Chemistry, toxicology & urinalysis

Contributed by Cody Orahoske, Ph.D.


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Antinuclear antibodies (ANAs) (ANA Patterns: International Consensus on Antinuclear Antibody (ANA) Patterns [Accessed 5 March 2025])

• Many systemic autoimmune rheumatic diseases are characterized by the presence of 1 or more autoantibodies
• Autoantibodies can target various cellular components, including the surface, cytoplasm, nuclear envelope, nucleus or cell cycle specific structures
• ANA can be detected in several autoimmune diseases, including systemic lupus erythematosus, Sjögren syndrome, systemic sclerosis, rheumatoid arthritis, idiopathic inflammatory myopathies, mixed connective tissue disease, lupus, lupoderma, sclerodermatomyositis, systemic vasculitis, undifferentiated connective tissue disease, Behçet disease and autoimmune syndromes induced by adjuvants
• A positive ANA test alone is not diagnostic of any autoimmune disease, as it can also be seen in patients with other clinical conditions and even in healthy individuals
• A negative indirect immunofluorescence (IIF) ANA result reduces the likelihood of some systemic autoimmune diseases but does not definitively rule them out

Systemic lupus erythematosus (Ann Rheum Dis 2019;78:736)

• Autoimmune disease involving multiple organs, in which injury is caused mainly by the deposition of immune complexes and binding of antibodies to various cells and tissues
• Autoimmune disease affecting multiple organs, where tissue injury primarily results from immune complex deposition and antibody binding to various cells and tissues
• Can present as acute or chronic injury, affecting the skin, joints, kidneys and serosal membranes
• Heterogeneous presentation, with patients exhibiting a wide range of clinical features
• Prevalence is ~1 in 2,500, with a significantly higher frequency in women (1 in 700; F:M = 9:1)
• Typically manifests in the second or third decade of life but can occur at any age
• Driven by immune complex mediated tissue injury, cytotoxic T cell activation and an overactive immune response
• Underlying mechanism is a failure to maintain self tolerance
• HLA-DQ locus has been linked to the production of ANA (small risk)
  • Lupus nephritis (Nat Rev Dis Primers 2020;6:7)
  • Caused by inflammatory response to immunogenic, endogenous chromatin and apoptosis derived macrovesicles

Sjögren syndrome (primary and secondary) (Ann Rheum Dis 2020;79:3)

• Chronic autoimmune disease characterized by dry eyes and dry mouth due to the destruction of the lacrimal and salivary glands
• Primary form, known as sicca syndrome, occurs as an isolated disorder
• Secondary form is associated with other autoimmune diseases, most commonly rheumatoid arthritis
• Marked by a reduction in tear and saliva production, resulting from lymphocytic infiltration and fibrosis of the lacrimal and salivary glands
• Exact underlying mechanism remains largely unknown
• Most commonly affects women in their fifth to sixth decade of life

Systemic sclerosis (scleroderma) (Ann Rheum Dis 2017;76:1327)

• Chronic inflammation is believed to result from autoimmunity, leading to widespread damage to small blood vessels and progressive interstitial and perivascular fibrosis affecting the skin and multiple organs
• Excessive fibrosis can occur throughout the body, impacting the skin, gastrointestinal tract, kidneys, heart, muscles and lungs
• The disease is thought to arise from 3 key processes: autoimmune responses, vascular damage and abnormal collagen deposition
• 2 major categories
  • Diffuse scleroderma
    • Widespread skin involvement with rapid progression and early visceral involvement
  • Limited scleroderma
    • Skin involvement is often confined to fingers, forearms and face
• Limited scleroderma can progress to CREST syndrome, a condition characterized by a combination of calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia

Rheumatoid arthritis (RA) (Lancet 2016;388:2023)

• Chronic autoimmune disorder that primarily targets the joints, leading to nonsuppurative proliferative and inflammatory synovitis
• Often progresses to articular cartilage destruction and, in some cases, ankylosis
• The autoimmune response is initiated by CD4+ helper T cells, with disease progression driven by autoantibodies targeting self antigens and cytokine mediated inflammation
• Key cytokines involved include IFNγ, IL17, RANKL, TNF and IL1
• Anticitrullinated peptide antibodies (ACPA) serve as diagnostic markers and can be detected in up to 70% of rheumatoid arthritis (RA) patients
• Rheumatoid factors (RF) are autoantibodies (IgM and IgA) that bind to the Fc region of IgG and are present in ~80% of affected individual

Inflammatory myopathies (polymyositis, dermatomyositis, inclusion body myositis) (J Intern Med 2016;280:8)

