laboratory diagnosis, autoimmune disease

1. Guide: Dr.(Mrs.)M.V.Jadhav

2.  This topic will be covered under the following heads.
1. Concepts of autoimmune diseases
2. Theories of autoimmune diseases
3. Introduction
4. Spectrum of autoimmune diseases
5. Historical events
6. Indications
7. Lab work up for autoimmune diseases
8. Algorithmic approach
9. Tissue diagnosis
10. Conclusion.

3.  Autoimmunity refers to the presence of antibodies or T
lymphocytes that react with self antigens and do not
necessarily imply that the development of self
reactivity has pathogenic consequences.
 For pathological autoimmunity ideally three
requirements should be met
 The presence of an immune reaction specific for some
self Ag or self tissue
 Evidence that such a reaction is not secondary to tissue
damage
 Absence of another well defined cause of disease.
Concept of Autoimmune Diseases

4. Mechanisms preventing autoimmunity
 Central tolerance:
 elimination of autoreactive lymphocytes in central
lymphoid organ
 Peripheral tolerance:
 Anergy: through the absence of the second
(costimulatory) signal during CD4 activation
 Generation of supressor cells
 Periperal deletion of autoreactive cells
Theories of autoimmune diseases

5.  Susceptibility genes – breakdown of self tolerance
 Environmental triggers - infectious agent
 molecular mimicry(cross reactivity between microbial
protein and host tissue)
 Superantigenic stimulation
 Microbial adjuvanticity
 Polyclonal B-cell activation
Mechanism of autoimmunity

6. Spectrum/classification of
autoimmune disorders:
organ specific
Thyroid gland
 Graves’ disease
 Hashimoto’s thyroiditis
Pancreas
 Type I DM
 Insulin resistant DM
Adrenal gland
 Autoimmune Addison’s disease
SKIN
 Pemphigus Vulgaris
 Pemphigus Foliaceus
 Dermatitis Herpetiformis
 Autoimmune alopecia
 Vitiligo
Genital tract
Immune mediated infertility
Hematological
 Auto Immune Hemolytic Anemia
 Autoimmune Thrombcytopenic Purpura
GIT
 Pernicious Anemia
 UC
 CD
 Celiac disease
Neurological
 Myasthenia Gravis
 Multiple Sclerosis
 Gullian Barre syndrome
Heart
 Acute Rheumatic Fever.
Liver
 Autoimmune hepatitis
 Primary billiary cirrhosis
 Primary sclerosing cholangitis

7.  SLE
 Rheumatoid Arthritis
 Scleroderma/CREST
 Dermatomyositis &
polymyositis
 Mixed connective tissue
diseases
 Antiphospholipid syndrome
 Sjogren Syndrome
 Overlap syndrome
 Systemic vasculitis
 Takayasu’s arteritis
 Giant cell arteritis
 Wegener’s granulomatosis
 PAN
 Churg –strauss syndrome
 Systemic
In middle of the spectrum – goodpasteur disease

8.  1904-Donath and Landsteiner described a complement
– dependent autohemolytic serum antibody
 1948 - landmark year in the history of SLE. American
clinical haematologists Malcolm Hargraves and Robert
Morton,with laboratory technician Helen Richmond,
first describe LE cell.
 1957-IF-ANA was designed by George Friou
Historical events:

9. Indications
 To establish or rule out the diagnosis(SLE)
 To support clinical diagnosis (AS)
 To follow the course of disease, particularly to suggest
that a flare is occuring
 To detect the other organ or system involvement
 To plan current & near future therapy for treatment
 To monitor the effectiveness of treatments.
 To detect the various complications related to therapy

10. Laboratory work up for
autoimmune diseases

11.  Clinical evaluation helps in deciding plan of lab
investigation.
 Clinical pointers-
 Systemic autoimmune diseases:
 Chronic illness
 Multisystem involvement
 Vague symptomatology
 Clinical overlap i.e. an individual with one specific syndrome
is more likely to develop a second symdrome.eg. High
incidence of pernicious anemia in individuals with
autoimmune thyroiditis.
 Local autoimmune disease:
 Chronic illness and unexplained organ failure.

