GUIDED BY

Respected S K Sharma Sir Lecturer Department of Hematology

PRESENTED BY Jasbir Kaur

B.SC. M.L.T-Part II

AUTOIMMUNE DISEASES
Autoimmune disease are the diseases in which the immune system makes mistakes and instead of mediating protective function becomes the agressor. In autoimmune disease immune system is unable to distinguish between self and nonself components of body

Organ-specific Autoimmune diseases

Antigens and autoimmunity restricted to specific organs in the body
Type I diabetes Goodpastures syndrome Multiple sclerosis Graves disease Hashimoto thyroiditis Myasthenia gravis

Systemic Autoimmune Disease

Antigens and autoimmunity are distributed in many tissues (systemic)
Rheumatoid arthritis Systemic lupus erythematosus Scleroderma Primary Sjogrenss syndrome polymyositis

LUPUS ERYTHEMATOSUS
Lupus Erythematosus is an autoimmune disorder which develops when the bodys own immune system that normally protects the body against invading infections,begins to attack patients own tissues. This occurs due to production of autoantibodies.

Types of Lupus Erythematosus

There are following four types of L.E Discoid Systemic Drug-induced Neonatal

PROBABLE CAUSE OF PRESENCE OF ANAs IN L.E

ANAs are autoantibodies to nuclear antigens The sustained presence of apoptotic cells and exposure of intracellular or nuclear antigens that are modified by the process of apoptosis may lead to production of autoantibodies to these antigens in susceptible people. Autoantibodies to nuclear antigens specially antibodies to chromatin or nucleosomes including antibodies to ds DNA are most characteristic of SLE

APOPTOSIS
SELF PROGRAMMED CELL DEATH

APOPTOSIS: Morphological events cell shrinkage organelle reduction mitochondrial leakage chromatin condensation nuclear fragmentation membrane blebbing & changes

Blebbing & Apoptotic bodies

Bleb The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by M s Apoptotic body

DISCOID LUPUS ERYTHEMATOSUS

SYN: Cutaneous lupus erythematosus DLE is a chronic skin condition which is characterized by inflammation and scarring type skin lesion which occur on the face,ears,scalp and other body areas Some patients with DLE

DRUG INDUCED LUPUS

10% of the incidence of SLE Clinical features are less severe than SLE. Results from hypersenstivity to certain medications like Chlorpromazine Hydralazine Isoniazid Methyldopa etc.

NEONATAL LUPUS

A rare disease affecting babies born to women with Systemic Lupus Erythematosus

SYSTEMIC LUPUS ERYTHEMATOSUS

Syn: Disseminated Lupus Erythematosus SLE is an autoimmune disorder results from tissue damage caused by pathogenic subsets of auto antibodies and immune complexes.

Systemic Lupus Erythematosus (SLE)

chronic inflammatory immune complex connective tissue disease Mainly affects kidneys,skin,spleen,CNS,heart etc. Affects mostly females of childbearing age specially during pregnancy. more common in African Americans, Hispanics, Asians

Auto antibodies found in S.L.E

a Antinuclear antibodies(ANF immunoflorescence) LE Cell(Anti DNA-histone) Double stranded DNA(ds DNA) Single stranded DNA Histone Nucleoprotein(soluble or particulate) Nuclear glycoprotein(Sm antigen) Extractable nuclear antigen(ENA) (Double stranded RNA) b Anticytoplasmic antibodies Mitochondria Microsomal

SOME IMPORTANT ANTIBODIES

Antinuclear antibodies (ANA) The best screening test for SLE. ANA are found in 95% of patients with SLE, often in high titer. However, ANA may be found occasionally in normals and sometimes in patients with other disorders, but usually in low titer.

Anti-ds-DNA antibody High titers of anti-ds-DNA antibodies are seem only in SLE.This test should be carried out on all patients suspected of having SLE.
.