• Dermatomyositis is characterized by damage to small blood vessels, which contributes to muscle injury
  • Vasculopathic changes may be observed in the nail folds, eyelids, gums and as capillary dropout in skeletal muscle
  • Disease involves elevated inflammatory markers and evidence of direct immunologic injury
• Polymyositis is an adult onset inflammatory myopathy similar to dermatomyositis but lacking distinctive cutaneous features
  • In the affected muscle, CD8+ cytotoxic T cells are present and vascular injury does not play a significant role
• Inclusion body myositis typically affects late adulthood, usually after the fifth decade of life
  • It presents with slow, progressive muscle weakness, most severe in the quadriceps and upper extremity muscles
  • Unlike dermatomyositis and polymyositis, inclusion body myositis is associated with abnormal protein aggregates

Mixed connective tissue disease (Immunotargets Ther 2023;12:79)

• Anti-U1 ribonucleoprotein is commonly found in mixed connective tissue disease and contributes to immune dysfunction, leading to the activation of both T and B cells
• Mixed connective tissue disease is a combination of disorders that share clinical features of systemic lupus erythematosus (SLE), systemic sclerosis and polymyositis
• It may present with Raynaud phenomenon, myositis and renal involvement

Antineutrophil cytoplasmic antibodies (ANCAs) (Nat Rev Rheumatol 2019;15:91)

• Antineutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that target antigens within the primary granules of neutrophils, lysosomes of monocytes and endothelial cells
• ANCAs are highly useful as diagnostic markers, with their titers generally correlating with clinical severity
• cANCA (cytoplasmic ANCA) targets proteinase 3, also known as PR3 ANCA
• pANCA (perinuclear ANCA) targets myeloperoxidase, renamed as MPO ANCA
• ANCAs can directly activate neutrophils, leading to the release of reactive oxygen species and proteolytic enzymes

ANCA associated vasculitis (AAV) (Nat Rev Dis Primers 2020;6:71)

• Granulomatosis with polyangiitis (GPA)
  • Formerly known as Wegener granulomatosis
  • Characterized by acute necrotizing granulomas in the upper or lower respiratory tract
  • Associated with necrotizing or granulomatous vasculitis, primarily affecting small and medium sized vessels
  • Focal necrotizing glomerulonephritis is common
  • Can present as localized or systemic
  • In most cases, PR3 ANCA is present
• Microscopic polyangiitis (MPA)
  • Also known as leukocytoclastic vasculitis
  • Necrotizing vasculitis affecting capillaries, venules and small arterioles
  • A key feature of MPA is that all lesions tend to be of the same age
  • Skin, mucous membranes, lungs, brain, heart, gastrointestinal tract, kidneys and muscle can all be involved
  • In most cases, MPO ANCA is present
• Churg-Strauss syndrome (eosinophilia, asthma and granulomas)
  • Small vessel necrotizing vasculitis associated with asthma, allergic rhinitis, lung infiltrates, peripheral eosinophilia, extravascular necrotizing granulomas and eosinophilic infiltration of vessels and tissues
  • Features include hyperresponsiveness to allergic stimuli, cutaneous involvement, gastrointestinal bleeding and renal disease
  • Can also involve myocardial eosinophilic infiltrates, leading to cardiomyopathy
  • In some cases, MPO ANCA may be present

Test System / Manufacturer	Document	Effective Date
Pharmacia & Upjohn Varelisa ReCombi ANA Profile (dsDNA, RNP, Sm, SSA / Ro, SSB / La, Scl-70, centromere, Jo-1)	K050625	4/26/2005
Sweden Diagnostics Varelisa ReCombi CTD Screen EIA Kit (dsDNA, RNP, Sm, SSA / Ro, SSB / La, Scl- 70, histone, centromere, ribosomal P, Jo-1)	K050967	7/6/2005
DiaSorin Anti-Nuclear Antibody (ANA) Screen Kit (manual / qualitative)	K932876	4/11/2006
Euroimmun ANA IFA: Hep-20-10 Kit (qualitative and semiquantitative)	K070763	6/20/2007
Varelisa ANA CTD Screen (dsDNA, RNP [RNP70 A C], Sm [B B' D], SSA / Ro [52, 60 kDa], SSB / La, Scl-70, CENP B, histone, ribosomal P protein, Jo-1)	K050967	7/12/2007
AESKULISA ANA HEP-2 (dsDNA, histones, SSA, SSB, Sm, SmRNP, Scl-70, Jo-1, centromere)	K081104	5/19/2008
TheraTest Laboratories EL-ANA / 7 (ssDNA, dsDNA, Sm, RNP / Sm, SSA, SSB, chromatin)	K051066	9/4/2008
TheraTest Laboratories EL-ANA/9 (ssDNA, dsDNA, Sm, RNP / Sm, SSA, SSB, Scl-70, chromatin)	K051066	9/4/2008
Phadia Varelisa ReCombi ANA Screen (dsDNA, U1RNP (RNP70 A C), Sm, SSA / Ro, SSB / La, Scl- 70, CENP B, Jo-1)	K083188	3/16/2009
AESKUSlides ANA-HEp-2	K120889	11/27/2012
Euroimmun ANA Screen ELISA (IgG) (dsDNA, histone, ribosomal P, SSA, SSB, Sm, SmRNP, Scl-70, Jo-1, centromere)	K131185	7/19/2013
Gold Standard Antinuclear Antibody (ANA) Screen ELISA Test (dsDNA, histone, ribosomal P, SSA [Ro60 and Ro52], SSB / La, Sm, SmRNP, Scl-70, Jo-1, centromere, lysed Hep2 extract)	K131330	1/30/2014
Euroimmun ANA IFA Biochip Test System	K131791	3/4/2014
INOVA NOVA Lite DAPI ANA Kit (manual fluorescence microscope)	CR150047	4/17/2015
INOVA NOVA View Automated Fluorescence Microscope (automated fluorescence microscope)	CR150047	4/17/2015
Euroimmun EUROPattern Microscope and Software (manual and semi-automated)	CR140252	4/27/2015
Inova Diagnostics HEp-2 External EB Kits (IFA)	CR150517	11/25/2015
TheraTest Laboratories EL-ANA Profiles (EL-ANA / 4, 2 plate kit; 5 plate kit; ssDNA, dsDNA, RNP / Sm, chromatin)	CR150571	1/7/2016
Image Navigator by Immuno Concepts Microscope and Software (manual and semi-automated fluorescence microscope)	CR160266	6/23/2016
HELIOS® Automated IFA System (instrument & software; manual and semi-automated)	CR150547	8/2/2016
IMMCO Diagnostics Inc. ImmuGlo HEp-2 Elite IFA (manual fluorescence microscope)	CR170488	6/8/2018
Zeus dIFine Digital Scanner and Software (instrument & software; manual and semi-automated)	CR200352	5/13/2022