12. Means to overcome the diagnostic
problem
 Choose appropriate tests according to clinical
presentation for cost effectiveness and to reduce
labour intensity.
 Algorithmic approach

13.  For multi systemic disorders like SLE there are
criteria for classification based on clinical and
laboratory data
 Meant for differentiation from other Rheumatic
diseases.
 Set forth in 1972,ammended in 1982and modified in
1993 & in 1997
 Widely used to facilitate the diagnosis of SLE
Clinical classification

14. 1997 Revised Criteria for classification of SLE
(1) Malar Rash
(2) Discoid Rash
(3) Photosensitivity
(4) Oral ulcers usually painless
(5) Arthritis( nonerosive involving 2 or more peripheral joints)
(6) Serositis ( pleuritis or pericarditis)
(7) Renal Disorder
-persistent proteinuria>0.5g/dl or >3+ or
-cellular casts (red cells, Hb, granular, tubular or mixed)
(8) Neurologic – seizures or psychosis without other causes
(9) Haematologic
-hemolytic anemia with reticulocytosis or
-leucopenia<4000/ul on 2 or more occasions or
-thrombocytopenia<1,00,000/ul in absence of offending drugs
-lymphopenia(<1500/ul)on 2 or more occasions
(10) Immunological disorder(Anti-dsDNA,Anti-Sm and antiphospholipid)
(11) ANA(an abnormal titre of ANA by immunoflorescence )
Person is said to have SLE if any 4or more of the 11 criteria present at any time in a
paient’s history.
Specificity – 95% sensitivity – 75%

15. The 1987 Revised Criteria for the Classification of RA
 1. Guidelines for classification
a) Four of seven criteria are required to classify a patient as having rheumatoid
arthritis (RA).
b) Patients with two or more clinical diagnoses are not excluded.
 2. Criteria
a) Morning stiffness: s lasting 1 h before maximal improvement.
b) Arthritis of three or more joint areas: At least three joint areas, observed by a
physician simultaneously, have soft tissue swelling or joint effusions, not just
bony overgrowth.
c) Arthritis of hand joints: Arthritis of wrist, metacarpophalangeal joint, or
proximal interphalangeal joint.
d) Symmetric arthritis:
e) Rheumatoid nodules:
f) Serum rheumatoid factor:
g) Radiographic changes: Typical changes of RA on posteroanterior hand and
wrist radiographs that must include erosions or unequivocal bony
decalcification localized in or most marked adjacent to the involved joints.
Criteria a–d must be present for at least 6 weeks. Criteria b–e must be observed by
a physician
Sensitivity – 91-94% & specificity-89%

16. Sjogren’s syndrome
The European Community Criteria require 4 of the following
6 items to be present for SS to be diagnosed:
 at least 1 specified ocular symptom;
 at least 1 oral symptom;
 abnormal findings on Schirmer's test or rose bengal stain;
 abnormal results on salivary gland biopsy;
 at least 1 abnormal result for unstimulated salivary flow
rate, sialography, or scintigraphy; and
 presence of at least 1 of the following antibodies,
rheumatoid factor, ANA, SS-A or SS-B

17.  Routine investigations - Urine examination
 Hematological investigations -routine & special
 Biochemical tests –S.proteins, LiverFunctionTests,
RenalFunctionTests ,Thyroid Function Tests.
 Demonstration of autoantibodies
 Serological tests -non specific- VDRL
 More specific serological tests like Rose-Waaler method, Latex
Agglutination, turbidometry , nephlometry , ELISA
 Immunoflourescence
 Study of HLA association
 Biopsy study . Routine H & E, immunoflourescence , electron
microscopy & immuno electro microscopy in kidney ,skin,
synovium ,liver etc
Lab investigation of autoimmune
disease

18. Irrespective of whether the autoimmune disease are
systemic or organ specific diagnostic problems are
unique.
 Diseases are present in combination.
 The lab testing is complex and often misunderstood.
 Clinical complexity of autoimmune disease is
proportional to the complexity of test results.
 Labour intensity of most of the tests
Problems with lab diagnosis of AID

19. ROLE OF
ROUTINE INVESTIGATIONS

20.  Reflection of renal pathology
 Proteinuria (indicative of glomerular filter malfunctioning )
 Active urine sediments ( indicative of active glomerular
damage- SLE , Vasculitis ).In such cases detailed renal
functions should be analyzed by biochemical tests.
 Hematuria
 Hemoglobinuria - occurs occasionally with an acute
exacerbation of hemolysis in AIHA( indicative of intravascular
hemolysis)
 Hemosiderinuria- an occasional finding in AIHA( indicative of
intravascular hemolysis)
 Oliguria /anuria-may develop in AIHA,lupus nephritis
Urine examination:

21.  Hemoglobin – can indicate anemia
 RBC indices
 Normocytic Normochromic- chronic disease per se.
 Macrocytic – pernicious anemia
 Features of AIHA -
 Autoagglutination
 Microspherocytes
 Polychromasia
 Schistocytes
 Tear drop cells
 Erythrophagocytosis by monocyte – occasionally.
Hematological investigations:

22.  WBC –
 Eosinophilia - 1000/ul in 80% pt of Churg strauss Syndrome
 PMN leucocytosis - Vasculitis ( PAN, Wegner granulomatosis)
 Normal or moderately decreased in AIHA
 CLL blood picture – secondary AIHA
 Leucopenia is very common in active SLE.
 Lymphopenia <1500/μl seen in SLE.
 Platelet count
 Normal, occasionally lowered sometimes sufficient to cause
purpura- AIHA
 Decreased in – ITP, SLE, RA , Scleroderma
 Increased in - Wegner granulomatosis

23.  Reticulocyte Count
 5 % to 30% - Hemolytic anemia but may be higher.
 It is indicative of increased production of RBC( as in
hemolytic anemia)

24.  Erythrocyte Sedimentation Rate :
 Indirect measure of the inflammatory process.
 Very important clue for systemic autoimmune diseases
monitoring and treatment efficacy.
 It indicates Acute Phase Response , returns to normal
during remission.
 ESR is influenced by a number of confounding variables
such as RBC shape , number , deformity , hence not as
specific as CRP.

