AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY

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AIMS OF THE LABORATORY TESTS OF CLINICAL
IMMUNOLOGY  

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AIMS OF THE LABORATORY TESTS OF CLINICAL
IMMUNOLOGY    a. diagnosis of inherited and
acquired immunodeficiencies b. diagnosis of
autoimmune and allergic diseases c. verification
of the immunological background of the disease I.
SEROLOGICAL TESTS 1.Immunoglobulins IgG IgG1-4
(infections, autoimmun dis.) IgA (infections,
autoimmun diseases) IgM (infections,
autoimmun diseases) IgD IgE
(allergy) 2. Acute phase proteins
-positive acute phase proteins C reactive
protein(CRP), serum amyloid
A (SAA complement factors, fibrinogen,
haptoglobin, procalcitonin, etc.
CRP! Elevated in acute
infections and autoimmune diseases (RA)
-negative acute phase proteins transferrin,
albumin 3. Complement factors C3, C4, C1INH
Congenital deficiencies (predisposition for
autoimmun diseases) Increased
consumption activity marker of SLE
HANO herediter angioneurotic oedema C1INH
deficiency
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4. Determination of the level of circulating
immune complexes 5. Measurement of natural
autoantibodies antibacterial antibody
titer isohaemagglutinin titer
(anti-A, anti-B)   6.
Measurement of cold agglutinins 7. Detection of
cryoglobulins
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8. AUTOANTIBODIES   Their types 1.natural
(physiologic) autoantibodies (IgM) 2.pathologi
c autoantibodies (IgG)   The benefit
of their verification - markers for diagnosis
and differential diagnosis,
- formation of subgroups, subtypes -
prognostic value estimation of
disease activity and efficacy of therapy
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9. AUTOANTIBODIES OF POLYSYSTEMIC AUTOIMMUNE
DISEASES   Rheumatoid factor a. Antinuclear
autoantibodies (ANA)-collective name of
antibodies directed

against various
nuclear antigens, detected by indirect
immunofluorescence. b. Anti-double stranded DNA
one of the diagnostic criteria of SLE. c.
Anti-histon antibodymarker of drug induced
SLE. d. Anti-chromatin (nucleosoma) antibody
directed against the complex of
ds-DNAhiston, marker of lupus nephritis. e.
Anti-extractable nuclear antigen (ENA) antibodies
directed against SS-A(Ro),SS-B(La),Sm,
U1-RNP, Scl-70, Jo-1 antigens. f. Anti-neutrophil
cytoplasmic antibodies (ANCA) c-ANCA
(cytoplasmic fluorescence) p-ANCA
(perinuclear fluorescence) atypic-ANCA
(atypic c p fluorescence) g. Antiphospholipid
antibodies anti-cardiolipin
anti-beta 2 glycoprotein I lupus
anticoagulans
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Autoantibodies in polysystemic autoimmune
diseases   Type of antibody Disease Anti-dsDNA
SLE (renal involvement) -histon
SLE(drug induced) -Sm SLE
-nucleosome
lupus nephritis -U1RNP MCTD, Raynauld
synd. -SS-A(Ro) Sjögren, RA, SLE
-SS-B(La) Sjögren, RA, SLE -Scl
70 PSS (renal involvement)
-Centromer PSS (CREST synd.)
-Nucleolar PSS -PM-1 PM, DM
-Jo-1 PM, DM PM serum CK elevation
ANCA vasculitis    
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Marker antibodies in polysystemic autoimmune
diseases
autoantibodies of ANA positivity x also
cytoplasmic positivity
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Organ specific autoantibodies

• Antithyreoidea antibodies
• - anti-thyreoglobulin (anti-TG)
• - anti-thyreoidea peroxidase (anti-TPO)
  Hashimoto-thyreoiditis
• - anti-thyreoidea stimulating hormon receptor
  (anti-TSH-receptor)
  
  
  
  Graves-Basedow disease
• Anti-pancreatic islet cell antibody (anti-ICA)
  I. type diabetes mellitus
• Anti-gastric parietal cell antibody aneamia
  perniciosa autoimmune gastritis
• Anti-adrenal-cortex (17,21-hydroxylase) antibody
  Addison disease
• Anti-glomerular basal membrane antibody
  Goodpasture syndrome
• Anti-smooth-muscle antibody autoimmune hepatitis
• Anti-liver-kidney-microsome (anti-LKM) antibody
  autoimmune hepatitis

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• Main laboratory tests of autoantibody
  determination
• - indirect immunofluorescence by microscopy
• positivity/negativity,
• pattern,titer, duration
• - enzyme linked immunosorbent assay
  (ELISA)(quantitative)
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• - immunhistology

