1. Common Rheumatologic Tests: Evaluation and Interpretation

11

Common Rheumatologic Common Rheumatologic Tests: Evaluation and Tests: Evaluation and

InterpretationInterpretation

DisclosuresDisclosures

• Nothing to discloseNothing to disclose

IntroductionIntroduction

• Immunologic laboratory testing in Immunologic laboratory testing in rheumatology is useful for rheumatology is useful for supporting or refuting a clinically supporting or refuting a clinically suspected diagnosissuspected diagnosis

• ““Shotgun approaches” or “screening Shotgun approaches” or “screening tests” often lead to false positives, tests” often lead to false positives, and further unnecessary and further unnecessary workups/referralsworkups/referrals

BackgroundBackground

• Defining attributes of a testDefining attributes of a test– SensitivitySensitivity– SpecificitySpecificity– Positive predictive valuePositive predictive value– Negative predictive valueNegative predictive value– Likelihood ratiosLikelihood ratios– Pretest and posttest probabilitiesPretest and posttest probabilities

Attributes of a testAttributes of a test

• SensitivitySensitivity– Proportion of patients with a disease Proportion of patients with a disease

who have a positive test resultwho have a positive test result

• SpecificitySpecificity– Proportion of patients without a disease Proportion of patients without a disease

who have a negative test resultwho have a negative test result

• Both sensitivity and specificity are Both sensitivity and specificity are independent of disease prevalenceindependent of disease prevalence


• Predictive valuePredictive value– likelihood of disease or lack thereof likelihood of disease or lack thereof

based on a positive or negative test based on a positive or negative test resultresult

– Negative predictive value (NPV)Negative predictive value (NPV)•True negative/(true negative + false True negative/(true negative + false

negative)negative)

– Positive predictive value (PPV)Positive predictive value (PPV)•True positive/(true positive + false positive)True positive/(true positive + false positive)

Predictive valuePredictive value

• Predictive value is significantly Predictive value is significantly affected by disease prevalenceaffected by disease prevalence– Predictive value of a positive Predictive value of a positive

rheumatologic test in patient with rheumatologic test in patient with polyarthralgias is likely to be higher in a polyarthralgias is likely to be higher in a rheumatology practice than in a family rheumatology practice than in a family physician’s officephysician’s office

– As pretest probability increases, so does As pretest probability increases, so does the clinical utility of a specific testthe clinical utility of a specific testLane, SK and Gravel, JR. American Family Physician. 65;6,1073,2002.


• Likelihood ratioLikelihood ratio– LR for a negative test result:LR for a negative test result:

(1-sensitivity)/specificity(1-sensitivity)/specificity

– LR for a positive test result:LR for a positive test result:sensitivity/(1-specificity)sensitivity/(1-specificity)

Likelihood ratioLikelihood ratio

• Provides additional measure by allowing Provides additional measure by allowing calculation of posttest probability based calculation of posttest probability based on pretest probability and test resulton pretest probability and test result

• Decision to use a test should be based Decision to use a test should be based upon whether posttest probability will upon whether posttest probability will be significantly different from the be significantly different from the pretest probability given a positive or pretest probability given a positive or negative test resultnegative test result

ACR AD HOC Committee. Arthritis Care and Research. 47:429, 2002.


• If a test has a high positive likelihood If a test has a high positive likelihood ratio (e.g., 10), and the test result is ratio (e.g., 10), and the test result is positive, then the posttest probability positive, then the posttest probability of the test will be greatly increasedof the test will be greatly increased

• If the likelihood ratio is only 1, then If the likelihood ratio is only 1, then no difference would be expected no difference would be expected between pretest and posttest between pretest and posttest probabilitiesprobabilities

Performance characteristics of Performance characteristics of specific ANAsspecific ANAs

AntigenAntigen ConditionCondition SensitivitySensitivity SpecificitSpecificityy

+ LR+ LR - LR- LR

Anti-dsDNA Anti-dsDNA AbAb

SLESLE 57%57% 97%97% 16.316.3 0.490.49

Anti-Sm AbAnti-Sm Ab SLESLE 25-30%25-30% highhigh ** **

Anti-Ro/Anti-Ro/SSA AbSSA Ab

Sjogren’s, Sjogren’s, SCLESCLE

8-70%8-70% 87%87% ** **

Anti-La/SSB Anti-La/SSB AbAb

Sjogren’s, Sjogren’s, SCLESCLE

16-40%16-40% 94%94% ** **

Scl-70Scl-70 SclerodermaScleroderma 20%20% 100%100% >25>25 0.80.8

AnticentroAnticentromeme

CRESTCREST 65%65% 99.9%99.9% 650650 0.40.4

Anti-U-3 Anti-U-3 RNPRNP

SclerodermaScleroderma 12%12% 96%96% 33 0.920.92Colglazier, CL et al. Southern Medical Journal.2005

Acute phase reactantsAcute phase reactants

• Heterogeneous group of proteins Heterogeneous group of proteins synthesized in liver in response to synthesized in liver in response to inflammationinflammation– FibrinogenFibrinogen– HaptoglobinHaptoglobin– C-reactive proteinC-reactive protein– Alpha-1-antitrypsinAlpha-1-antitrypsin

Acute phase protein Acute phase protein responseresponse

Adapted from Gitlin JD, Colten HR in Pick E, Landy M [eds]: Lymphokines.14;123,1987.