25.  ANA is clearly responsible for the characteristic LE
bodies and LE cells.
 Although the auto antibodies cannot penetrate the
healthy cells , they can attack the nuclei of damaged
cells.
 LE factor is an IgG antibody directed against
deoxyribonucleoprotein
 This antibody acts on the damaged nucleus producing
swelling & homogenisation of nuclear material-LE
bodies.
 LE bodies phagocytosed by neutrophils – K/as LE cell
LE cell test

26.  LE cell test is easy to perform but difficult to standardize
and impossible to titrate or quantitate.
 LE cell test is positive in 50 to 75% of patients , but not as
frequently positive as an ANA so negative test does not rule
out the SLE.
 +ve,test not itself diagnostic of SLE but only confirms
clinical diagnosis.
 LE cells are demonstrated in SLE, chronic hepatitis,
rheumatoid arthritis and other collagen disorders.
 Sensitivity & Specificity less than ANA.
 Sensitivity -75%
 In tissue sections LE bodies can be seen principally in renal
glomerulus and interstitium as round basophilic structures
also called as hematoxylin bodies.
 Although found in 20% cases they are pathognomic of SLE

27. LE cell Hematoxylin body

28.  Coombs’ test -AIHA
 Direct Antiglobulin Test ( DAT )
The direct antiglobulin test (DAT) detects sensitized red
cells with IgG and/or complement components c3b and
c3d in vivo.
Types of AHG (Qualitative & Quantitative):
 Polyspecific “broad spectrum”(antiIgG & antiC ):
positive test indicate presence of auto antibodies . Not
necessarily diagnostic.
 Monospecific: AntiIgG only type ,antiC3d only type.

29.  In vivo coating of red cells with IgG and/or complement
may occur in:
 Autoimmune hemolytic anemias
 Drug induced hemolytic anemias( secondary AIHA )
 Pattern of reactivity in autoimmune hemolytic anemia
Anti -IgG Anti –C3d Type of AIHA
+ + WAIHA
+ - WAIHA
- + CHD,PCH,
WAIHA

30.  Indirect Antiglobulin Test (IAT)
 The IAT is done to determine the presence of sensitization of
red cells with IgG and/or complement in vitro in the following
conditions:
 Screening and detection of unexpected antibodies in serum.

31. Biochemical tests

32.  Serum Bilirubin
 In AIHA –1-3mg/dl but is sometimes persistently normal despite
continuing hemolysis.
 In AIH- May be Normal or 3-10mg/dl in severe cases
 In PBC,PSC- N/ mild elevated
 To diagnose Autoimmune hepatitis various parameters Of LFT are
included in the diagnostic criteria & scoring system .
 Ratio of increased Alkaline Phosphatase to Aminotransferase
 Total serum Globulin, Y- globulin
 In PBC – cholestatic type abnormality
 Increased GGT & Alkaline Phosphatase
 Mild elevation in ALT & AST
 In PSC-
 Increased Alkaline Phosphatase
 Increased Aminotransferase
LFT - AIHA, AIH,PBC,PSC

33. RFT
 Urine R & M-to detect protein,sugar,ketones & casts in SLE & vasculitis to
know the renal involvement.
 GFR & Sr creatinine level to predict severity of kidney involvement.

34. Serological tests

35. Non specific serological tests
 In resourse poor setting
 Detect Antibodies against the phosholipid β2
glycoprotein complex by using cardiolipin antigen in
syphilis patients.
 SLE patients having antiphospholipid antibodies so
gives +ve result.
VDRL

36.  It is widely used for assessing disease activity in
autoimmune disorders , because it is rarely elevated
persistently without continued inflammatory
response.
 It is fastest rising acute phase reactant and returns to
normal quickly following successful therapies
 Indicates aggressiveness of disease and are an indirect
measure of proinflammatory cytokines IL-1,IL-6 &
TNF.
 It has no confounding factors
 It is a good prognostic & follow up marker.
 Methods: Radialimmunodiffusion,
 Electroimmunodiffusion
 Nephelometry
C- Reactive Proteins

37. Radial immunodiffusion. The patient's serum is placed in the wells cut out of the
agarose gel. The agarose gel contains antibodies to the test antigen (CRP in this case). The
antigen diffuses into the agarose and forms precipitin rings.The diameter of the rings is
directly proportional to the amount of antigen in the serum and is quantified by
comparing the size of the ring to that of simultaneously tested standards.

