Specimen Collection

3.5 mL SST Serum (Preferred)

4 mL Plain Serum

250 uL Microsample Serum
Reference Intervals

Normal < 1:80

Turnaround Time: Between 3 days and 5 days

Turn around time is working days as Virology and Immunology do not routinely work weekends or public holidays.

Diagnostic Use and Interpretation

Antinuclear antibodies (ANA) react with antigens located in the cell nucleus. The determination of an ANA frequently plays a key role in the differential diagnosis of connective tissue and autoimmune disease. Virtually all cases of systemic lupus erythematosus (SLE) have a positive ANA and therefore a negative ANA has a strong negative predictive value for ruling out SLE. ANA are not useful for monitoring the activity of SLE and should only be ordered for diagnostic purposes.

ANA are typically non-specific and, particularly at low titre, can be found in many conditions and in healthy (particularly older) persons. It can be found at high titre in a number of autoimmune conditions but is only diagnostically useful in a limited number of these conditions. The exception is the cell line used at LabPLUS which is termed Hep-2000 (as opposed to the conventional Hep-2). The Hep-2000 line is transfected with SSA antigen which is expressed as a very specific pattern that easily allows it's recognition by trained personnel.

Please note: From 22 January 2018, homogeneous and speckled ANA patterns will be checked for the presence of DFS70 antibody and the results reported in Concerto under the ANA header. DFS70 antibody will produce a typically high titre ANA pattern that could be interpreted as clinically significant but, to date, no SARD association has been identified for DFS70 antibody.

DFS antibody will be automatically tested if the appropriate ANA result is observed and cannot be requested separately.

An ANA should therefore generally be ordered if any of the following conditions are being considered:

1. SLE (primary or drug induced)
2. Mixed connective tissue disease (MCTD) or overlap syndrome
3. Systemic sclerosis, limited or diffuse
4. Inflammatory myositis (Polymyositis, Dermatomyositis)
5. Sjogren's Syndrome
6. Autoimmune hepatitis
7. Juvenile Idiopathic Arthritis

Therefore ANA should generally only be ordered in the following clinical situations when they are not explained otherwise:

1. Systemic inflammatory signs that are not related to infection
2. Mouth ulcers, hair loss
3. Chronic suggestive rashes or erythema (photosensitive, urticarial)
4. Inflammatory arthritis
5. Sicca Symptoms (Xerostomis, Xerophthalmia)
6. Unexplained serositis (pleuritis, pericarditis)
7. Possible autoimmune liver disease
8. Glomerular disease (haematuria, casts)
9. Myositis (raised CK and weakness)
10. Skin thickening
11. Raynaud's phenomenon
12. Interstitial lung disease
13. Leukopenia, lymphopenis or haemolytic anaemia not otherwise explained

ANA should not be ordered in the following situations:

1. For monitoring a connective tissue disease
2. Screening for CTD when someone already has an autoimmune condition
3. 'Tiredness'
4. Other forms of arthritis
5. Gout
6. OA and lower back pain
7. Other symptoms/signs that remain unexplained

Usually ANA specimens are automatically analysed for antibodies to extractable nuclear antigens (ENA) and double stranded DNA but this pattern of analysis is often modified due to requestor preference.

No diagnosis can be based on a single ANA result, with other clinical and laboratory criteria required for a definitive diagnosis. Therefore, even high titre ANAs should be assessed in conjunction with other clinical data.

Certain drugs e.g. anticonvulsants, anti-tuberculous, anti-tumour necrosis factor agents, and antihypertensive drugs may induce a lupus-like condition resulting in high ANA titres. This type of lupus usually goes into sustained remission following removal of the triggering medication.

It is important to understand that ANAs can predate the onset of clinically detectable disease, in some cases by many Years.

The following has been adapted from the International Consensus on ANA patterns (ICAP) and outlines nuclear, mitotic and cytoplasmic patterns that may be observed either individually or in combination.

[www.anapatterns.org]

ANA HEP-2000 CELL LINE PATTERNS

Nuclear	Mitotic	Cytoplasmic
Homogenous	Centrosome/Centromere	F-Actin-like
Speckled	Intercellular bridge/Midbody	Mitochondrial antibody-like
Dense fine speckled	Mitotic spindle apparatus	Ribosomal-P-like
Nuclear dots (ND)		Jo-1-like
Nuclear membrane (NM)
Nucleolar
Cell-cycle

The patterns in the above diagram can be seen both independently and in combination. When seen in combination, all patterns are reported.

All cytoplasmic patterns observed require specific second round testing to confirm the presence of the suspected antibody

For both NM and ND patterns second round specific testing for both gp210 (NM) and sp100 (ND) antibodies will be performed.

Nuclear patterns

Homogeneous

The pattern is associated with SLE or drug-induced LE. It can also be seen in other autoimmune conditions such as autoimmune hepatitis.

Speckled

This pattern may be associated with connective tissue disease but also other non-specific chronic inflammatory conditions.