Common markers of Common markers of inflammationinflammation

• ESRESR– Measures distance (in mm) that RBCs Measures distance (in mm) that RBCs

fall within specified tube (Westergren or fall within specified tube (Westergren or Wintrobe) over 1 hourWintrobe) over 1 hour

– Indirect measure of changes in acute-Indirect measure of changes in acute-phase reactants and quantitative Igsphase reactants and quantitative Igs

– Decreases by ~50% in 1 week after Decreases by ~50% in 1 week after inflammation resolvesinflammation resolves

Mechanism of elevated ESRMechanism of elevated ESR

• If higher concentration of If higher concentration of asymmetrically charged acute-phase asymmetrically charged acute-phase protein or hypergammaglobulinemia protein or hypergammaglobulinemia occurs, dielectric constant of plasma occurs, dielectric constant of plasma increases and dissipates inter-RBC increases and dissipates inter-RBC repulsive forces, leads to closer repulsive forces, leads to closer aggregation of RBCs, so they fall aggregation of RBCs, so they fall faster, and cause ESR elevationfaster, and cause ESR elevation

Hobbs, K in West, S. Rheumatology Secrets,2002

Noninflammatory conditions Noninflammatory conditions with elevated ESRwith elevated ESR

• AgingAging

• Female sexFemale sex

• ObesityObesity

• PregnancyPregnancy

Rule of thumbRule of thumb

• Age-adjusted upper limit normal for Age-adjusted upper limit normal for ESRESR– Male: age/2Male: age/2– Female: (age + 10)/2Female: (age + 10)/2

Causes of markedly elevated Causes of markedly elevated ESRESR

• ESR >100ESR >100– Infection, bacterial (35%)Infection, bacterial (35%)– CTD (GCA, PMR, SLE, vasculitides (25%)CTD (GCA, PMR, SLE, vasculitides (25%)– Malignancy: lymphomas, myeloma, etc Malignancy: lymphomas, myeloma, etc

(15%)(15%)– Other causes (25%)Other causes (25%)

Hobbs, K in West, S. Rheumatology Secrets,2002.

Causes of extremely low Causes of extremely low ESRESR

• ESR ~ 0mm/hrESR ~ 0mm/hr– AgammaglobulinemiaAgammaglobulinemia– Afibrinogenemia/dysfibrinogemiaAfibrinogenemia/dysfibrinogemia– Extreme polycythemia (Hematocrit Extreme polycythemia (Hematocrit

>65%)>65%)– Increased plasma viscosityIncreased plasma viscosity

Approach to elevated ESRApproach to elevated ESR

• Complete H&PComplete H&P• Routine labs (CBC, CMP, UA)Routine labs (CBC, CMP, UA)• Up-to-date cancer screening/health Up-to-date cancer screening/health

maintenancemaintenance• Repeat ESRRepeat ESR• If still elevated without other associationIf still elevated without other association

– Consider SPEP, CRPConsider SPEP, CRP– Recheck in 1-3 months (up to 80% Recheck in 1-3 months (up to 80%

normalize)normalize)

C-reactive protein (CRP)C-reactive protein (CRP)

• Pentameric proteinPentameric protein– Trace concentrations in human plasmaTrace concentrations in human plasma– Highly conserved over hundreds of Highly conserved over hundreds of

millions of years of evolutionmillions of years of evolution– Properties of recognition and activationProperties of recognition and activation

•Activates classic complement pathwayActivates classic complement pathway

•Modulates behavior of phagocytic cells (both Modulates behavior of phagocytic cells (both inflammatory and non-inflammatory inflammatory and non-inflammatory influence)influence)

CRPCRP

• Acute phase reactant produced by Acute phase reactant produced by liverliver– Response to IL-6, other cytokinesResponse to IL-6, other cytokines

• Rises and falls quicklyRises and falls quickly– Elevation within 4 hr of tissue injuryElevation within 4 hr of tissue injury– Peak at 24-72 hrPeak at 24-72 hr– Half-life ~18 hrHalf-life ~18 hr

Rule of thumbRule of thumb

• CRP <0.2 mg/dL: normalCRP <0.2 mg/dL: normal

• CRP 0.2-1.0 mg/dL: indeterminate CRP 0.2-1.0 mg/dL: indeterminate (may be seen in smoking, DM)(may be seen in smoking, DM)

• CRP >1.0 mg/dL: inflammatoryCRP >1.0 mg/dL: inflammatory

• Levels > 10mg/dL suggest bacterial Levels > 10mg/dL suggest bacterial infection (up to 85%), or possibly infection (up to 85%), or possibly systemic vasculitis, metastatic cancersystemic vasculitis, metastatic cancer

Morely JJ, et al. Ann N Y Acad Sci;389,1982.

