APPROACH TO A PATIENT WITH POSITIVE ANA LEVELS

CHAIRPERSON:- Dr. H. S. SandhuSpeaker:- Dr. Prabhpreet Gill

ANA Anti-nuclear antibodies (ANAs, also known as

anti-nuclear factor or ANF) are autoantibodies directed against various contents of the cell nucleus.

ANA is not a routine investigation, done to evaluate any body function or failure.

It is a measure of on going autoimmune process with/without related autoimmune disease and hence, needs strong suspicion based on proper history, thorough clinical evaluation and ruling out other common causes, leading to patients distress before labeling it an autoimmune disease.

AUTOIMMUNITY AND AUTOIMMUNE DISEASES

Autoimmunity represents the end result of the breakdown of one or more of the basic mechanisms regulating immune tolerance.

The essential feature of A/I disease is presence of tissue injury caused by the immunological reaction with self antigens.

Autoimmunity on the other hand refers merely the presence of a/b or T-lymphocytes that react with self antigens which may/maynot develop any pathological consequences. This self reactivity may be either the cause or the consequence of an on going pathological process.

A/I DISEASE IS AN INTERACTION B/W GENES, ENVIRONMENT AND IMMUNE REGULATION

ARE POSITIVE ANA LEVELS ALWAYS ASSOCIATED WITH ILLNESS ? No. ANAs can be found in approximately 5% of the

normal population, usually in low titers (low levels). These people usually have no disease. Titers of lower than 1:80 are less likely to be significant. (ANA titers of less than or equal to 1:40 are considered negative.) Even higher titers are often insignificant in patients over 60 years of age.

Ultimately, the ANA result must be interpreted in the specific context of an individual patient's symptoms and other test results. It may or may not be significant in a given individual.

ANA TESTING Fluorescent Antinuclear Antibody Test or FANA. An ANA report has three parts: positive or negative. if positive, what’s the titre. the pattern of fluorescence.

Serum from the patient's blood specimen is added to microscope slides which have commerically prepared cells on the slide surface. If the patient's serum contains antinuclear antibodies (ANA), they bind to the cells (specifically the nuclei of the cells) on the slide.

A second antibody, commercially tagged with a fluorescent dye, is added to the mix of patient's serum and commercially prepared cells on the slide. The second (fluorescent) antibody attaches to the serum antibodies and cells which have bound together. When viewed under an ultraviolet microscope, antinuclear antibodies appear as fluorescent cells.

If fluorescent cells are observed, the ANA (antinuclear antibody) test is considered positive.

A titre is determined by repeating the positive test with serial dilutions until the test yields a negative result. The last dilution which yields a positive result (fluorescence) is the titre which gets reported. For example, if a titre performed for a positive ANA test is:1:10 positive1:20 positive1:40 positive1:80 positive1:160 positive1:320 negative

The reported titre will be 1:160.

SIGNIFICANCE OF FLUORESCENCE

HOMOGENOUS PATTERN(TOTAL NUCLEAR FL.)

RIM/PERIPHERAL PATTERN (A/B DIRECTED AGAINST DSDNA )

SCATTERED PATTERN(CENTROMERE) A/B AGAINST CENTROMERE

SCATTERED PATTERN (NUCLEOLAR) A/B AGAINST VARIOUS PROTEINS

ANA IS POSITIVE IN :- Systemic lupus erythematosus (lupus or SLE)

- over 95% Progressive systemic sclerosis (scleroderma)

- 60-90% Rheumatoid Arthritis - 25-30% Sjogren's syndrome - 40-70% MCTD - 95% Juvenile arthritis - 15-30%

Other causes:-, autoimmune hepatitis polymyositis and dermatomyositis Addison disease Idiopathic thrombocytopenic purpura Hashimoto's thyroiditis Autoimmune haemolytic anaemia Type I diabetes mellitus

SINCE ANA IS TO BE EVALUATED INDIVIDUALLY FOR EACH PATHOLOGY, SO HERE IS APPROACH FOR COMMON CAUSES OF ANTINUCLEAR ANTIBODY POSITIVITY

35 YRS OLD FEMALE,P/W DISCOID AND MALAR RASH , JOINT PAINS AND G.MALAISE

SLE If clinical features like:- malar rash, discoid

rash, photosentivity, serositis, proteinuria, sezuire /psychosis, anemia/leucopenia /thrombocytopenia, oral ulcers, arthritis are present then go for ANA testing along with routine investigations.

Key points before approach:-ANA test is positive in 95% of the patients(sensitive marker). Its specificity is 70%.

ANA may be positive 2-3 years before symptoms appear. So pt should be followed 6 monthly.