38.  RF are autoantibodies ( usually IgM )directed against the Fc
portion of the IgG molecule.
 IgG & IgA RF also detected. IgA RF related with more severe
disease & erosion.
 Found in more than 2/3rds of adults with Rheumatoid
Arthritis.
 Found in 5% healthy persons.
 Not specific for Rheumatoid Arthritis.
 Other conditions associated with RF are :-SLE ,
Vasculitis,Sjogren’s Syndrome, Chronic Liver Disease ,
Hepatitis B , Hepatitis C , Sarcoidosis , Interstitial Pulmonary
Fibrosis , Leprosy , Syphilis , SABE , Visceral Leishmaniasis ,
Schistosomiasis , Malaria & tuberculosis.
 Sensitivity- 80%,
 Specificity is compromised(72%)
Rheumatoid Factor

39.  It has prognostic significance because patients with high titres tend
to have more severe & progressive disease with extraarticular
manifestations.
 In summary a test for the presence of RF can be employed to confirm
the diagnosis in individuals with a suggestive clinical presentation
and , if present in high titre, to designate patients at risk for severe
systemic disease.
 Techniques for estimating RF :
 Rose Waaler method ( sheep RBC agglutination)-reported as titre eg.
1:32 , 1: 64
 Latex agglutination
 Turbidometry
 Nephlometry-reported in IU
 ELISA

40.  RA is associated with several autoantibodies, which can serve
as diagnostic and prognostic markers other than RF
• Antifillagrin antibodies [ACPA];
a) Antikeratin antibodies (AKA)
b) Antiperinuclear factor (APF)
c) anti-CCP [anti-cyclic-citrullinated-peptide])
d) Anti-Sa antibodies, targeting citrullinated vimentin
• Anti-RA33. .
IgM RF APF IIF Anti-CCP2ELISA Anti-CCP 3 ELISA
Sensitivity (%) 66–85 60 69 74
Specificity (%) 72 95 95 -98 96
•.
Recent advance in diagnosis of RA

41.  Anti-CCP antibodies can be detected in 50% of patients with
early RA, at a time when RF is negative, allowing for improved
diagnosis and early specific treatment
 It is rarely found in other clinical conditions.
 It may better discriminate between patients with rheumatoid
arthritis and patients with polyarticular manifestations
associated with hepatitis C virus infection.
 Now many authors concluded that it is a useful test to
confirm a diagnosis of RA .

42. Anti Nuclear Antibody
Antinuclear antibodies are antibodies that are directed
against several nuclear antigens and can be grouped into
four categories-
• Antibodies to DNA
• Antibodies to histone
• Antibodies to nonhistone proteins bound to RNA
• Antibodies to nucleolar antigens

43.  Antinuclear antibody (ANA) test a marker of the
autoimmune process that is positive with a variety of
autoimmune diseases. It may be positive in
 systemic lupus erythematosus,
 Sjögren’s syndrome,
 rheumatoid arthritis,
 autoimmune hepatitis,
 primary biliary cirrhosis,
 alcohol-related liver disease, and
 hepatitis B.

44. Method Antigen Source Sensitivity
Immunofluorescence microscopy Tissue sections; cell lines Sensitive assay, often used for
screening
Double immunodiffusion
(Ouchterlony)
Tissue and cell extracts Requires precipitin reaction; high
specificity but not very sensitive
Counterimmuno-electrophoresis Tissue and cell extracts Increased sensitivity and speed as
compared with immunodiffusion
procedure
Immunoblotting, Western blot Cell extracts Very sensitive; permits detection of
antibodies against soluble and
insoluble antigens
Dot blot, linear blot (line
immunoassays)
Purified native or recombinant
antigens
Qualitative assay, average
sensitivity
ELISA Purified native or recombinant
antigens
Very sensitive; quantitative; high
throughput; can determine
antibody class; low cost
Microsphere multiplexed assay Purified native or recombinant
antigens
Very sensitive (compares to
ELISA); semiquantitative; rapid;
expensive proprietary technology
Methods for Detection of Autoantibodies
to Nuclear and Intracellular Antigens

45. INDIRECT IMMUNOFLORESCENCE
 Hep2 Cells ( immortalized, derived from human larynx carcinoma
(human epithelioma type 2 - clone ATCC CCl 23) fixed on glass
slides.
 Incubated with diluted patients' sera with a titer of 1 / 80 in pH 7.2
phosphate buffered saline (PBS) for 30 minutes in a moist
chamber at room temperature.
 Then, the plates are washed twice for 10 minutes in PBS
and incubated for 30 minutes with secondary antibody
conjugated with antigamaglobulina human fluorescein
isothiocyanate (FITC) in the dark at room temperature.
 After incubation, slides are washed in PBS and mounted with
buffered glycerin and cover slip.