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• BENEFITS OF THE LABORATORY MEASUREMENTS IN
  CLINICAL IMMUNOLOGY
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• 1. DATA FOR DIAGNOSIS
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• a.) immunodeficiencies
• - serology IgG, IgA, IgM, IgE, C3, C4, level of
  natural autoantibodies
• - test of phagocytes phagocytosis, chemotaxis,
  killing assay,
• chemiluminescence measurements (phagocytosis
  killing)
• - cell surface antigens on lymphocytes CD3, CD4,
  CD8, CD56, CD19
• active immunization Pneumococcus vaccination,
  measurement of
• antibody
  titers.
•  

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• b.) Antimicrobial, reactive inflammations
• serology We (augmented), increases in the levels
  of C reactive protein,
• immune complex, IgG, IgA, IgM,
  IgE, C3, C4
• cellular tests increased white cell count,
  granulocytosis, lymphocytosis,
• elevated phagocytic activity
• increased expression of activation markers of T
  cells HLA-DR, CD25,
• CD30, CD38, CD45RO, CD69
• c.) Allergy
• - serology increase in the serum levels of total
  and allergen specific IgE
• - cellular test allergen induced lymphocyte
  proliferation
• d.) autoimmune diseases
• - serology organ specific autoantibodies in
  organ specific diseases,
• marker autoantibodies in polysystemic
  diseases

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2. MEASUREMENTS FOR FOLLOWING UP THE
THERAPY   a.) following up the immunosuppression
antibody levels b.) following up the
immunoreactivity proliferation response
(blast transformation) cytokine
production active immunization Pneumococcus
vaccination c.)
determination of the effect of desensibilisation
in allergy d.) measurement of the effect of
immunosubstitution IVIG therapy  
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3. LABORATORY PARAMETERS AS CRITERIA FOR
DIAGNOSIS   a.) immunodeficiencies b.)
antibodies-organ specific autoantibodies
-marker antibodies in polysystemic autoimmune
diseases e.g.
anti-U1RNP MCTD, cAnCA Wegener
granulomatosis SLE anti-dsDNA,
anti-Sm c.) demonstration of atypic
immunoglobulins monoclonal components in
myeloma multiplex
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• 4. LABORATORY PARAMETES AS TOOLS FOR FORMATION OF
  SUBGROUPS OR VERIFICATION OF RISK FACTORS
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• Seropositive and seronegative RA (HLA-B-27)
• Antiphospholipid syndrome, primer or associated
  with SLE (thrombosis, abortions).
• Anti-Jo1 and the synthetase syndrome
  anti-histidyl-tRNA synthetase positivity
  pulmonary fibrosis (PM/DM)
• Bronchial asthma, PM/DM sensitive or resistant to
  glucocorticosteroids
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• 5. VERIRIFICATION OF OVERLAP IN ASSOCIATED
  AUTOIMMUNE DISEASES
•  
• Simultaneous occurence of various marker
  antibodies SLE-Ss, SLE-PM/DM, PSS-Ss
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• 5. RELATIONS BETWEEN THE LABORATORY PARAMETERS
  AND THE ACTIVITY OF DISEASES
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• Definition of diagnosis by laboratory tests.
• Differentiation of the active and inactive states
  of the diseases by laboratory tests
• -         identification of the autoantibody at
  least once
• -         antibody titer and disease activity
  anti-ds-DNA-SLE,
• c-ANCA-vasculitis
• 
  antiphospholipid syndroma
• Demonstration of activation markers in
  lymphocytes
• Cytokine measurements in samples from biopsy
• Molecular genetic methods, demonstration
  of oncogens, etc

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TYPES OF LABORATORY METHODS USED IN CLINICAL
IMMUNOLOGY
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SEROLOGICAL TESTS   1. QUANTITATIVE MEASUREMENTS
OF PROTEINS IN SERUM, PLASMA, URINE AND
LIQUOR  - radial immunodiffusion
(Mancini-test) - turbidimetry - nephelometry - 
electrophoresis   2. TOTAL SERUM COMPLEMENT
ACTIVITY (CH50) haemolytic test   3. PASSÍVE
HAEMAGGLUTINATION - anti-bacterial antibodies