A few patients develop ANA +vity with in year of symptom onset, repeated testing may thus be useful.

ANA – lupus may exits but is very rare in adults and is usually associated with other a/b like anti Ro and anti dsDNA.

Ant-dsDNA and anti-sm are specific to SLE but only anti-dsDNA correlates with disease activity and with occurrence of glomerulonephritis. A titre of 1:10 is considered +ve. So, it has a prognostic value. It is +ve in 60-80% with active lupus.

Anti-ds-DNA antibody is not found in drug-induced lupus.

Others a/b :- anti-RNP, anti-Ro and anti-La (not specific for SLE, seen in sicca syndrome. Ass with dec risk of lupus nephritis.), anti-phospholipids, anti-platelet, anti-erythrocyte, anti-ribosomalP (CNS lupus)

DRUGS CAUSING LUPUS Anti arrhythmics:- procainamide,

disopyramide, propafenone Anti hypertensives:- ACE(I)/captopril ,

B#/acebutolol/atenolol, diltiazem, hydralazine

Anti thyroid drug:- propylthiouracil Neuroleptics like chlorpromazine, lithium Anti epileptic :-carbemazapine, phenytoin Anti tubercular:- isoniazide, rifampicin Others :- sulphasalazine, lova/simvastatin,

IFN, TNF (I),mino/tetracycline, pencillamine, tiotropium bromide inhaler, ophthalmic timolol etc

DRUG INDUCED LUPUS

Predominant in caucasians, Appear during therapy with certain

medications. Less female susceptability than SLE. Rarely

involves kidneys and/or brain. Rarely ass with anti-dsDNA(review diagnosis

if +ve) , commonly ass with ant-histone a/b (over 95%)

Resolves after several weeks of discontinuation of medications

NOTE

Except for hydralazine where incidence of DIL is 5-7%, rest all drugs though commonly used, rarely causes SLE. Hence, treatment history is very important before specific diagnosis.

Things to be kept in mind :- pt should not be symptomatic before start of these drugs.

Symptoms should take atleast any time b/w 3 weeks to 2 years to appear.

Pt is most likely anti-histone +ve and always anti-dsDNA –ve…….

42 YEARS FEMALE P/W LOSS OF WRINKLES, SKIN TIGHTENING AND…….

RAYNAULDS PHENOMENON I.E. SINCE 2-3 YEARS SHE COMPLAINS, ON EXPOSURE TO COLD /STRESS, HER HANDS GO

PALE

THEN TURN BLUE

RED

NOW SHE DEVELOPED DIGITAL GANGRENE AND SEVERE ISCHAEMIC PAIN

SYSTEMIC SCLEROSIS Furter ,Check for features of ILD/PAH, proteinuria, IHD

or conduction abnormalities. These all finding favour PSS.

Antinuclear antibodies are present in the sera of over 90% of patients with systemic sclerosis. Antibody to native DNA is either lacking or present in extremely low titer.

50-96% of pts with lSS have detectable anticentromere antibody. In contrast, anticentromere antibody is found in less than 10% of individuals with diffuse scleroderma.

B/w 20-40% of individuals with systemic sclerosis and diffuse scleroderma have serum antibody reactive with an extractable nuclear antigen of 70 kDa termed Scl-70. The antigen has been definitely characterized as DNA topoisomerase I, involved in the initial uncoiling of supercoiled DNA prior to transcription.

PT P/W RHEUMATOID NODULES

FIXED JOINT DEFORMITIES

RHEUMATOID ARTHRITIS

With clinical suspicion of RA, we go for routine lab investigations, ESR, CRP, RA and ANA.

Levels of ESR and CRP are usually high in rheumatoid arthritis and may be good indicators of the extent of disease activity at any given time organs in the body.

The majority of people have a positive RF(80%) result , during periods of active disease.

A positive ANA result indicates an unusually active immune system.

About 40% of people with rheumatoid arthritis have a positive ANA result.

In the first few months of onset of rheumatoid arthritis, these immunologic tests may be negative, and, in some patients, they are always negative.

ANA levels stand no where in diagnosis and prognosis of RA as against RF, ant-cyclic citrullinated a/b(mostly +ve in seronegative RA), CRP and ESR.

FELTY SYNDROME

This is a variant of seropositive RA with SM, LAP, granulocytopenia and/or thrombocytopenia.

ANA is more commonly +ve in FS than in RA. Specific antinuclear antibodies identified are

SS-A or SS-B. ANCA is seen in the sera of 33% of patients

with Felty's syndrome and is absent in RA.