46.  Reading is done with a fluorescence microscope,
 The extent of staining is determined by dilution of serum
(1:100, 1:400 and 1:1600) in order to establish the endpoint.
 The antibody patterns and the titres (ie the dilution at which
the staining was still visible) are reported.
 Pattern of nuclear fluorescence suggests the type of
antibody present in the patients serum

47. Clinical significance of a positive ANA?
• Low titre ANAs (ie 1:40 or 1:100) - usually not
clinically relevant.
• High titre ANA (particularly 1:1600) -greater
clinical significance

48.  Four Basic Patterns
(1)Homogenous or Diffuse nuclear staining pattern
most commonly indicates antibodies to chromatin,
histones,and occasionally double stranded DNA
(2)Rim or Peripheral staining pattern
most commonly indicates antibodies to dsDNA
(3)Speckled pattern
there is presence of uniform or variable sized
speckles. This is the most commonly observed pattern
of flouresence and therefore the least specific. It indicates
the presence of antibodies to non-DNA nuclear constituent
eg.Sm antigen, ribonucleoprotein, SS-A,SS-B

49. (4)Nucleolar pattern
there is presence of a few discrete spots of
fluorosence in the nucleus and represents antibodies to
nucleolar RNA.This pattern is reported most often in
patients of systemic sclerosis.

50. “homogenous” pattern of a positive ANA

51. “speckled” pattern of staining is more characteristic of the presence
of autoantibodies to extractable nuclear antigens, particularly ribonucleoprotein.
This pattern is not very specific, but may be seen with “mixed
connective tissue disease” without serious renal or pulmonary diseases.

52. The “nucleolar pattern” of staining in which the bright fluorescence is
seen within the nucleoli of the Hep2 cells. This pattern is more suggestive
of progressive systemic sclerosis

53. Sometimes when performing the ANA test, the substrate cells demonstrate
particular patterns of staining. This is the “rim” pattern that is more
characteristic of SLE.

54.  The flourescence patterns are not absolutely specific for the type
of antibody because many autoantibodies may be present in
combinations of patterns .
 The immunoflourescence test for ANA is positive in virtually
every patient with SLE hence this test is sensitive but it is not
specific because patients with other autoimmune diseases also
frequently score positive.
 5% to 15% of normal individuals have low titres of these
antibodies.
 The incidence increases with age
 Detection of antibodies to specific nuclear antigens require
specialized techniques.
 Antibodies to dsDNA antibodies & Smith(Sm) antigen are
virtually diagnostic of SLE.
 Correlations-
 High titres of dsDNA are usually associated with active renal
disease.
 High titres of anti SS-B (la) are associated with low risk of nephritis.

55. Antinuclear Antibodies in various Autoimmune Diseases
Diseases, % Positive
Nature of Antigen Antibody
System
SLE Drug-
induced
LE
Systemic
Sclerosis
--Dfffuse
Limited
Sclero-
derma.
-CREST
Sjogren
Syndrome.
Inflammatory
Myopathies
Many nuclear
antigens
Generic ANA >95 70-90 70-90 70-90 50-80 40-60
Native DNA Anti-ds DNA 40-60 <5 <5 <5 <5 <5
Histones Antihistone 50-70 >95 <5 <5 <5 <5
Core proteins of
small nuclear
particles
Anti-Sm 20-30 <5 <5 <5 <5 <5
Ribonucleoprotein. Nuclear RNP 30-40 <5 15 10 <5 <5
RNP SS-A(Ro) 30-15 <5 <5 <5 70-95 10
RNP SS-B(La) 10-15 <5 <5 <5 60-90 <5
DNA topoisomerase
I
Scl-70 <5 <5 28-70 10-14 <5 <5
Centromeric
protieins
Anticentromer
e
<5 <5 22-36 90 <5 <5
Histidyl-t-RNA
synthetase
Jo-1 <5 <5 <5 <5 <5 25

56. Cellular
antigen
Disease associated with
antibodies to the
antigena
Method used for
detection of
antibodies to the
antigen
Nuclear
immunofluorescen
pattern
DNA-histone
complex
(nucleoprotein)
SLE, RA, CAH IFA Homogeneous,
Peripheral
dsDNA SLE IFA, RIA, ELISA,
hemagglutination
Peripheral
SsDNA SLE, drug-induced SLE,
CAH, infectious
mononucleosis, RA
IFA, RIA, ELISA,
hemagglutination
Homogeneous
Histones SLE, drug-induced SLE IFA, ELISA Homogeneous
nRNP SLE, MCTD IFA,CIEP,
hemagglutination,
immunodiffusion
c
,
Speckled
Sm SLE IFA, CIEP
immunodiffusion,
Speckled
Antinuclear Antigen–Antibody Systems in Rheumatic Disease

