RHEUMATIC DISEASE LABORATORY TESTING

BASICS OF LAB TEST ORDERING

Pitfalls: Misdiagnosis Permanent labeling

of patient Needless additional

testing Inappropriate therapy

and/or referral Expense Overlooking the

correct diagnosis NEEDLESS ANXIETY

FOR PATIENT!!!

Intent: Establish a

diagnosis Determine prognosis Monitor disease

activity Indicate organ

involvement Monitor stage of

disease Indicate disease

mechanisms Guide treatment Monitor toxicity

BASICS OF LAB TEST ORDERING

“We checked an ANA, ESR, & RF to look for connective tissue disease…..”

TWO BY TWO TABLE

DIS (+)

DIS(-)

TEST (+)

A (TP)

B (FP)

PPV =A/A+B

TEST(-)

C (FN)

D (TN)

NPV =D/C+D

SENS =TP/TP+FN

SPEC = TN/FP+TN

DISEASE PREVALENCE & PREDICTIVE VALUES

SNOUT

A test with highSeNsitivity is excellentFor ruling OUT a dx…

“SNOUT”

Sensitivity is true posover true pos plusfalse negatives

SPIN

A test with a highSPecificity is great forRuling IN a dx

“SPIN”

Specificity is true negative over true negative plus false positive

TYPES OF LABS1. High sensitivity labs - SNOUT:

good for “screening” or “ruling out” ESR (inflammation) ANA (lupus – but NOT “CTD”)

2. High specificity labs - SPIN: good for “diagnosing” or “ruling in”

ds-DNA (lupus) Scl-70 (PSS) Jo-1 (ILD in polymyositis) C-ANCA (WG)

TYPES OF LABS3. Disease activity labs:

How is treatment working ? CPK complement levels (lupus) ds-DNA (lupus) ESR, CRP (RA, GCA, PMR)

4. Prognosis labs: Should I be aggressive early with meds?

RF (RA) ds-DNA antibody (SLE) Citrulline antibody (RA)

“THE NET” – looking for organ damage and mimickers UA with micro

glomerulonephritis CBC

anemia chronic disease Lipids

accelerated atherogenesis w/ inflammation Hep B & Hep C

extrahepatic disease features. TSH CPK Chem Panel and LFT’s ? Vitamin D (fibromyalgia)

ESR

ESR The king of non-specific lab tests

but very sensitive Infection / malignancy/CTD all raise

the ESR Westergren method used at

NMCSD (up to 120 mm/hr) Useful for “ruling out” systemic

inflammatory disease

ESR Non-inflammatory factors that raise ESR

Aging Female Sex Obesity Pregnancy Anemia

Markedly Low ESR (0 mm/hr) Afribinogenemia/dysfibrinogenemia Agammaglobulinemia Extreme polycythemia (HCT > 65) Increased plasma viscosity

ESR Adjunctive Testing

Good history and physical Repeat test 1-3 mo? Prior ESRs? CRP SPEP, Quantitative Immune Globulins Age Appropriate Malignancy Screen

CRP (“Can’t refuse prednisone”) Produced as an acute-phase reactant by the liver in response

to IL-6 and other cytokines Seen in sera of patients with pneumococcal pneumonia in

1930 (protein could precipitate C-polysaccharide of pneumococcus)

Can recognize phosphocholine, other phospholipids Can activate complement (classic) Can bind to and modulate the behavior of phagocytic cells in

both pro and anti-inflammatory ways Quicker rise and fall than ESR Sensitive but not diagnostic of any particular condition “CRP rises with infection and ESR rises with CTD’s….” (not

always true!!!) Normal less than 1.0 mg/dl > 8-10 mg/dl, think bacterial infection, systemic vasculitis, or

widely metastatic cancer

CRP

OTHER ACUTE PHASE REACTANTS Polyclonal Immunglobulins (SPEP) Alkaline phosphatase Transaminases Fibrinogen Haptoglobin Serum amyloid A Platelet count Ferritin Alpha-1 antitrypsin Decreased albumin (“negative” APR)

COMPLEMENT A system of interacting serum proteins that

function sequentially as initiators, regulators, and effectors of cell lysis and inflammation

Provide innate defense against microbes and a “complement” to humoral immunity

Biologic cascade in which, by limited proteolysis, one component activates the next, causing rapid and robust amplification of the system

Critical to normal processing of immune complexes

Causes host tissue damage in antibody-mediated autoimmune syndromes

COMPLEMENT Measurement useful if:

concern for inherited deficiency states which can pre-dispose to infectious and rheumatic syndromes

immune-complex mediated disease

COMPLEMENT

COMPLEMENT

CLASSICAL:

Ag-Ab complexes such as SLE

ALTERNATIVE:

EndotoxinIgADrugs

COMPLEMENT

COMPLEMENT REGULATION C1 inhibitor – [prevents C1s from activating

C4 and C2] deficiency -> hereditary angioneurotic edema

and chronic C4 activation/consumption with resulting low C4

C3 and C4 nephritic factors Autoantibodies against alternative and classical

pathway C3 convertases, respectively Stabilizes antibody, increasing half-life causing

excessive C3 cleavage Secondary deficiency of C3

ROUTINE COMPLEMENT LEVEL TESTING

CH 50 reflects a functionally intact classic pathway;

good screen for deficiency state (C1-9) C3

useful screen for BOTH classic and alternative pathways

C4 depressed with activation of classic pathway or

in patients with angioneurotic edema C5-9

terminal attack complex

DIMINISHED COMPLEMENT LEVELS

partial or complete inherited deficiency C1q inhibitor deficiency -> hereditary

angioedema and low C4 C2 or C4 deficiency -> SLE C3 deficiency -> recurrent infections (bacterial) C5a inhibitor deficiency -> FMF C5-9 deficiency -> recurrent neisserial

infxn

immune complex consumption (SLE / post-strep GN, cryoglobulinemia, SBE,

membranoproliferative GN, Sjogren’s, other vasculitides)

RHEUMATOID FACTOR

RHEUMATOID FACTOR Hemagglutinating

activity noted in 1940, associated with RA in 1949

Heterogeneous family of IgM abys directed against IgG

RF response transiently associated with many infectious diseases (TB, SBE, syphillis, HCV)

RF response more permanent with chronic diseases

1-4% of healthy whites, 10-25% age > 70 , 30% some NA Indians

80% of RA pts (+) 80-90% of SS (+) 70% of chronic HCV (+) Both Types II and III

cryoglobulins worse prognosis and

more aggressive RA Not specific for RA No correlation with RA

disease activity

RHEUMATOID FACTOR

RHEUMATOID FACTOR

Elevated RF (ChRONIC immune stimulation) Ch- chronic disease (esp hepatic

and pulmonary) R- rheumatoid arthritis O- other connective tissue disease N- neoplasms (lymphoproliferative

diseases, esp after XRT, chemo) I – Infections C - cyroglobulins

ANTI-CITRULLINE AB ELISA available against CCP (cyclic

citrullinated peptide) – one of the anti-filaggrin antibodies

Very specific, not very sensitive (SPIN) Utility may be in defining early and

aggressive RA, prior to development of RF Useful in RF (+) positive conditions that

mimic RA Sjogren’s Hep C cryoglobulinemia

ANA ANA’s are directed against a variety of

nuclear antigens in serum of patients with/without rheumatic disease as well as in normal persons

The ANA IS NOT a useful screen for rheumatic disease (nor is the RF) not sensitive enough (SNOUT) not specific enough (SPIN)

The ANA IS a useful screen for SLE is very sensitive-95% (SNOUT) Offers less in area of specificity (SPIN)

ANA many false positives

5% of healthy controls pos at 1:160 dilution 10-15% of healthy controls pos at 1:80 dilution 30% of healthy controls pos at 1:40 dilution positive with SBE / age / liver disease / thyroid dz

should be done on HEp2 cell line pattern is important and helps w/ dx titer doesn’t correlate with disease activity a negative ANA “rules out” SLE most of the

time

ANA TESTINGPre-testAssessment

ANA Your Action

Low likelihood

< 1:80> 1:160

IgnoreObserve; look elsewhere

Moderatelikelihood

< 1:80> 1:160

Observe; look elsewhereDisease specific antibodies

Highlikelihood

negativepositive

Fill out consult to RheumDisease specific antibodies

ANA PATTERNS

NUCLEOLAR NUCLEOLAR

RIM RIM

SPECKLEDSPECKLED

HOMOGENEOUSHOMOGENEOUS

ANA PATTERNS

NUCLEOLAR Against RNA Nonspecific Associated with

PSS PM-DM SLE vasculitis

RIM or PERIPHERAL Against DNA Specific for SLE Should trigger

check for ds-DNA SLE More active

disease Nephritis

ANA PATTERNS

CENTROMERE Associated with

CREST Calcinosis Raynaud’s Esophageal

Dysmotility Sclerodactyly Telangeictases

ANA PATTERNS

HOMOGENEOUS Against DNA &

histone Nonspecific Associated with

SLE Drug induced lupus RA Vasculitis PM-DM

SPECKLED Against the various

ENA’s “More” specific-

SPIN Associated with

SLE Sjogren’s PSS PM-DM RA

SPECIFIC ANA’s

ds-DNA Nearly 100%

specific for SLE Correlates well

with disease activity

Probably pathogenic

Moderate Sensitivity

SPECIFIC ANA’sSmith (Sm)