Serum protein electrophoresis Serum protein electrophoresis (SPEP)(SPEP)

• Quantifies the acute-phase responseQuantifies the acute-phase response– Increase in alpha-1 and -2 zones (alpha-Increase in alpha-1 and -2 zones (alpha-

1 antitrypsin and haptoglobin)1 antitrypsin and haptoglobin)– Increase in beta-gamma area Increase in beta-gamma area

(fibrinogen and CRP)(fibrinogen and CRP)– Decrease in pre-albumin, albumin, and Decrease in pre-albumin, albumin, and

the beta zone (transferrin)the beta zone (transferrin)

Normal SPEPNormal SPEP

erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG

SPEP- acute inflammationSPEP- acute inflammation


SPEP- Polyclonal SPEP- Polyclonal gammopathy gammopathy


Antinuclear antibodies Antinuclear antibodies (ANA)(ANA)

• Initial LE test in 1940sInitial LE test in 1940s– Incubate bare nucleus with pt’s serum, Incubate bare nucleus with pt’s serum,

allowing ANAs to bind to nucleusallowing ANAs to bind to nucleus– Then add normal PMNs and if sufficient Then add normal PMNs and if sufficient

Ab have bound to nucleus, nucleus is Ab have bound to nucleus, nucleus is opsonized and PMNs engulf the materialopsonized and PMNs engulf the material

– LE cell is PMN containing phagocytosed LE cell is PMN containing phagocytosed nucleusnucleus

LE CellLE Cell

Current ANA measurementCurrent ANA measurement

• Fluorescence microscopyFluorescence microscopy– HEp-2 cells (derived from human epithelial HEp-2 cells (derived from human epithelial

tumor cell line) incubated with pt’s serumtumor cell line) incubated with pt’s serum– Fluoresceinated Ab added, binds to pt’s Fluoresceinated Ab added, binds to pt’s

Abs bound to nucleusAbs bound to nucleus– Amount of ANA determined by dilution of Amount of ANA determined by dilution of

the pt’s serum - the greater the dilution the pt’s serum - the greater the dilution (titer) at which nuclear fluorescence (titer) at which nuclear fluorescence detected, the higher the ANA concentration detected, the higher the ANA concentration

ANAANA

• Arbitrary definition of positive ANA is Arbitrary definition of positive ANA is the level that exceeds that seen in the level that exceeds that seen in 95% of the population95% of the population

• Titers usually “positive” at 1:40 to Titers usually “positive” at 1:40 to 1:801:80

• Clinically significant titers (with HEp-Clinically significant titers (with HEp-2 cells) ~1:1602 cells) ~1:160

ANAANA

• High sensitivity in SLE, but poor High sensitivity in SLE, but poor specificityspecificity

• Positive ANA has predictive value of Positive ANA has predictive value of only 11% (positive LR =2.2)only 11% (positive LR =2.2)

• ANA found in 5-10% of pts without ANA found in 5-10% of pts without CTDCTD– Healthy pts, chronic infections (e.g., Hep Healthy pts, chronic infections (e.g., Hep

C), multiple meds, etc.C), multiple meds, etc.

ANAANA

• ConditionCondition– SLESLE– Drug induced lupusDrug induced lupus– MCTDMCTD– Autoimmune liver Autoimmune liver

dzdz– Sjogren’s syndromeSjogren’s syndrome– PolymyositisPolymyositis– RARA

• % ANA-positive% ANA-positive– 99%99%– 95-100%95-100%– 95-100%95-100%– 60-100%60-100%– 75-90%75-90%– 30-80%30-80%– 30-50%30-50%

Adapted from Hobbs, K in West, S Rheumatology Secrets, 2002.

ANAANA

• ConditionCondition– Multiple sclerosisMultiple sclerosis– Pts with silicone Pts with silicone

breast implantsbreast implants– Healthy relatives of Healthy relatives of

pts with SLEpts with SLE– NeoplasmsNeoplasms– Normal elderly (>70 Normal elderly (>70

yrs)yrs)

• % ANA-positive% ANA-positive– 25%25%

– 15-25%15-25%

– 20%20%

Adapted from Hobbs, K in West, S. Rheumatology Secrets, 2002

ANAANA

• Is the ANA a good screening test for SLE?Is the ANA a good screening test for SLE?– If >5% of normal U.S. population has positive If >5% of normal U.S. population has positive

ANA, then over 12.5 million “normal” people ANA, then over 12.5 million “normal” people in U.S. are ANA positivein U.S. are ANA positive

– Prevalence of SLE is only ~1/1000, so only Prevalence of SLE is only ~1/1000, so only 250,000 individuals with SLE and positive 250,000 individuals with SLE and positive ANAANA

– If entire population was screened, more If entire population was screened, more normal individuals would be detected with normal individuals would be detected with positive ANA than SLE pts. by ~50:1positive ANA than SLE pts. by ~50:1

Hobbs, K. in West, S Rheumatology Secrets. 2002..

