA
Function:
 Protein stimulating polymerase III
Associated diseases:
 (70 - 95 %)Sjögren’s Syndrome
 (10 - 20 %)SLE