RNA/protein complex involved in processing messenger RNA

More severe disease Speckled ANA Higher prevalence in AA & Asians Moderate sensitivity (30%) Highly specific for SLE

SPECIFIC ANA’s

RNP (U1 RNP) Ribonucleoprotein Speckled ANA Diagnostic of

MCTD if: High titer No other ENA’s Features of SLE,

PM, RA, and PSS RF (+)

HISTONE High sensitivity

and low specificity Drug induced

lupus gives anti-histone alone

SLE gives anti-histone along with ds-DNA

Also seen in RA

SPECIFIC ANA’sRo/SS-A

Protein-RNA complex found in both nucleus and cytoplasm

Sjogren’s and lupus The rare ANA-neg

lupus Subacute cutaneous

lupus (a very photosensitive lupus)

Neonatal SLE and congenital heart block

La/SS-B Protein-RNA complex

found in both nucleus and cytoplasm

Sjogren’s and lupus The rare ANA-neg

lupus May protect against

renal disease Usually also have

antibodies to Ro/SS-A

Ro/SS-A & La/SS-B

SPECIFIC ANA’sSCL-70

Anti-topoisomerase I

Found in 20% of patients with PSS

VERY specific NOT sensitive Lung disease Associated with

nucleolar ANA pattern

Jo-1 Anti-histidyl tRNA

synthetase A cytoplasmic

protein Found in 30% of

PM/DM patients – often with lung involvement/ILD

Highly specific

CRYOGLOBULINS

CRYOGLOBULINSA group of serum Ig’s with conformationalchange in the cold:

Precipitate or gel on cold exposure

Phenomenon reversible with rewarming

Found in variety of clinical situations

Other labs: RF, low complement, QIG, ESR

TYPE I Single monoclonal Ig

or light chain TYPE II (essential)

“mixed” – a monoclonal directed against polyclonal IgG (rheumatoid factor activity)

TYPE III “polyclonal” – no

monoclonal Ig also RF activity

CRYOGLOBULINS Type I (myeloproliferative disorders)

Myeloma, lymphoma, Waldenstrom’s Type II

Infections (majority HCV and some HBV), CTD (Sjogren’s, lupus),

Type III CTD, Infection lymphoproliferative disorder

CRYOGLOBULINS “Essential mixed cryoglobulinemia” is

now recognized as driven by chronic Hep C in most cases.

Clinical features: Acrocyanosis / digital necrosis (type I) Palpable purpura (type II and III) Livedo reticularis Raynaud’s Arthritis / GN / peripheral neuropathies

HLA B-27 Class I MHC cell surface marker found in 6-8% of NA whites, 3-4% of NA

AA’s, 18-50% of Haida Indians 90-100% of AS pts/lesser frequency in other

spondyloarthropathies NOT a “screening” test for the SNSA’s

IBD-associated arthritis – 50% Ankylosing spondylitis (AS) – 90% Reactive / Reiter’s – 80% Psoriatic arthritis – 50% w/ spine; 15% peripheral

HLA B-27

ANTIPHOSPHOLIPID AB’s lupus anticoagulant

1. prolonged PTT or PT or final common pathway (DRVVT)

2. Failure to correct by mixing patient plasma w/ nml plasma

3. Correction with addition of excess phospholipid or platelets

4. Ruling out other coagulopathies anticardiolipin antibodies

via ELISA antibodies against beta-2 glycoprotein

Via ELISA

NEJM 7 Mar 2002

ANCAC-ANCA and P-ANCA

Originally defined by indirect IF staining of neutrophils C-ANCA

Cytoplasmic IF pattern

P-ANCA Perinuclear IF pattern

ANCA (“C3PO”)C-ANCA Ab against PR3

(proteinase-3) (found in neuts/monos)

Wegener’s granulomatosis

C-ANCA usually found in widespread WG

C-ANCA seen less frequently with limited WG

Correlates with dz activity in 60% of cases

P-ANCA Ab against MPO

(myeloperoxidase), elastase, lactoferrin, others

Churg-Strauss MPAN Crescentic GN (pauci-

immune GN) Drug-induced (PTU,

minocycline, hydralizine) IBD SLE / RA/HIV No correlation with

disease activity

ARTHROCENTESIS NORMAL/NONINFLAMMATORY (0-2,000 WBC)

Transparent / < 25% POLYS osteoarthritis / AVN / sympathetic effusion

INFLAMMATORY (2,000-60,000 WBC) Translucent / > 50% POLYS RA / SLE / crystal / spondyloarthropathies

PURULENT (80,000-100,000 WBC) Infection / predominantly POLYS

SUMMARY

No lab test is as good as your history and physical exam

No lab test “screens” for CTD’s Disease pattern recognition is far

more helpful than any serology or test Know the SENS and SPEC of lab tests

for different diseases Say “NO” to laboratory panels

QUESTIONS!!!