ANAANA

• Clinical value of ordering an ANA test Clinical value of ordering an ANA test can be dramatically enhanced when can be dramatically enhanced when there is a reasonable pre-test there is a reasonable pre-test probability of an autoimmune probability of an autoimmune diseasedisease

ANA patternsANA patterns

• Homogeneous (diffuse)Homogeneous (diffuse)– SLE, drug-induced SLE, other diseasesSLE, drug-induced SLE, other diseases


• Rim (peripheral)Rim (peripheral)– SLE, autoimmune hepatitisSLE, autoimmune hepatitis


• SpeckledSpeckled– SLE, MCTD, Sjogren’s, Scleroderma, SLE, MCTD, Sjogren’s, Scleroderma,

other dzother dz


• NucleolarNucleolar– Scleroderma, hepatocellular carcinomaScleroderma, hepatocellular carcinoma


• CentromereCentromere– Limited scleroderma (CREST)Limited scleroderma (CREST)

Drug-induced ANAsDrug-induced ANAs

• Common drugs that cause positive Common drugs that cause positive ANAsANAs– ProcainamideProcainamide– HydralazineHydralazine– PhenothiazinesPhenothiazines– DiphenylhydantoinDiphenylhydantoin– IsoniazidIsoniazid– QuinidineQuinidine

Lupus or ANA profileLupus or ANA profile

• If screening ANA is positive and If screening ANA is positive and additional info needed to further additional info needed to further delineate type of autoimmune delineate type of autoimmune diseasedisease

• In extremely rare instances, ANA In extremely rare instances, ANA may be negative but SS-A antibodies may be negative but SS-A antibodies may be detected in pts. with an SS-A may be detected in pts. with an SS-A associated diseaseassociated disease

Lupus ProfileLupus Profile

dsDNdsDNAA

RNPRNP SMSM SS-ASS-A SS-BSS-B CENTROMERECENTROMERE

SLESLE 60%60% 30%30% 30%30% 30%30% 15%15% RareRare

RARA (-)(-) (-)(-) (-)(-) RareRare RareRare (-)(-)

MCTDMCTD (-)(-) >95%>95% (-)(-) RareRare RareRare RareRare

ScleroderSclerodermama

(-)(-) Low Low titertiter

(-)(-) RareRare RareRare 10-15%10-15%

CRESTCREST (-)(-) (-)(-) (-)(-) (-)(-) (-)(-) 60-90%60-90%

Sjogren’sSjogren’s (-)(-) RareRare (-)(-) 70%70% 60%60% (-)(-)

Hobbs, K. in West, S Rheumatology Secrets. 2002.

Lupus ProfileLupus Profile

• Antibodies to dsDNA are associated with lupus Antibodies to dsDNA are associated with lupus nephritis, and often parallel disease activitynephritis, and often parallel disease activity

• Antibodies to SS-A/Ro and SS-B/La are Antibodies to SS-A/Ro and SS-B/La are commonly associated with Sjogren’s syndrome commonly associated with Sjogren’s syndrome

• Anti-Ro/SSA antibodies increase risk for Anti-Ro/SSA antibodies increase risk for neonatal lupus/congenital heart block (CHB), neonatal lupus/congenital heart block (CHB), especially when in conjunction with anti-La/SSB especially when in conjunction with anti-La/SSB Ab Ab – Overall risk is ~5%Overall risk is ~5%

Antibodies to ribonuclear Antibodies to ribonuclear protein (RNP)protein (RNP)

• Target is spliceosomal snRNPs in Target is spliceosomal snRNPs in nucleoplasmnucleoplasm

• Seen in SLE, scleroderma, mixed Seen in SLE, scleroderma, mixed connective tissue disease (MCTD)connective tissue disease (MCTD)

• High levels very suggestive of MCTDHigh levels very suggestive of MCTD– MCTD is overlap disease with features of MCTD is overlap disease with features of

SLE, scleroderma, and polymyositisSLE, scleroderma, and polymyositis

Anticentromere and SCL-70 Anticentromere and SCL-70 AbAb• Anticentromere AbAnticentromere Ab

– up to 98% pts with limited scleroderma up to 98% pts with limited scleroderma (CREST)(CREST)

– 22-36% pts with diffuse scleroderma22-36% pts with diffuse scleroderma

• Anti-SLC70 (anti-topoisomerase I)Anti-SLC70 (anti-topoisomerase I)– 22-40% pts with diffuse scleroderma22-40% pts with diffuse scleroderma

• longer disease duration, association with longer disease duration, association with cancer, pulmonary fibrosis, digital pitting cancer, pulmonary fibrosis, digital pitting scars, cardiac manifestationsscars, cardiac manifestations

Anti-dsDNA Ab prior to Dx of Anti-dsDNA Ab prior to Dx of SLESLE

• Serum from 130 SLE patientsSerum from 130 SLE patients– 55% had anti-dsDNA Ab prior to SLE Dx55% had anti-dsDNA Ab prior to SLE Dx– Mean onset of Ab 2.7 years prior to DxMean onset of Ab 2.7 years prior to Dx

(range <1mo-9.3 years)(range <1mo-9.3 years)– 58% of cases with at least 2 positive 58% of cases with at least 2 positive

samples had significant rise in anti-samples had significant rise in anti-dsDNA within 6 months of DxdsDNA within 6 months of Dx

M. R. Arbuckle, et al. Scandinavian Journal of Immunology 54 (1-2) , 211–219.

Evaluation of pt with positive Evaluation of pt with positive ANA and generalized ANA and generalized arthralgiasarthralgias• H & P - any signs of CTD?H & P - any signs of CTD?

