Stillwell’s Board Shots- Lab

Testing for the Rheumatic

Diseases
Mark Stillwell, MD, FACP
 Lab Testing for the Rheumatic Diseases-
Introduction
• I. Introduction-
• A. Autoimmunity involves the loss of normal homeostasis, such that the organism produces an abnormal response to its own
tissue. The hallmarks of autoimmune diseases generally involve the presence of self-reactive T cells, autoantibodies and
inflammation.
• B. Synthesis of acute phase reactants occurs as a response to pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-alpha.
• C. This presentation will go over markers that may be seen with autoimmune diseases, ***but it is important to know that
labs alone never diagnose an autoimmune disease, criteria for diagnosis usually depend on a combination of criteria, including
symptoms, signs plus labs......e.g. a positive ANA alone does not mean a patient has SLE......and a patient that has a positive
ANA may not have any autoimmune disease at all. Various autoimmune diseases often have several positive autoimmune
markers that crossreact with other autoimmune disorders, and it is common to see diseases have symptoms/signs of more
than one disorder (called overlap syndromes).
• D. Obtaining the following basic tests are always important in evaluation of patients with suspected autoimmune diseases:
• 1. CBC/manual diff- leukopenia and thrombocytopenia (both usually mild) are common to see in many autoimmune
diseases (**severe pancytopenia can even be seen, as in the variant of RA called Felty’s syndrome, associated with
splenomegaly). Anemia is common, often an anemia of chronic disease, but even hemolytic anemia is seen in some
autoimmune disorders.
• 2. CMP- important to look for renal and liver dysfunction. It is also common to find elevated globulins (with globulins >
albumin, rather than the normal albumin > globulins. This is usually a polyclonal elevation of globulins).
• 3. UA with microscopic- looking for evidence of proteinuria, hematuria or glomerulonephritis (RBC casts).
 Lab Testing for the Rheumatic Diseases- Acute
Phase Reactants
• II. Acute Phase Reactants- One of the body’s internal responses to
inflammation, infection, or other major insult is to synthesize proteins that are
involved in the body’s response. These proteins are called “positive” acute
phase reactants and increase in inflammatory circumstances, the best known is
the *erythrocyte sedimentation rate (ESR). Many are synthesized by the liver,
including *C-reactive protein (CRP, an opsonin on microbes), fibrinogen &
prothrombin (trap invading microbes in clots and cause chemotaxis),
haptoglobin (binds hemoglobin, inhibiting microbe iron uptake), serum amyloid
A protein (recruits immune cells to inflammatory sites), *ferritin (binds iron,
inhibiting microbial iron uptake), complement factors (opsonization,
chemotaxis), ceruloplasmin (oxidizes iron, facilitating ferritin, inhibiting
microbial iron uptake), mannan-binding lectin (complement activation), alpha 2-
macroglobulin (inhibits fibrinolysis) and alpha 1-antitrypsin (downregulates
inflammation).......”negative” acute phase reactants decrease in inflammation
(theory- done to save amino acids for production of positive acute phase
reactants), and include albumin and transferrin.
 Lab Testing for the Rheumatic Diseases- Acute
Phase Reactants
• II. Acute Phase Reactants-
• A. ESR- is the rate at which RBCs descend with gravity in anticoagulated whole blood
in a standardized 100 mm tube in 1 hour. This can now be done in automated
analyzers.
• 1. In an inflammatory state, elevated fibrinogen in the blood causes RBCs to stick
to each other forming stacks called “rouleaux”......which settle faster due to increased
density.
• 2. Increases with systemic and localized inflammation (autoimmune diseases) and
infection (especially deep-seated bacterial infections such as infective endocarditis,
osteomyelitis and abscesses), malignant neoplasms (especially lymphoma and
myeloma), tissue injury/ischemia (MI, CVA) and trauma .....”normal” is usually < 20
mm/hour and < 15 mm/hour for men......for men or women > 50 yo some consider
normal <30 mm/hr
• 3. Also increases with age, pregnancy, anemia (less resistance by other RBCs),
ESRD, obesity (increased IL-6 release by adipose tissue) and is slightly higher in
women.
How ESR is read
Source: johnyfit.com
ESR on middle tube is 18
Source: medicine.mcgill.ca
ESR is 18 in this situation, read at one hour
Source: slideplayer.com
 Lab Testing for the Rheumatic Diseases- Acute
Phase Reactants
• II. Acute Phase Reactants-
• B. CRP- is a ring-shaped pentameric protein of hepatic origin that
increases in response to IL-6 secretion from macrophages and T cells, in
response to inflammation. It then binds lysophosphatidylcholine
expressed on the surface of dead or dying cells and bacteria in order to
activate the complement system via C1q.