Dense Fine Speckled

This pattern description is only used when the presence of DFS70 antibody has been confirmed by specific second round testing. When the pattern occurs in isolation it is non-specific and not associated with systemic autoimmune disease.

Centromere

This pattern is associated with patients who have CREST syndrome which is a variant of PSS.

Nuclear Dots

This pattern can be sub-divided into few (1-6) and many (6-20) discrete dots per interphase cell. In either case, second round testing for sp100 antibodies will occur as, in the correct clinical context this antibody has a strong association with PBC. In PBC patients who are sp100 antibody positive there is an increased rate of disease progression. Apart from PBC, ND's can be seen in both connective tissue disorders and other inflammatory disorders.

Nuclear Membrane

The pattern is associated with a number of autoimmune mediated diseases, namely AIH, PBC, SLE and APS. An antigen that can give rise to a NM pattern is gp210. The antibody directed against gp210 is specific for PBC and , PBC patients who are gp210 antibody positive, typically have a more aggressive and severe form of the disease.

Nucleolar

Sub-division of the 'nucleolar' staining is possible into 'nucleolar homogeneous', 'nucleolar clumpy' and 'nucleolar speckled'. This sub-classification requires dedicated staff and digital imaging capacity which LabPLUS currently doesn't have so pattern reporting will be 'Nucleolar'. The sub-classification implies the following antigens with their disease associations:

Nucleolar Homogeneous: PM-ScL Myositis -scleroderma overlap (up to 50%)

PSS, Myositis (5-10%)

Nucleolar Clumpy: Fibrillarin High specificity for PSS but low (5%) frequency

Nucleolar Speckled: RNA polymerase 1 High specificity for PSS (up to 20%) with particular association with the diffuse cutaneous form with cardiac and renal involvement

Cell cycle

Cell-cycle dependant patterns are observed and reported in three variants:

(a) Cell-cycle dependant (non-CENP-F/PCNA): This pattern has no disease association

(b) CENP-F-like: These antibodies have been associated with neoplasms, most notably lung and breast cancer

(c) PCNA - like: These antibodies have a very high specificity for SLE but are seen in low frequency (approximately 5%). They have also been reported in patients with chronic HBV and / or HCV infections.

Mitotic patterns

Centrosome / Centriolar

This is a rare non-specific pattern but has been observed in patients with Raynaud's syndrome, CREST, PSS, undifferentiated connective tissue diseases and in some post-infectious neurological syndromes

Intercellular bridge / Midbody /MSA-2

This is a rare pattern and is limited to patients with PSS and Raynaud's syndrome

Mitotic Spindle Apparatus / NuMA / MSA-1

This is a clinically non-specific pattern, but has been reported in patients with a variety of autoimmune diseases including SLE, Sjogren?s syndrome, Sharp?s syndrome and Polyarthritis

Cytoplasmic patterns

F-Actin

A component of smooth muscle (SMA), F-Actin antibodies have a high specificity for autoimmune hepatitis type 1. The preferred method for SMA detection is IIF using mouse stomach substrate as opposed to ANA testing. In the setting of a reactive SMA result (typically at high titre) further specific second-round testing would be required for confirmation of the presence of F-Actin antibodies.

Mitochondrial antibodies (AMA)

Presumptive AMA cytoplasmic-based IIF patterns are confimed by line immunoassay (LIA). There are two reactive outcomes namely

(a) AMA-M2: These are the 'classical' AMA subunit [2] antibodies that have a very high diagnostic specificity for PBC (>95%)

(b) The recombinant fusion protein 3E-BPO which also has high specificity for PBC (>90%)

It should be noted that is cases of suspected PBC the appropriate serological test is for Tissue antibodies rather than ANA

Ribosomal-P

This cytoplasmic - based pattern is specific for SLE. At LabPLUS, suspected cases of Ribo-P reactivity require confirmation by second - round specific antibody testing using either luminex methodology and / or LIA. Ribo-P antibody results are reported under the ENA group.

Please note that only approximately 30% of Ribo-P antibody patients will demonstrate cytoplasmic staining so the appropriate test for this antibody is ENA.

Jo-1

Jo-1 antibody is considered a diagnostic marker for inflammatory myositis. This cytoplasmic-based pattern is seen in 24-30% of patients with idiopathic myositis / overlap syndrome and is often associated with interstitial lung disease (even with mild or no myopathic features). At LabPLUS, suspected cases of Jo-1 reactivity require confirmation by second - round specific antibody testing using either luminex methodology and / or LIA. Jo-1 antibody results are reported under the ENA group.

Please note that only approximately 30% of Jo-1 antibody patients will demonstrate cytoplasmic staining so the appropriate test for this antibody is ENA.

Contact Information

For further information contact the laboratory, (09) 307 4949 ext 22103 or:
Associate Professor Rohan Ameratunga , Immunopathologist: Locator 93-5724,

Dr Richard Steele , or The LabPLUS Immunology Team