• If ANA titer If ANA titer >> 1:160, consider lupus 1:160, consider lupus profileprofile

• Other possible tests: CBC, CMP, C3, Other possible tests: CBC, CMP, C3, C4, SPEP, RF, ESR, UA, lupus C4, SPEP, RF, ESR, UA, lupus anticoagulant, anticardiolipin anticoagulant, anticardiolipin antibodyantibody

Antiphospholipid antibodiesAntiphospholipid antibodies

• Heterogeneous group of Ab that bind Heterogeneous group of Ab that bind to plasma proteins, have affinity for to plasma proteins, have affinity for phospholipid surfacesphospholipid surfaces– Anticardiolipin Ab (ACL)Anticardiolipin Ab (ACL)– Lupus anticoagulant (LAC)Lupus anticoagulant (LAC)– Beta 2-glycoprotein IBeta 2-glycoprotein I


• ACL measured by ELISA assay for ACL measured by ELISA assay for IgG, IgM, and IgA isotypesIgG, IgM, and IgA isotypes

• LAC measured by phospholipid-LAC measured by phospholipid-dependent screening test, if dependent screening test, if prolonged, add 1:1 mix with normal prolonged, add 1:1 mix with normal plasma - if no correction, LAC present plasma - if no correction, LAC present

• Beta 2-glycoprotein I measured by Beta 2-glycoprotein I measured by ELISAELISA


• Conditions with positive aPL Conditions with positive aPL – ~8% normal population~8% normal population– chronic infections e.g., HIV, Hep Cchronic infections e.g., HIV, Hep C– Medications e.g., phenothiazines, Medications e.g., phenothiazines,

hydralazine, phenytoin, procainamide, hydralazine, phenytoin, procainamide, quinidinequinidine

– ~20% pts. with systemic vasculitis~20% pts. with systemic vasculitis– ~15% pts. with recurrent miscarriage~15% pts. with recurrent miscarriage– ~50% pts. with SLE~50% pts. with SLEHansen, KE. in West, S Rheumatology Secrets, 2002.


• ~50% pts. with SLE and aPL will ~50% pts. with SLE and aPL will develop a thrombotic eventdevelop a thrombotic event

• ~3-7% pts. per year who have aPL will ~3-7% pts. per year who have aPL will experience a new thrombotic eventexperience a new thrombotic event

• Overall positive predictive value of an Overall positive predictive value of an aPL for future CVA, venous thrombosis, aPL for future CVA, venous thrombosis, or recurrent MC is between 10-25%or recurrent MC is between 10-25%

Hansen, KE. in West, S. Rheumatology Secrets, 2002.

CryoglobulinsCryoglobulins

• Immunoglobulins that precipitate in Immunoglobulins that precipitate in cold temperaturescold temperatures

• May cause hyperviscosity or vasculitisMay cause hyperviscosity or vasculitis

• Symptoms include fatigue, Symptoms include fatigue, arthralgias/arthritis, cutaneous arthralgias/arthritis, cutaneous vasculitis or purpura, neuropathies, vasculitis or purpura, neuropathies, visceral organ involvement, and visceral organ involvement, and digital ischemiadigital ischemia

CryoglobulinsCryoglobulins

• Type I- Monoclonal Ig (IgG or IgM)Type I- Monoclonal Ig (IgG or IgM)– Lymphoproliferative disordersLymphoproliferative disorders

• Type II- Monoclonal IgM directed Type II- Monoclonal IgM directed against polyclonal IgGagainst polyclonal IgG– Majority associated with Hepatitis CMajority associated with Hepatitis C

• Type III- Mixed polyclonal IgG and IgMType III- Mixed polyclonal IgG and IgM– Connective tissue diseases, chronic Connective tissue diseases, chronic

infectionsinfections

Anticytoplasmic Antibodies Anticytoplasmic Antibodies

• Often more helpful in diagnosis than Often more helpful in diagnosis than antibodies against nuclear antigensantibodies against nuclear antigens

• Seen with multiple autoimmune Seen with multiple autoimmune diseases and several forms of diseases and several forms of vasculitisvasculitis

Anticytoplasmic antibodiesAnticytoplasmic antibodiesDiseaseDisease CytoplasmicCytoplasmic

AntigenAntigenFrequencyFrequency

PolymyositisPolymyositis tRNA synthetase tRNA synthetase (anti-Jo-1, etc)(anti-Jo-1, etc)

20-30%20-30%

SLESLE Ribosomal PRibosomal P 5-10%5-10%

Wegener’s Wegener’s granulomatosisgranulomatosis

Serine Serine proteinase-3proteinase-3

(in neutrophils)(in neutrophils)

90%90%

Microscopic Microscopic polyarteritispolyarteritis

MyeloperoxidaseMyeloperoxidase

(in neutrophils)(in neutrophils)70%70%

Primary biliary Primary biliary cirrhosiscirrhosis

MitochondriaMitochondria 80%80%

Hobbs, K. in West, S. Rheumatology Secrets. 2002.