• 1. Normal levels vary between 0.8 and 3.0 mg/L
• 2. *A more sensitive and accurate reflection of the acute phase
response than ESR....CRP goes up quicker with inflammation and comes
down faster with response to therapy.
• 3. Usually the traditional CRP is used in evaluation of inflammatory
diseases. There is a “high sensitivity CRP” available, but it is generally
used in cardiac disease evaluation.
CRP is an annular pentameric protein
Source: wikipedia.org
 Lab Testing for the Rheumatic Diseases- ANA
• III. Anti-Nuclear Antibodies (ANA)
• A. ANA- *95-98% of SLE patients are ANA+.......*but finding a positive ANA is common in most other
autoimmune diseases....*therefore very sensitive, but not specific, for SLE.
• 1. Done by indirect immunofluorescence using dilutions of patient serum overlaid onto a substrate such as
immortalized laryngeal epithelial cancer HEp-2 cells,
• 2. **ANA staining patterns:
• a. Homogenous/Diffuse pattern- the entire nucleus is stained, staining DNA and histone proteins......a
nonspecific test for many autoimmune processes except this what is seen with drug-induced lupus (ssDNA and
histone)
• b. Speckled pattern- staining of fine or course speckles throughout the nucleus indicating antibodies against
many antigens:
• 1) U1 RNP- > 90% of those with MCTD
• 2) Sm- 30% of those with SLE (specific for it, not sensitive)
• 3) Ro (SS-A)- seen in 60% of Sjogren’s, and seen in subacute cutaneous lupus syndrome, neonatal lupus
and ANA-negative lupus
• 4) La (SS-B)- seen in 50% of Sjogren’s and 15% of SLE
• 5) SCL-70- 40% of those with scleroderma
• 6) PM-1- can be seen in poly- and dermatomyositis
• 7) Jo-1- can be seen with polymyositis
 Lab Testing for the Rheumatic Diseases- ANA
• III. Anti-Nuclear Antibodies (ANA)
• A. ANA-
2. ANA staining patterns:
• c. Nucleolar pattern- stains RNA polymerase I, seen in scleroderma
• d. Centromere pattern- seen in 75% with CREST
• e. Peripheral (rim) pattern- stains dsDNA, specific for SLE, but not sensitive, as only
seen in 50% with SLE
• f. Nuclear dot pattern- stains mitochondria, seen in primary biliary cirrhosis
• g. Cytoplasmic pattern- stains mRNA, seen in primary biliary cirrhosis
• 3. **Titers of </= 1:80 are often of questionable significance, as 30% of the population
is positive at 1:40.....5% of the population has a titer of 1:160, while only 1% of the
population has autoimmune disease.
• 4. **Common to find positive ANA tests in first degree relatives of those with
autoimmune diseases, even if they themselves do not have an autoimmune
disease.....**so a positive ANA, even at titer of >/= 1:160 does not necessarily mean the
patient has an autoimmune disease.
 ANA patterns- a. homogenous, b. speckled, c. nuclear dots, d. nucleolar, e. centromere, f. nuclear membrane, g. cytoplasmic,
h. negative/nonspecific Source: researchgate.net
ANA- a positive test does not necessarily mean disease exists
Source: slideplayer.com
ANA is positive in many diseases
Source: the-rheumatologist.org
ANA patterns
Source: mdedge.com
 Lab Testing for the Rheumatic Diseases- RF
• IV. Rheumatoid Factor (RF)
• A. An *autoantibody (usually *IgM, but can be IgG, IgA, IgE or IgD) to
the Fc portion of IgG (i.e. actually an antibody to an antibody). RF and
IgG form immune complexes that contribute to the disease process.
• B. Titer of 1:40 or higher seen in 70-80% of those with **RA and 50-
70% of those with **Sjogren’s syndrome.......the higher the titer, the
higher the probability of finding destructive articular disease.
• C. Also common in *infective endocarditis (seen in 50% of cases). Can
also be seen in EBV infection, Parvovirus infection, SLE,
cryoglobulinemia, Syphilis, TB and sarcoidosis.
 Lab Testing for the Rheumatic Diseases- Anti-
DNA Antibodies
• V. Anti-DNA antibodies
• A. Anti-single stranded DNA antibodies (Anti-ssDNA) are not generally useful
in the diagnosis and management of SLE, but can be found in *drug-induced
lupus, RA and in healthy relatives of those with SLE.
• B. Anti-double stranded DNA antibodies (Anti-dsDNA) are useful in the
diagnosis and management of **SLE.