57. Cellular
antigen
Disease associated with
antibodies to the
antigena
Method used for
detection of
antibodies to the
antigen
Nuclear
immunofluores
cenpattern
SS-A/ Ro
SS-B/ La
SLE,RA, Sjögren's
syndrome
RAIFA on human
tissue culture cell
lines,CIE
immunodiffusion,
Speckled
(cytoplasm and
nucleus)
Scl-70 Scleroderma(dcSSc) IFA,
immunodiffusion
Speckled
centromere CREST variant(lcSSc) IFA on human tissue
culture cell lines
Centromere
Nucleolar
(RNA)
Scleroderma IFA Nucleolar
PM-l, PM-Scl Polymyositis-scleroderma
overlap
IFA,immunodiffusion ? Nucleolar,
speckled
Jo-1, Jo-2 Polymyositis IFA,immunodiffusion ? speckled

58. Antineutrophil Cytoplasmic
Antibody(ANCA)
 Detection methods-
 IFM- most sensitive
 ELISA
 Radioimmunoassay
 Western blotting
 Dot blotting
 flow cytometry,
 Immunoprecipitation,
 capture enzyme immunoassay techniques

59. Method for IFM
 Isolated normal human neutrophils are used as substrate.
 These neutrophils are cytocentrifuged against multiwell glass
slides, fixed with 99% ethanol, and then incubated with
dilutions of patients' sera.
 Slides are stained with a fluorescein-labeled polyspecific
antihuman immunoglobulin (Ig) conjugate.
 Slides are read on a fluorescence microscope using IFM,
 There are three patterns of neutrophil staining:
 c-ANCA,
 perinuclear ANCA (p-ANCA),
 ‘atypical patterns’

60. C-ANCA P-ANCA
Most ethanol-fixed neutrophils show perinuclear
and nuclear fluorescence without cytoplasmic
staining
Most ethanol-fixed neutrophils show finely
granular, cytoplasmic fluorescence with
central accentuation.There is no nuclear
staining.

61.  The antigenic specificity of c-ANCA is proteinase 3 (PR-3) & of p-
ANCA is myeloperoxidase (MPO)
 ANCA patterns should be confirmed with more specific tests for PR-3
and MPO ELISA .
 Anti-PR3 reactivity is most often seen in
 active Wegener's granulomatosis
 polyarteritis nodosa
 but may be seen in idiopathic crescentic glomerulonephritis or Churg–
Strauss syndrome.
 MPO-ANCA is most often seen in
 polyarteritis nodosa,
 idiopathic crescentic glomerulonephritis,
 Churg–Strauss syndrome; however, it may be seen in Wegener's
granulomatosis.
 P-ANCA also reported in about 75-80% of patients with active
ulcerative colitis or primary sclerosing cholangitis& about 75% of
patients with chronic active hepatitis.
 A negative ANCA should not be used to exclude disease

62.  Antibodies to phospholipids are identified in SLE in three ways:
 Serologically false-positive test for syphilis by a positive VDRL test,
which is a flocculation assay using carbon particles coated with cholesterol
, lecithin , cardiolipin
 The lupus anticoagulant assay , which is a prolongation of the
kaolin partial thromboplastin time that is not corrected by
normal plasma.
 Cardiolipin immunoassay with the use of cardiolipin or other
negatively charged phospholipids as antigens and β2-glycoprotein I as co-
factor.
 Newly developed assays for β2-glycoprotein I autoantibodies seem
to bring specificity to the findings since it was described that the β2-
glycoprotein is the specific epitope for anti-phospholipid autoantibodies.
Antiphospholipid Antibodies

63. Aso titre & anti DNAase B
 Supportive evidence of a preceding streptococcal infection
within the last 45 days for diagnosis of rheumatic fever &GN
 It is ordered when the symptoms emerge, usually in the weeks
following a sore throat or skin infection.
 ASO titre >200 unit
 Anti DNAse B >300 or 350 unit significant
 ASO titre high found in acute rheumaic fever but in GN titres
are low.
 Anti DNAse B useful in case of acute GN for retrospective
diagnosis of streptococcal pyoderma in which ASO titre is
much of less value.
 Methods:
 Latex agglutination test
 Immunoturbitimetric assay

64. Complement assay
 SLE is the most common rheumatic disease associated with low serum
complement levels, especially C3, C4, and C1q,
 The monitoring of serial complement levels provides a useful
parameter of disease activity.
 complement levels often may fall prior to a flare of disease activity,
and patients who appear well but have falling complement levels
should be followed more frequently for signs of disease activity.
 Similarly, serum complement levels (C3, C4, or CH50) are useful in
monitoring the response to treatment.
 Other diseases in which serum complement levels may be reduced-
 vasculitis associated with rheumatoid arthritis and Sjögren's
syndrome;
 essential mixed cryoglobulinemia;
 polyarteritis nodosa.