Anti-neutrophil cytoplasmic Anti-neutrophil cytoplasmic Antibodies (ANCA)Antibodies (ANCA)

• C-ANCAC-ANCA– Most commonly seen in Wegener’s Most commonly seen in Wegener’s

granulomatosis, microscopic granulomatosis, microscopic polyarteritis, rarely Churg-Strauss polyarteritis, rarely Churg-Strauss vasculitisvasculitis

ANCAANCA

• P-ANCAP-ANCA– seen in multiple diseases as well as seen in multiple diseases as well as

vasculitisvasculitis

P-ANCAP-ANCA

• MPO positiveMPO positive– Microscopic Microscopic

polyarteritispolyarteritis– Pauci-immune GNPauci-immune GN– Churg-Strauss Churg-Strauss

vasculitisvasculitis– Drug-induced Drug-induced

syndromessyndromes

• MPO negativeMPO negative– Ulcerative colitisUlcerative colitis– Autoimmune Autoimmune

disease disease – HIVHIV– Chronic infections Chronic infections

or neoplasms (rare)or neoplasms (rare)

ANCA ANCA

• If pt. tests positive to ANCA, If pt. tests positive to ANCA, evaluation of specific antigen testing evaluation of specific antigen testing for MPO and PR3 should be for MPO and PR3 should be undertakenundertaken

• If C-ANCA is not against PR3 or P-If C-ANCA is not against PR3 or P-ANCA is not against MPO, must ANCA is not against MPO, must consider causes other than vasculitisconsider causes other than vasculitis

Rheumatoid factor Rheumatoid factor

• Autoantibody directed against the Fc Autoantibody directed against the Fc (constant) region of an IgG molecule(constant) region of an IgG molecule– Multiple isotypes, including IgM, IgG, Multiple isotypes, including IgM, IgG,

IgA, and IgEIgA, and IgE– IgM RF is routinely measured using latex IgM RF is routinely measured using latex

agglutination titers, nephelometry, and agglutination titers, nephelometry, and ELISAELISA

Rheumatoid factorRheumatoid factor

• Very low levels normal, but higher Very low levels normal, but higher production secondary to chronic production secondary to chronic immune stimulationimmune stimulation

• RF positive in ~80% of patients with RF positive in ~80% of patients with RARA

• Multiple other causes of positive RFMultiple other causes of positive RF

Conditions associated with a Conditions associated with a positive rheumatoid factorpositive rheumatoid factor

• Rheumatologic Rheumatologic diseasesdiseases– RA (80-85%)RA (80-85%)– Sjogren’s (75-95%)Sjogren’s (75-95%)– MCTD (50-60%)MCTD (50-60%)– Scleroderma (20-30%)Scleroderma (20-30%)– Sarcoidosis (15%)Sarcoidosis (15%)– Polymyositis (5-10%)Polymyositis (5-10%)

• Non-rheumatologic Non-rheumatologic conditionsconditions– Chronic hepatitis Chronic hepatitis – Pulmonary diseasePulmonary disease– NeoplasmsNeoplasms– AgingAging– Cryoglobulinemia Cryoglobulinemia

(40-100%)(40-100%)– InfectionsInfections

• AIDS, Mono, TB, AIDS, Mono, TB, syphilis, parasites, syphilis, parasites, endocarditisendocarditis

Adapted from Kathryn Hobbs, from Rheumatology Secrets, 2002, p.60.

Frequency of RF positivity in Frequency of RF positivity in normal populationnormal population

• AGEAGE– 20-60 years20-60 years– 60-70 years60-70 years– >70 years>70 years

• Frequency of +RFFrequency of +RF– 2-4%2-4%– 5%5%– 10-25%10-25%

Adapted from Kathryn Hobbs in West, S. Rheumatology Secrets, 2002.

Anti-CCP antibodiesAnti-CCP antibodies

• ELISA assay based on filaggrin from ELISA assay based on filaggrin from human skin or synthetic citrullinated human skin or synthetic citrullinated peptidespeptides

• Target amino acid in filaggrin is Target amino acid in filaggrin is citrulline, a post-translationally citrulline, a post-translationally modified arginine residuemodified arginine residue

• High specificity and moderate High specificity and moderate sensitivity for RAsensitivity for RA

Anti-CCP antibodiesAnti-CCP antibodies

• Sensitivity 68% for RASensitivity 68% for RA

• Specificity 98% for RASpecificity 98% for RA

• Can be seen in active TB, other CTDCan be seen in active TB, other CTD

• Clinical implicationsClinical implications– Predictive of more aggressive disease Predictive of more aggressive disease

with more progressive joint damage with more progressive joint damage

Early antibody production as Early antibody production as indicator of future disease?indicator of future disease?

• Longitudinal study of 79 RA patientsLongitudinal study of 79 RA patients– ~50% produced anti-CCP Ab and/or IgM-~50% produced anti-CCP Ab and/or IgM-

RF prior to onset of diseaseRF prior to onset of disease– Positive results occurred median of 4.5 Positive results occurred median of 4.5

years (range 0.1-13.8) before symptom years (range 0.1-13.8) before symptom onset onset

– Elevated levels of either IgM-RF or anti-Elevated levels of either IgM-RF or anti-CCP may imply high risk for CCP may imply high risk for development of RAdevelopment of RAM. J. Nielen, et al. Arthritis Rheum 50:380, 2004.