• 1. *Titer correlates with SLE activity.
• 2. *Specific (95%) for SLE, but only found in 50-70% of cases...only rarely
found in other autoimmune disorders.
• 3. High titers found in those with active lupus glomerulonephritis.
• 4. Occasionally seen in drug-induced lupus.
• 5. Uses a target antigen *Crithidia lucilae, a flagellated protozoan with a
dsDNA-containing organelle called a kinetoplast.
 Lab Testing for the Rheumatic Diseases- Anti-
Smith Antibodies (Anti-Sm)
• V. Anti-Smith (Anti- Sm) antibodies
• A. An antibody to extractable nuclear antigen (ENA), like anti-RNP,
anti-Ro, anti-La, Anti-SCL-70, anti-centromere and anti-Jo-1. They
react against 7 proteins in U1, U2, U4 and U5 ribonuclear proteins.
• B. Specific (55-100%) even when patient is in remission, but not
sensitive (30%), for **SLE.
• C. If present, these are associated with poor outcomes in patients
with lupus nephritis, and is *specific for lupus nephritis.
 Lab Testing for the Rheumatic Diseases- Anti-
U1 Ribonucleoprotein Antibodies (Anti-RNP)
• VI. Anti-RNP antibodies
• A. Also an antibody to extractable nuclear antigen (ENA), reacts
to 3 proteins associated with U1 ribonuclear protein in U1 RNA.
• B. Specific and sensitive (90-100%) for **mixed connective tissue
disease (MCTD) in high titers......sometimes seen in low titers in
other autoimmune diseases.
 Lab Testing for the Rheumatic Diseases- Anti-
SCL-70 Antibodies (Anti-SCL-70)
• VII. Anti-SCL-70 antibodies (also known as anti-topoisomerase I)
• A. Also an antibody to extractable nuclear antigen (ENA).
• B. Seen in 28-70% of **scleroderma cases (when positive, often
correlates with more severe disease) and 10-18% of CREST and
25% of SLE patients.......note: scleroderma = progressive systemic
sclerosis
• C. When positive in scleroderma patients, it predicts those at
higher risk for diffuse cutaneous and interstitial lung involvement.
 Lab Testing for the Rheumatic Diseases- Anti-
Ro (SS-A) and Anti-La (SS-B) Antibodies
• VIII. Anti-Ro (SS-A) and Anti-La (SS-B) antibodies
• A. Also antibodies to extractable nuclear antigen (ENA).
• B. Anti-Ro (SS-A) seen in 60-75% of **Sjogren’s and 25% of SLE
cases
• C. Anti-La (SS-B) seen in 30-40% of **Sjogren’s and 10% of SLE
cases.
 Lab Testing for the Rheumatic Diseases- Anti-
Centromere Antibodies
• IX. Anti-Centromere antibodies
• A. Also antibodies to extractable nuclear antigen (ENA).
• B. Positive in > 60-80% of **CREST cases.......with > 98%
specificity........note: CREST = calcinosis, Raynaud’s phenomenon,
esophageal dysmotility, sclerodactyly and telangiectasia syndrome.
 Lab Testing for the Rheumatic Diseases- Anti-
PM-1 Antibodies
• X. Anti-PM-1 antibodies
• A. Directed against inner membrane of mitochodria.
• B. Positive in some cases of *polymyositis and
*dermatomyositis....and especially in polymyositis-scleroderma
overlap (67%).
 Lab Testing for the Rheumatic Diseases- Anti-
Histone Antibodies
• XI. Anti-Histone antibodies
• A. Directed against histone protein subunits.
• B. Positive in 90-100% of **drug-induced lupus cases......and
some with RA/Felty’s syndrome.
 Lab Testing for the Rheumatic Diseases- Anti-
Citrullinated Protein Antibodies (CCP)
• XII. Anti-CCP antibodies
• A. Directed against citrullinated peptides and proteins.
• B. Positive in the **majority of RA patients (70-78% sensitive,
similar to RF and *88-96% specific, more specific than RF).
• C. May also be found in JRA, psoriatic arthritis, SLE, myositis
syndromes and Sjogren’s syndrome.
 Lab Testing for the Rheumatic Diseases- Anti-
Neutrophil Cytoplasmic Antibodies (ANCA)
• XIII. ANCAs- mainly IgG antibodies that react with cytoplasmic granules of neutrophils
and monocytes.