ANAs
ANAs can be divided into:
those directed against dsDNA those directed against ssDNA those directed against histones those directed against non-histone nuclear proteins : nucleic acid-protein complexes

L.E CELL(LUPUS ERYTHROMATOSUS CELL)

. LE cell appear as a neutrophil in the cytoplasm of which a large homogenous mass I.e LE body is present. The nucleus of the ingested leucocyte appear to be wrapped around ingested LE body.

The LE cell is usually a polymorpho nuclear neutrophil (rarely a monocyte or an eosinophil) that has ingeted the altered nucleus of another leukocyte

HISTORY OF L.E CELL

The L.E cell phenomenon was first described by Hargraves , Richmond and Morton in 1948 in bone marrow. Haserick,Lewis and Bortz in 1950 showed that L.E cell could be produced by incubating bone marrow with gamma globulin from an L.E patient. Mischer and Fauconnect in 1954 suggested that it is gamma globulin representing an antibody to a component of the cell nucleus

LE CELL PHENOMENON REQUIRE Four components:1. 2. 3. 4. Antinuclear antibody (LE factor) Nuclear protein material Complement Actively phagocyte neutrophils

L.E FACTOR
LE factor is .an antinuclear factor (ANF) present in the serum of patients with lupus erythematosus. . antibody to nucleoprotien, chiefly histone, . acts in the presence of complement as an opsonizing agent; . rendering the sensitized nuclei which leads to the formation of LE body

PROPERTIES OF L.E FACTOR

Ig G in nature ( globulin)-Incomplete antibody (7S) Stable at 56C-60C for 15 mins but destroyed at 65C Storage stable-It is stable in serum stored at refrigerator temperature and in the frozen state. Needs nucleoprotein to act upon and lyses it. Acts upon any cell which has nucleus e.g hepatic, splenic, renal, cancerous cells, leucocytes and nuclei of various animal cells. Demonstrated in body fluids and in cord blood of children whose mother is suffering from SLE.

L.E CELL PHENOMENON

Trauma to leucocytes Incubation phase
L.E factor causes lysis of leucocyte nuclei Formation of L.E body

Formation of L.E cell

Chemotaxis Engulfing of L.E body

DEMONSTRATION OF L.E CELL

LE cell
Direct method By using clotted blood By using defibrinated blood indirect method By using patient serum normal leukocytes

DIRECT METHOD
1. BY USING CLOTTED BLOOD

{BY USING PATIENT SERUM + PATIENT LEUCOCYTES}

REQUIREMENTS:- Wintrobe tube, centrifuge SPECIMEN:- Clotted blood

PROCEDURE:1. Obtain blood by veni puncture. Transfer 5 ml to a plain dry tube and leave at room temperature for 2-3hours after blood clots. 2. After blood clots, rim the clot and transfer the serum and clot to a special sieve and mash through the sieve with the pestle. 3. Collect the strainings in a petri dish. Fill one or more wintrobe hematocrit tube with the material collected. 4. Incubate wintrobe tube at 37C for 2 hours. 5. Centrifuge tube at approx 1000 rpm for 5-10 mins. 6. Discard supernatant serum, make 3 or 4 smears from buffy coat and stain with a Romanowsky stain. 7. Examine the slide carefully for the presence of LE cell.

2. BY USING DEFIBRINATED BLOOD

This method was described by Zinkham and coleny. Sample Defibrinated blood. a. By rotating blood with glass beads b. By swirling blood in a conical flask containing a glass rod having capillaries fused at its lower end PROCEDURE:1. Collect about 2-3 ml of blood from the patient and transfer about 2 ml of the blood into a 75*12 mmglass test tube (or a vial) containing four to six glass beads. 2. Seal the tube with tightly fitting rubber bung. 3. Rotate the preparation at about 33 RPM at 20C for 30 mins to allow defibrination. 4. Place it at 37C for 10 mins.

5. Transfer the content of the tube to a wintrobe hematocrit tube. 6. Centrifuge the wintrobe tube for 10 mins at 150200 g(1200RPM) 7. Remove serum and take buffy coat and make 2-3 buffy coat films. 8. Allow the films to dry. 9. Stain with Romanowsky stain and look for LE cell.