55YRS FEMALE P/W GRITTY SENSATION IN EYES, B/L PAROTID SWELLING, DRY TONGUE & SORE THROAT

SJOGREN’S SYNDROME H/o recurent eye and chest infection +nt.

ANA- ANA +

other confirmatory tests specific antinuclear a/b

About 70% of Sjögren’s patients have a positive ANA test

result. 70% pts are positive for SS-A and 40% are positive for SS-B.

Negative tests donot r/o diagnosis, which can be further confirmed on H/p of parotid glands (show CD+4 lymphocytic infiltrates.) Positive shimers I test and Rose bengal test.

Others are:-Parotid sialography, salivary scinctigraphy or unstimulated whole salivary flow less than 1.5ml in 15 min.

AUTOIMMUNE HEPATITIS One has to rule out genetic disorders, viral markers,

drug toxicity and alcohol abuse, before suspecting AIH.

Factors favouring AIH:- 1. Female sex 2. Predominant AT elevation 3. Presence and elevation of gammaglobulins levelsThen we go for ANA levels, if +ve in high titers in

absence of antimicrobial a/b and viral markers, we strongly suspect AIH(type-1, most common, seen in adults).

If is ANA –ve, we investigate for smooth ms and LKMI a/b.(+ve in type 2)

An association with extrahepatic autoimmune diseases such as rheumatoid arthritis, autoimmune thyroiditis, ulcerative colitis, diabetes mellitus and a family history of autoimmune or allergic disorders has been reported in AIH.

ANA IN PBC

ANA are also detected in PBC, they frequently display unique immunofluorescence patterns such as nuclear dots (i.e., SP100) or a nuclear ring-like pattern(Laminins, gp210). While in autoimmune hepatitis the predominant ANA pattern is a homogeneous or speckled immunofluorescence appearance.

Specific a/b are AMA.

35 yrs old male Pt p/w gum bleeding and multiple small petechiae all over body more so on LL. H/o loose stools +nt 3 wks ago, for which he took t/t. lab investigations shows mild aneamia and thrombocytopenia.

PBF :- DECREASE IN PLATELET NUMBER AND INCREASED SIZE OF PLATELETS

ITP After ruling out any drug induced or current

infection as the cause of thrombocytopenia, we go for BM examination(which revealed inc in megakaryoblasts), we label the pt as ITP

To further classify it into , 1’ or 2’ look for HIV, HCV infection and ANA levels,

if neg then it 1’ ITP.ANA levels are +nt in 25-32% of 2’ ITP.

MISCARRIAGE IN PREGNANCY There are five reasons for miscarriage which

have been identified: Cause :- Infection   1%, 1. Anatomy abnormal   5-10% 2. Progesterone level low   20% 3. Chromosome abnormal :Primary

miscarrier (no live births)   7%    :Secondary

miscarrier (one or more live births)   50% 4. Immune mechanisms   50% 5. Unknown   15%

When the immune system is the cause of miscarriage, the chances of mother having a successful pregnancy without treatment after three miscarriages is 30%, after four miscarriages 25%, and after 5 miscarriages 5%.

Some of the autoantibodies in sequence are :- Antiphospholipid antibodies.

Antinuclear antibodies. Natural killer cells. Cytotoxic B-cells.. Most ANA+ women with recurrent miscarriages

do not have clinical signs of SLE.The miscarriage rate in SLE patients is much

higher than that of the general population.

Women with ANA are treated with prednisone, that does not cross the placenta easily and is also broken down by enzymes in the placenta so that the fetus is exposed to only trace amounts. Additionally, the body produces the equivalent of 8 mg per day of this corticosteroid. When indicated, Prednisone should be started prior to conception.

MCTD Mixed connective tissue disease (MCTD) is a

systemic, autoimmune connective tissue disease. Clinically, patients exhibit varied combinations of features common to other autoimmune diseases such as systemic lupus erythematosus (SLE), polymyositis, rheumatoid arthritis, or systemic sclerosis (scleroderma). MCTD is often referred to as an overlap syndrome.

ANA are +ve in over 95% of pt. A/b specific to it are anti-snRNP 70 a/b (90%).

SUMMARY ANA is an indirect fluorescent antibody test for the

semi-quantitative detection of antinuclear antibody in human serum.

Diagnosis cannot be made on the basis of antinuclear antibody detection alone. The physician must interpret these results inconjunction with the patient's history and symptoms, the physical findings, and other diagnostic procedures.

Treatment should not be initiated on the sole basis of a positive test for antinuclear antibodies.

Clinical indications, other laboratory findings, and the physician's clinical impression must be considered before any treatment is initiated.