• A. Initial testing screens sera for two general immunofluorescence staining patterns:
• 1. **Cytoplasmic ANCA (cANCA)- mainly antibodies against proteinase-3, most
often seen in Wegener’s granulomatosis (granulomatosis with polyangiitis, 90%
+).........c-ANCA (atypical form, is seen in 85% of cystic fibrotics).
• 2. **Perinuclear ANCA (pANCA)- mainly antibodies against to myeloperoxidase
(MPO), most often seen in microscopic polyangiitis (necrotizing small vessel vasculitis
with necrotizing granulomas), crescentic glomerulonephritis and Churg-Strauss
syndrome (eosinophilic granulomatosis with polyangiitis) ....also seen in other types of
vasculitis, IBD, SLE, JRA and RA......up to 80-90% with primary sclerosing cholangitis
are positive also.
• B. *Some common forms of vasculitis tend to usually be ANCA-negative however
(e.g. polyarteritis nodosa, Henoch-Schoenlein purpura, cryoglobuliemia and giant
cell/temporal arteritis)
ANCA patterns
Source: mdedge.com
ANCA and vasculitis
Source: aafp.org
ANCA and vaculitis
Source: aafp.org
ANCA-associated diseases
Source: slideshare.net
ANCA positivities
Source:slideshare.net
 Lab Testing for the Rheumatic Diseases- Anti-
Jo-1 Antibody
• XIV. Anti-Jo-1 Antibody-
• A. A myositis specific autoantibody directed against histidyl-tRNA
synthetase that catalyzes the binding of the histadine to tRNA
during its tRNA protein synthesis.
• B. Found in patients with the *inflammatory myopathies (20-30%
of polymyositis........may also be seen in dermatomyositis and
inclusion body myositis) and in idiopathic pulmonary fibrosis (60-
70%). Those who are positive for anti-Jo-1 with myositis tend to
have worse muscle and lung involvement than those negative.
 Lab Testing for the Rheumatic Diseases- Anti-
Mitochondrial Antibody (AMA)
• XV. Anti-Mitochondrial Antibody (AMA)-
• A. Autoantibodies against primarily mitochondria in liver cells.
• B. Seen in 95-98% of people with **primary biliary
cirrhosis.....and can be seen in other liver diseases (e.g. 34% with
autoimmune hepatitis.....occasionally in viral hepatitis, and non-
alcoholic fatty liver disease) and other autoimmune systemic
diseases (e.g. SLE).
 Lab Testing for the Rheumatic Diseases- Anti-
Smooth Muscle Antibody (ASMA)
• XVI. Anti-Smooth Muscle Antibody (ASMA)-
• A. Autoantibodies against actin, troponin and tropomyosin in
protein found in smooth muscles.
• B. 50-80% sensitive and 90% specific for **autoimmune hepatitis.
Also can be seen in **primary sclerosing cholangitis (20-50%
sensitive) and Hepatitis C.
 Lab Testing for the Rheumatic Diseases- Anti-
Liver-Kidney Microsomal Antibody(ALKMA)
• XVII. Anti-Liver-Kidney Microsomal Antibody (ALKMA)-
• A. Type I- seen in *autoimmune hepatitis type 2.....and 10% of
Hepatitis C patients.
• B. Type II- seen in drug-induced hepatitis.
• C. Type III- seen in chronic hepatitis of autoimmune
polyendocrine syndrome....and in Hepatitis D.
 Lab Testing for the Rheumatic Diseases- Anti-
Saccharomyces Cerevisiae Antibody (ASCA)
• XVIII. Anti-Saccharomyces Cerevisiae Antibody (ASCA)-
• A. IgG and IgG antibodies to baker’s yeast used to help distinguish
Crohn’s Disease from Ulcerative Colitis.
• 1. If ASCA+ and pANCA-......**more likely CD (70% sensitive)
• 2. If ASCA- and pANCA+.......more likely UC
• B. May also be seen in primary sclerosing cholangitis (44%).....can
also be present in autoimmune hepatitis and primary biliary
cirrhosis.
 Lab Testing for the Rheumatic Diseases- Celiac
Disease Panel
• XIX. Celiac Disease Panel-
• A.* Tissue Transglutaminase IgA positive in 93-98% with 98%
specificity.
• B. *Deamidated Gliadin IgA and IgG (95% and 50% sensitive
respectively). The IgG may be important in the diagnosis in IgA deficient
patients.
• C. *IgA Endomysial antibody is 90-95% sensitive and 99-100% specific
• D. Total IgA antibody is also done. IgA deficiency can be seen in Celiac
Disease, making above tests falsely negative.
• E Genetic testing- HLA-DQ2 and HLA-DQ8........found in 30-35% of the
population, so positive test certainly does not mean they have celiac
disease, but negative testing makes current or future celiac disease very
unlikely.