ComplementComplement

• Cascade of proteins activated by Cascade of proteins activated by many agents, including immune or many agents, including immune or antigen-antibody complexesantigen-antibody complexes

• May be decreased due toMay be decreased due to– Increased consumption (proteolysis)Increased consumption (proteolysis)

• Increased levels of circulating immune Increased levels of circulating immune complexes activate classical pathwaycomplexes activate classical pathway

– Decreased productionDecreased production•Hereditary deficiency or liver diseaseHereditary deficiency or liver disease

Hereditary complement Hereditary complement deficienciesdeficiencies

• May see SLE-like disease with May see SLE-like disease with deficiencies in C1-C4deficiencies in C1-C4

• Terminal complement (C5-9) Terminal complement (C5-9) deficiencies lead to recurrent deficiencies lead to recurrent infectionsinfections

• Deficiency in C1 INH leads to Deficiency in C1 INH leads to angioedema (hereditary or acquired)angioedema (hereditary or acquired)

Diseases associated with low Diseases associated with low complement levelscomplement levels

• Rheumatic diseasesRheumatic diseases– SLE, systemic vasculitis, cryoglobulinemia, SLE, systemic vasculitis, cryoglobulinemia, RA (rare)RA (rare)

• GlomerulonephritisGlomerulonephritis– Post streptococcal and Post streptococcal and

membranoproliferativemembranoproliferative

• Infectious diseasesInfectious diseases– Bacterial sepsis, SBE, Hepatitis B, other Bacterial sepsis, SBE, Hepatitis B, other

viremias, parasitemiasviremias, parasitemias

Complement level Complement level assessmentassessment

• C3 and C4 generally decreased with C3 and C4 generally decreased with increased disease activity in SLEincreased disease activity in SLE

• Decreased levels may predict Decreased levels may predict impending disease flaresimpending disease flares– C4 lowers before C3 and remains lower C4 lowers before C3 and remains lower

longerlonger

• CH50 not useful as disease activity CH50 not useful as disease activity markermarker

Serum uric acid levelsSerum uric acid levels

• Age- and sex-dependentAge- and sex-dependent

• Concentration levels rise with Concentration levels rise with puberty in males and menopause in puberty in males and menopause in femalesfemales

• Age of onsetAge of onset– Peak for males: 40-50 yearsPeak for males: 40-50 years– Peak for females: >60 yearsPeak for females: >60 years


• HyperuricemiaHyperuricemia– > 7.0 mg/dL in males> 7.0 mg/dL in males– >6.0 mg/dL in females>6.0 mg/dL in females

• 24 hour urine collection24 hour urine collection– Urate >800 mg/24 hrs suggests Urate >800 mg/24 hrs suggests

overproductionoverproduction– Urate <800 mg/24 hrs suggests Urate <800 mg/24 hrs suggests

underexcretionunderexcretion


• Important considerationsImportant considerations– Only 15% of pts. with hyperuricemia Only 15% of pts. with hyperuricemia

develop goutdevelop gout– If uric acid level>10mg/dL, risk If uric acid level>10mg/dL, risk

increases to 30-50%increases to 30-50%– In ~10% of patients with acute gout, In ~10% of patients with acute gout,

serum uric acid levels are normalserum uric acid levels are normal•Need joint aspiration and polarized light Need joint aspiration and polarized light

microscopy to diagnose with certaintymicroscopy to diagnose with certainty

Asymptomatic Asymptomatic hyperuricemiahyperuricemia

• Treatment indicationsTreatment indications– Acute overproduction e.g., tumor lysis Acute overproduction e.g., tumor lysis

syndromesyndrome– Severe hyperuricemia e.g., uric acid Severe hyperuricemia e.g., uric acid

levels >12mg/dL levels >12mg/dL •Risk of uric acid nephrolithiasis is ~50%Risk of uric acid nephrolithiasis is ~50%

HLA-B27 HLA-B27

• SensitivitySensitivity– ~95% for AS~95% for AS– ~80% for Reactive Arthritis~80% for Reactive Arthritis– ~70% for SpA associated with psoriasis~70% for SpA associated with psoriasis– ~50% for SpA associated with IBD~50% for SpA associated with IBD– ~70-84% for uSpA~70-84% for uSpA

Shmerling RH. Geriatrics;51:22, 1996.

HLA-B27HLA-B27

• SpecificitySpecificity– Low given prevalence is ~8% in Low given prevalence is ~8% in

Caucasian populationCaucasian population

• In patients with inflammatory back In patients with inflammatory back pain, HLA-B27 positivity yields pain, HLA-B27 positivity yields – 20-fold increased risk of SpA 20-fold increased risk of SpA – 15-fold higher risk of radiological 15-fold higher risk of radiological

sacroiliitissacroiliitisBraun J, et al. Arthritis Rheum;41:58, 1998.