Reference: U.S. Food and Drug Administration: CLIA - Clinical Laboratory Improvement Amendments [Accessed 5 March 2025]

• Immunofluorescence is a powerful detection method widely used in clinical laboratories for various applications, including fluorescence polarization immunoassays, fluorescence resonance energy transfer, direct immunofluorescence and indirect immunofluorescence
• These techniques are employed to analyze biomarkers in serum, plasma, urine and tissue
• Indirect immunofluorescence specifically utilizes a secondary detection antibody that binds to the antibody antigen complex
• After this interaction, the slide is examined under a fluorescence microscope
• If the antibody antigen complex is present, the detection antibody binds to it, emitting fluorescence
• Observed fluorescent patterns are correlated with specific markers, aiding in disease diagnosis

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A 42 year old woman presents with Raynaud phenomenon, skin tightening on her fingers and difficulty swallowing. Laboratory testing reveals a positive antinuclear antibody (ANA) with a titer of 1:1280. The ANA pattern seen in the image shown above is observed. Which of the following diagnoses is most likely?

1. Mixed connective tissue disease
2. Rheumatoid arthritis
3. Sjögren syndrome
4. Systemic lupus erythematosus
5. Systemic sclerosis

E. Systemic sclerosis. The centromere pattern seen here in the ANA test is associated with limited systemic sclerosis, particularly in patients with clinical signs such as Raynaud phenomenon and skin tightening. While systemic sclerosis can present with other patterns like homogeneous, speckled and nucleolar, in this case, the centromere pattern is most often associated with limited systemic sclerosis. Answer D is incorrect because systemic lupus erythematosus (SLE) typically presents with a homogenous or peripheral staining pattern rather than centromere. Answer C is incorrect because Sjögren syndrome typically shows an ANA pattern associated with SSA or SSB antibodies, not centromere. Answer A is incorrect because mixed connective tissue disease usually shows a speckled or nucleolar pattern rather than centromere. Answer B is incorrect because rheumatoid arthritis typically does not present with a high titer ANA and is more commonly associated with anti-CCP antibodies.

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Reference: Immunofluorescence for ANA and ANCA

A 55 year old woman presents with complaints of fatigue, pruritus, abdominal pain and dryness. Upon physical examination it is noted that she has mild jaundice and abdomen tenderness but no signs of organomegaly or palpable masses. The lab results show a positive ANA (pictured above) and her liver function test showed elevated alkaline phosphatase and bilirubin levels. Which of the following diagnoses is most likely?

1. Mixed connective tissue disease
2. Primary biliary cholangitis
3. Rheumatoid arthritis
4. Systemic lupus erythematosus
5. Systemic sclerosis

B. Primary biliary cholangitis. The multiple nuclear dots pattern seen in the ANA test is frequently associated with primary biliary cholangitis, a condition that often presents with fatigue, pruritus and liver dysfunction. Answer D is incorrect because systemic lupus erythematosus (SLE) typically presents with a homogenous or speckled ANA pattern rather than multiple nuclear dots. Answer E is incorrect because systemic sclerosis usually shows a centromere or nucleolar pattern, not multiple nuclear dots. Answer A is incorrect because mixed connective tissue disease is more commonly associated with a speckled or centromere pattern. Answer C is incorrect because rheumatoid arthritis typically does not present with a high titer ANA and is more often associated with anti-CCP antibodies, not a multiple nuclear dots pattern.

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Reference: Immunofluorescence for ANA and ANCA