65.  CA divided into 2 types:
 Quantitative Antigenic assay
 By Radioimmunodiffusion method
 Most widely used for C3 & C4
 Detect as little as 10ug/ml complement protein in serum
 Other more sensitive & accurate methods- ELISA ,nephelometry
 Functional assay
 By CH50 – measure the total hemolytic activity of serum,
defined as a amount of complement required to lyse 50% of
sheep RBCs.
 AH 50 – measure of alterative pathway of complement system
defined as amount of complement or dilution of serum that lyse
50% of rabbit RBCs

66.  Utility
Reduces labour intensity
Increases cost effectiveness
 Steps
 Clinical evaluation
 Choice of appropriate tests
 Choice of further tests based on the results of basic tests.
Algorithmic approach

68. Association of HLA with
Disease
Disease HLA Allele
Ankylosing
spondylitis
B27
Graves disease DR3
Celiac disease DQ2
Rheumatic arthritis DR4
Chronic active
hepatitis
DR3
Primary Sjogren
syndrome
DR3
Type-I diabetes DR3,DR4,DQ8
Goodpasture’s
syndrome
DR2
Association of
HLA with Disease
Disease HLA Allele
Myesthenia gravis B8, DR3 with DR1
Sjogren syndrome DR3
Hashimoto
thyroiditis
DR 5
Multiple sclerosis DR2
Pemphigus vulgaris DR4
Dermatitis
herpatiformis
DR3
Psoriatic vulgaris Cw6

69. Organ -specific Autoimmune diseases
Organ Disease Autoantibody Detection methods
Thyroid gland
Autoimmune thyroiditis Thyroid peroxidase
Thyroglobulin
ELISA:IIFM- unfixed monkey thyroid
IIFM-methanol-fixed monkey thyroid ;
passive hemagglutination; latex
agglutination.
Graves disease TSH receptor Radioreceptor assay;cAMP bioassay
Adrenal gland
Addisons disease Adrenocortical IIFM on unfixed monkey or human
adrenal cortex
Parathyroid gland
Parathyroid Parathyroid
endothelial protein
IIFM on unfixed monkey or huuman
adrenal cortex

70. ……..contd
Pancreas
Type-I Diabetes mellitus Islet cells :
Insulin-associated :
Antiglutamic acid
decarboxylase :
IIFM on human pancreas,Bld gp ‘O’
Radioimmunoprecipitation
ELISA,RIA,immunoblotting
Muscle
Dermatomyositis/
polymyositis
PM-1, Jo-1 ELISA; immunodiffusion
Gastrointestinal tract
Atrophic gastritis
Pernicious anemia
Ulcerative colitis
Crohn’s disease
Gastric parietal cell
Intrinsic factor
Salivary duct cells
Gastric parietal cell
Colon;
Lipopolysaccharide
DNAase sensitive
p-ANCA associated
ASCA
IIFM—mouse stomach/kidney
Radoactive B12 binding assay
IIFM—unfixed human salivary gland
IIFM—human,monkey,mouse,rat
gastric mucosa subtrate.
IIFM—human or rat colon ;
hemagglutination.
IIFM—ethanol fixed neutrophils
ELISA

71. …..contd
Celiac disease Tissue transglutaminase
IgA endomysial
Gliaden
ELISA
IIFM—monkey esophagus
ELISA
Liver
Autoimmune hepatitis Smooth muscle
Liver-kidney microsomal
ANA
IIFM-mouse stomach/kidney
IIFM-mouse stomach;ELISA
Hep-2 cells
Primary biliary cirrhosis
Primary sclerosing
cholangitis
Mitochondrial(m2)
P-ANCA
IIFM—mouse stomach/kidney;
ELISA
IIFM—ethanol
Neurologic
Myasthenia gravis
Demyelinating diseases
Achr
Myelin;tubulin;myelin
basic protein; myelin-
associated glycoprotein
Immunoprecipitation of iodine
125 α-bungarotoxin-conjugated
Achr; ELISA
IIFM—mammalian spinal
cord;ELISA;immunobloting.

72. …….contd
Kidneys
Anti—GBM disease Glomerular and lung
basement membrane
ELISA
Skin
Pemphigus
Pemphigoid
Dermatitis
herpetiformis
ICS(desmoglobins)
BMZ(hemidesmosomal
proteins)
Tissue transglutaminase
IgA endomysial
DIFM on skin biopsy
IIFM on monkey/guinea pig .
DIFM on skin biopsy
IIFM on monkey esophagus
ELISA
Monkey esopahgus