- rheumatoid factor
(Waaler-Rose)   4. INDIRECT IMMUNOFLUORESCENCE  
- antinuclear antibodies - organ specific
antibodies - ANCA (anti neutrophil cytoplasmic
antibody)   5. ELISA (enzyme linked immunosorbent
assay)   - autoantibodies - allergen specific
IgE
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CELLULAR MEASUREMENTS   Tests for phagocytes
microba induced chemiluminescence
(phagocytosiskilling) monocyte-granulocyte
functions
Fc, complement carbohydrate
receptor dependent methods  Tests
for the functions of lymphocytes
Proliferation (blast transformation)
induced by mitogen
NK cell activity 51Cr release from tumor
cells  Measurement of CD antigens (flow
cytofluorimetry) -  differentiation
antigens on the cell surface,
-  subpopulations of lymphocytes CD3 T
cells CD4 helper T cells CD8
cytotoxic T cells CD19 B cells CD56
NK cells  
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Measurement of immunoglobulins
Turbidimetry Light absorbtion
Nephelometry Light scattering
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Age related changes in the serum amounts of
immunoglobulins
Immunoglobulins g/l
IgG
IgA
IgM
months
age
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a1 globulins b1b2 globulins g globulins
Albumin a2 globulins
1 2 3 4 5 6 7 8 9
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PROTEINOGRAM OCT 18 93 2/1 ECH 1 AGE 0 SEX
TOTAL PROTEINS 61.0 G/L A/G 1.33 NAME 0/0 G/L N
ORM G/L Albumin 57.1 34.9 52-65 34-42 Alpha
1 3.6 2.2 2-4.5 1-3 Alpha 2 14.6 8.9 10-15 6-9 Bet
a 12.5 7.6 6-13 4-9 Gamma 12.2 7.4 10-19 6-13
N
Albumin
Albumin a1 zone a1 antitrypsin, orosomucoid, a1
antichymotrypsin a2 zone haptoglobin,
ceruleoplasmin, Gc globulin, a2 macroglobulin, a
lipoprotein b1 zone transferrin hemopexin b2
zone blipoprotein, C3 complement g zone IgG,
IgA, IgM, (IgD, IgE)
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with b mercaptoethanol reduction correct typing
of IgM K cryoglobulin. (To be confirmed after
isolation of the cryoprecipitate).
ELP G A M K
L
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ELP G A M K L
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Image of biclonal gammpathy
Presence of two Lambda monoclonal bands
confirmed l one with anti heavy chains (g, a
or m) l the other, more anodic, with anti Lambda
free light chain. Conclusion presence of a
Lambda Ig monoclonal component with more anodic
Lambda free light chains.
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Ur
Ser
Albumin
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Ur
Ser
IgG Transferrin
Albumin
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Ur
Ser
Albumin
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Hemolytic measurement of classical complement
pathway activation (CH50)
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Passive hemagglutination (Waaler-Rose test)
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Indirect immunofluorescence
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Principle of ELISA

• Antigen
• Antigen antibody complex
• Antigen antibody complex conjugate
• Antigen antibody complex conjugate
  substrate coloured product

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ELISA (anti-cardiolipin IgG)
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Combined ELISA (anti-Sm, anti-RNP)
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class of positivity (1-4)
Food panel
Inhalation panel
negative control positive control wheat saybean co
rn hazelnut peanut cow milt egg white cod
(fish) tomato orange
timoty grass mugwort ragweed, short rye,
cultivated birch Alternaria alternata
(mold) Dermatophagoides pteronissimus Dermatophago
ides farinae dog epithelium cat epithelium
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Allergen specific IgE
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CELLULAR MEASUREMENTS   1 Determination of
phagocytic activity in whole blood on the basis
of free radical (chemoluminescence) production
2. Determination of phagocytic activity of
separated monocytes and neutrophils   3.
Blastic transformation of stimulated
lymphocytes   4. NK cell activity   5.
Determination of CD antigens on the surface of
white cells by flow cytometry
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cpm
minutes
Chemiluminescence of stimulated (Mannozym) and
non stimulated (PBS) pheripheral phagocytes
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Fluorescence particles phagocytosed by neutrophil
granulocytes
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Lymphocyte blastic transformation

• Activation of lymphocytes (blastic
  transformation)
• by specific antigen
• aspecific mitogen
• T cell mitogen concavalin A (ConA),
• phytohemagglutinin (PHA)
• B cell mitogen lipopolysaccharid (LPS)
• T-B cell mitogen pokeweed mitogen (PWM)
• Evaluation of activation (blast formation) on the
  basis of 3H-thymidin uptake
• Evaluation of changes Index of proliferation (IP)

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Measurement of NK cell activity
Target cells 51Cr labeled K562 tumor cell
line Effector cells lymphocytes of
patients Evaluation a.) 51Cr release
determination in the supernatants of cell
culture from the patients and healthy
controls b.) comparison of the chrome
release in the two systems
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Distribution of leukocyte subpopulations
healthy control
dominance of lymphocytes
sample with debris
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Determination of CD3/CD19 positive cells using
monoclonal antibodies conjugated with different
stains
CD19 cells
CD3 cells
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Determination of CD4 and CD8 cells
CD8 cells
CD4 cells
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Measurement of activated T lymphocytes
CD3/HLADR cells
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Determination of CD20/CD5 double positive cells
healthy control
CD20/CD5 double positive cells
Patient with B-CLL
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