Synovial fluid analysisSynovial fluid analysis

• Studies to performStudies to perform– Gram stain and cultureGram stain and culture– Total leukocyte count with differentialTotal leukocyte count with differential– Polarized microscopyPolarized microscopy

Synovial fluid analysisSynovial fluid analysisFluid typeFluid type AppearanAppearan

ceceTotal Total WBCWBC

Count/Count/mmmm33

%PMNs%PMNs

NormalNormal Clear, Clear, viscousviscous

0-2000-200 <10%<10%

Non-Non-inflammatorinflammatoryy

Clear to Clear to sl. turbidsl. turbid

200-2000200-2000 <20%<20%

InflammatoInflammatoryry

Slightly Slightly turbidturbid

2000-2000-50,00050,000

20-70%20-70%

PyarthrosisPyarthrosis TurbidTurbid >50,000>50,000 >70%>70%

Adapted from Spencer, RT in West, S. Rheumatology Secrets, 2002

Synovial fluid analysisSynovial fluid analysis

• Noninflammatory joint effusionsNoninflammatory joint effusions– OA, joint trauma, mechanical derangement, OA, joint trauma, mechanical derangement,

AVNAVN

• Inflammatory synovial fluidInflammatory synovial fluid– Multiple rheumatic disordersMultiple rheumatic disorders– Infectious arthritis Infectious arthritis

• PyarthrosisPyarthrosis– Joint sepsisJoint sepsis– Pseudosepsis in gout, reactive arthritis or RAPseudosepsis in gout, reactive arthritis or RA

Polarized light microscopyPolarized light microscopy

GoutGout PseudogoutPseudogout

Crystal Crystal Monosodium Monosodium urate (MSU)urate (MSU)

Calcium Calcium pyrophosphate pyrophosphate dihydrate dihydrate (CPPD)(CPPD)

ShapeShape NeedleNeedle Rhomboid or Rhomboid or rectangularrectangular

BirefringenceBirefringence NegativeNegative PositivePositive

Crystal color Crystal color parallel to axisparallel to axis

YellowYellow BlueBlueAdapted from Spencer, RT in West, S. Rheumatology Secrets, 2002

CPPD and MSU crystalsCPPD and MSU crystals

ConclusionsConclusions

• Immunologic laboratory tests facilitate Immunologic laboratory tests facilitate diagnosis and provide information diagnosis and provide information regarding specific disease manifestations, regarding specific disease manifestations, disease activity and prognosis disease activity and prognosis

• Clinical utility of laboratory evaluation can Clinical utility of laboratory evaluation can be enhanced by the employment of be enhanced by the employment of evidence-based guidelinesevidence-based guidelines

• A thorough history and physical A thorough history and physical examination remain the best screening examination remain the best screening and diagnostic tools and diagnostic tools

References

1. ACR AD HOC Committee on Immunologic testing in the rheumatic diseases: an introduction. Arthritis Care and Research. August 15, 2002, Vol. 47, No. 4 pp.429-433.

2. Gitlin JD, Colten HR: Molecular biology of the acute phase plasma proteins. In Pick E, Landy M [eds]: Lymphokines. Vol. 14. San Diego, Academic Press, 1987, pp 123-153.)

3. Morley JJ, Kushner I: Serum C-reactive protein levels in disease. Ann N Y Acad Sci 389:406-418, 1982.

4. Macy EM, Hayes TE, Tracy RP: Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 43:52-58, 1997.

5. Morely JJ, et al. Serum C-reactive protein levels in disease. Ann N Y Acad Sci 1982;389:406-418.

6. M. R. Arbuckle, J. A. James, K. F. Kohlhase, M. V. Rubertone, G. J. Dennis, J. B. Harley (2001) Development of Anti-dsDNA Autoantibodies Prior to Clinical Diagnosis of Systemic Lupus Erythematosus. Scandinavian Journal of Immunology 54 (1-2) , 211–219.

7. M. J. Nielen, et al. Specific Autoantibodies Precede the Symptoms of Rheumatoid Arthritis. A Study of Serial Measurements in Blood Arthritis Rheum 2004,50:380-386.

8. Shmerling RH. Rheumatic disease: choosing the most useful diagnostic tests. Geriatrics 1996;51:22-6,29-30,32.

9. Braun J, Bollow M, Remlinger G et al. Prevalence of spondylarthropathiesin HLA-B27 positive and negative blood donors. .Arthritis Rheum 1998;41:58–67.

References

10. Sheldon, J. Laboratory testing in autoimmune diseases. Best Pract Res Clin Rheum. 2004; 18;3; 249-69.

11. Dorner, T and Hansen, A. Autoantibodies in normals- the value of predicting rheumatoid arthritis. Arthritis Res and Therapy.2004;6,5.

12. Lane, SK and Gravel, JW. Clinical utility of common serum rheumatologic tests. American Family Physician. 2002; 65,6.

13. Harris, E et al. Kelley’s textbook of rheumatology, Ed. 7th Ed., 2006.14. Kavanaugh, A and ACR AD HOC committee. Guidelines for immunologic

laboratory testing in the rheumatic diseases: anti-DNA antibody testing. Arthritis Care and Res. 2002;47;5;546-55.

15. Shojania, K. Rheumatology:2. What laboratory tests are needed? CMAJ 2000;162 (8):1157-63.

16. Zochling, J et al. The current concept of spondyloarthropathies with special emphasis on undifferentiated spondyloarthropathies. Rheumatology (Oxford) 2005;44:1483.

17. Schellekens GA, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrillunated peptide. Arthritis and Rheumatism 2000;42:155-163.

Documents

1. Common Rheumatologic Tests: Evaluation and Interpretation