73. AMA in rat kidney in PBC
diffuse IF of renal tubules, including DT
has a homogeneous cytoplasmic appearance
Anti LKM in mouse kidney
There is finely granular cytoplasmic IF in PCT
not DT
combined Anti SMA &
ANA using mouse stomach
DIFmicroscopy reveals granular
IgA IF in the tips of dermal papillae &
to a lesser extent, along the BM region
DIF microscopy reveals intense
linear C3 deposition along the BM regionDIF shows linear/granular C3
deposition on the cell surfaces (ICS) of
mainly basal keratinocytes without BM
immunofluorescence

74. Biopsy
 Sites
 Depending upon affection
- Kidney
- Skin
- Salivary gland
- Muscle,
- Synovium
- Liver
- Temporal aretery
 Processing
 H&E
 IF(direct & indirect)
 Electron Microscopy & Immuno Electron Microscopy for localisation of deposits
Tissue diagnosis

75. - Done in SLE, PAN, Churg-Strauss syndrome, GN
- In SLE Biopsy indicated to plan current & future therapy .
-WHO classification of Lupus Nephritis
(1)Normal/Minimal abnormality
(2)Pure Mesangial Alterations
(3)Focal Proliferative Glomerulonephritis
(4)Diffuse Proliferative Glomerulonephritis
(5)Membranous Glomerulonephritis
(6)Advanced Sclerosing Glomerulonephritis
KIDNEY BIOPSY

77. Skin biopsy
-DLE
-Scleroderma
-Pemphigous vulgaris
-Pemphigoid
-Dermatitis herpatiformis
scleroderma
epidermal atrophy, vacuolar alteration of
basal keratinocytes, marked thickening of the
BM, perivascular and interstitial lymphocytic
inflammation, and melanin incontinence
Dermal sclerosis. The skin is thickened due to marked expansion of the
dermis. Thick bundles of densely packed collagen replaces skin appendages

78. Suprabasal split Subepidermal split Subepidermal blister with neutrophilic abscess

79. Salivary Gland Biopsy
-in Sjogren Syndrome
• Clusters of lymphocytes and plasma cells composed of atleast 50 of these
mononuclear cells are to be carefully searched in close association to relatively intact
exocrine glands ,ducts or vessels.These clusters of mononuclear cells called foci are
quantitated in a 4mm sq area and reported as the focus score.
• Chisholm and Mason grading- focus score greater than 1 /4mm2 in a labial
salivary gland is regarded as the most disease-specific diagnostic criterion for
the salivary component of SS.

80. Dermatomyositis Polymyositis PAN
Muscle biopsy
Perifascicular fiber atrophy
Perivascular & perifascicular
Lymphocytic infiltrate
Both necrotic and regenerating muscle
fibres are scattered throughout the
fascicle .
Inflammatory cells are found in fascicle
circumferential panmural necrotizing
arteritis of a small muscular artery

81. SYNOVIAL BIOPSY
IN RHEUMATOID ARTHRITIS
Synovial hyperplasia & heavy lymphoplasmacytic infiltrate in RA

82.  Depending upon the site of affection,biopsy is taken
from nerve,temporal artery,prostate,testis,lung in
Vasculitis

83. Autopsy findings
 Necrotizing granulomatous lesion in middle & small
sized blood vessels
 E/o vasculitis – thickened & thrombosed vessels
- Infarct in organs
• Libman sacks endocarditis in mitral & tricuspid valve
• E/o pleuritis & pericarditis in case of RA
• Flea beaten kidney/ large white kidney

84.  With better understanding of pathology of
autoimmune diseases , the lab testing has become
more complex & is likely to continue to evolve.
 Goal of lab testing is to
 establish the diagnosis
 Predict the prognosis & disease course
 Monitor the treatment
all in a cost effective manner.
 Good understanding of testing positive result patterns
and disease association is required to attend this goal.
Conclusion

85. Antigen Disease Association
Homogenous and Diffuse
DNA-histone complex (nucleosome) SLE (60%)
Drug-induced lupus (95%)
Peripheral Rim
dsDNA SLE
Speckled
RNA polymerase types II and III Systemic sclerosis
RNP MCTD (100%)
Scl-70 Systemic sclerosis (15%-70%)
Sm SLE (25%-30%)
SS-A Sjögren's syndrome (8%-70%)
SLE (35%-40%)
SS-B Sjögren's syndrome (14%-60%)
SLE (15%)
Nucleolar
Nucleolar RNA, RNA polymerase 1 Systemic sclerosis
Pm-scl Polymyositis
Centromere
CENP Limited scleroderma

86.  Robbins & Cotran Pathological Basis of Disease ; 7th edition
 Henry’s Clinical Diagnosis and management by lab
methods;21st edition
 Harrison’s principles of Internal Medicine;16th edition
 De Gruchy’s Clinical Hematology in Medical Practice;5th
edition
 WHO Transfusion Medicine technical methods
 Lever’s Histopathology of the Skin
 Rosai & Ackerman’s Surgical Pathology; 9th edition
 Dacie & Lewis Practical Hematology ; 10th edition
 Internet
Bibliography