 Lab Testing for the Rheumatic Diseases-
Muscle Enzymes
• XX. Muscle enzymes- these increase in the autoimmune and drug-
induced myositis illnesses (e.g. polymyositis, dermatomyositis, inclusion
body myositis)
• A. CPK- especially the CPK MM isoenzyme.....the most classic one
followed in these disorders.
• B. Aldolase
• C. LDH- especially LDH 5 > LDH 4 isoenzymes
• D. Troponin- will increase with skeletal muscle issues, but mainly used
in cardiac disease (MI, myocarditis).
• E. Transaminases- AST (SGOT) much more than ALT (SGPT), normally
used to evaluate hepatocellular issues, also go up with muscle
inflammation/injury (GGT- gamma-glutamyl transpeptidase is much
more specific for the liver).
 Lab Testing for the Rheumatic Diseases-
Complement.....CH50, C3, C4
• XXI. Complement-
• A. CH50 or CH100 (Total hemolytic complement)- functionally assesses the entire classic complement
pathway (C1-C9), measured by ability to lyse antibody-coated sheep RBCs.
• 1. Low level-
• a. Extremely low- suggests a complement pathway deficiency.
• b. Mildly low can be seen with activation of the complement pathway, with consumption of
complement by immune complex formation quicker than the components can be replaced by the liver,
such as in *immune complex disease with SLE and glomerulonephritis.....also can be seen with
malnutrition.
• 2. High level- can occur with an acute phase inflammatory response.
• B. C3 and C4
• 1. Low level- common to see in *SLE with immune complex formation (and complement
consumption). Also seen in other diseases with immune complex formation such as antiphospholipid
syndrome, mixed cryoglobulinemia, Sjogren’s syndrome, membranoproliferative or post-streptococcal
glomerulonephritis and infective endocarditis. In SLE, normalization of complement levels is a good
sign.
• 2. High level- see in inflammatory states, as complement is an acute phase reactant.
 Lab Testing for the Rheumatic Diseases-
Antiphospholipid Syndrome Tests
• XXII. Complement-
• A. Antiphospholipid syndrome tests- often suspected in patients with autoimmune,
thrombotic or miscarriage issues often in the face of a baseline prolonged aPTT
(usually > 3 seconds) and a normal PT/INR. Antiphospholipid antibody testing panels
include variations of the following:
• 1. *Lupus anticoagulant- sample initially tested for aPTT or dilute Russell Viper
Venom Time (RVVT), and if prolonged a mixing test with normal pooled serum is
done......if the aPTT/RVVT remain prolonged it suggests an inhibitor is present. This is
then followed by confirmatory studies demonstrating phospholipid dependence on
the inhibitor.
• 2. *Anti-cardiolipin antibodies (IgG, IgM, IgA)
• 3. Anti-beta-2-glycoprotein-1 antibodies (IgG, IgM, IgA)
• 4. Anti-prothrombin antibodies (IgG, IgM)
• 5. False-positive RPR (i.e. positive non-treponemal test such as RPR with negative
treponemal test such as FTA-ABS)
Lupus anticoagulant
Source: slideplayer.com
Example of one method to test for Lupus Anticoagulant
Source: oncohemakey.com
 Lab Testing for the Rheumatic Diseases- Basic
Tests
• XXIII. Basic tests-
• A. CBC/diff- some element of *leukopenia or *thrombocytopenia common to
see in the autoimmune syndromes.....as is anemia of chronic disease or
sometimes hemolytic anemia. More pronounced abnormalities can be seen in
those with *Felty’s syndrome (RA + Splenomegaly + Pancytopenia).
• B. CMP- common to have *globulin elevations due to chronic inflammation, so
the normal albumin > globulin ratio is often reversed in those with autoimmune
diseases, instead revealing globulin > albumin. Specific serum protein
electrophoresis (SPEP) or immunoelectrophoresis (SIE) will usually show the
elevated globulins to be *polyclonal. Albumin may be low due to malnutrition
or chronic disease with protein wasting or renal protein losses.
• C. UA with micro- with nephropathy, proteinuria can be seen and with
glomerulonephritis RBC casts can be seen. With chronic kidney disease
increased waxy, granular and hyaline casts may also be seen.
Lab Testing for the Rheumatic Diseases-
Miscellaneous Summary Slides (from various
sources)
Source: pinterest.com
Source: semanticscholar.org
Source: townsendletter.com
Source: neuroendoimmune.wordpress.com
Source: thyroidpharmacist.com
ENAs
Source: jpma.org.pk