DIRECT METHOD

INDIRECT METHOD

BY USING PATIENT SERUM AND NORMAL LEUCOCYTES

REQUIREMENTS:A. Patient serum B. Normal leucocyte suspension PROCEDURE 1. Take 5-10 drops of patient serum in a 75*12 mm glass test tube or a vial containing 3-4 glass beads. 2. Add to it equal volume of normal leucocyte suspension. 3. Seal the tube with tightly fitting rubber bung. 4. Rotate the preparation at 33 rpm at 20C for 30 mins .

5. Place the tube at 37C for 10-15 mins.

6. Transfer the content to wintrobe hemotocrit tube. 7. Centrifuge at 150-200 g for 10 mins. 8. Remove serum and make buffy coat films. 9. Air dry and stain with Romanowsky stain.

STAINING OF PREPARED BUFFY COAT FILM FOR L.E CELL

LEISHMAN STAINING
MATERIALS REQUIRED

1. Previously prepared buffy coat film. 2. Staining rack. 3. Leishman stain

PROCEDURE

1. Place the buffy coat film in staining rack and pour Leishman stain on the slide and wait for 1-2 mins for fixation by methanol.

2. Dilute with double the volume of stain by phosphate buffer of pH 6.8 and mix by blowing. 3. Allow to stand for 15-20 mins for staining. 4. Wash the stain just by lifting the slide and washing under slow running tap water. 5. Clean the backside of film and let it dry. 6. Observe the film under microscope.

OBSERVATION
L.E. cell is usually a neutrophillic leucocyte distended by an intra cytoplasmic homogenous red purple body i.e L.E body and the phagocytic cell,,appears large than normal. The inclusion in the L.E cell is homogenous and redder than the usual color of unaltered chromatin.

L.E CELLS

IMPORTANT POINTS
Slides should be examined for at least 10 mins before a negative report is given. In addition to L.E cells extra cellular material may also seen. The material should not be considered significance unless the characteristic L.E are also seen.

INTERPRETATION
Systematic Lupus Erythematosus (75% cases). Lupoid hepatitis Drug reactions Rheumatoid Arthritis (3.6% patients) 90% of SLE cases are in women, usually of child bearing age

CAUSES OF FALSE NEGATIVE AND FALSE POSITIVE L.E CELL

FALSE ve RESULTS

1. Hypocomplementemia (complement necessary). 2. Leucopenia

FALSE +ve RESULTS

1. PRESENCE OF TART CELL

TART CELL
Tart cell is a monocyte rarely a neutrophil which has phagocytosed another cell or the nucleus of another cell. The phagocytosed material most often resembles a lymphocyte nucleus, in which case a definite nuclear pattern can be seen.

Presence of Tart cell

Tart cells often associated with Leucoagglutinins Drug reactions

DIFFERENCE BETWEEN L.E CELL AND TART CELL

Tart cell usually maintains an intact chromatin pattern of phagocytized nucleus. whereas in L.E cell,the L.E body shows no evidence of nuclear structure and appear as an opaque homogenous mass.

PRECAUTIONS
1. 2. 3. 4. 5. Sample. Differentiation from Tart cell. Proper leucocyte trauma. Time given for L.E body phagocytosis. Precaution at the time of making buffy coat film.

DEMONSTRATION OF ANTI NUCLEAR ANTIBODIES BY TECHNIQUES OTHER THAN L.E PHENOMENON

Indirect Immuno florescence ANA

ADVANTAGES OF IMMUNOFLORESCENCE TECHNIQUE OVER L.E CELL PREPARATION A. B. C. L.E phenomenon is positive in only about 75-80% of the cases of SLE, the indirect immuno florescence test is positive in almost 100% of the cases. A negative florescence test rules out lupus erythematosus. L.E test can become negative in the course of steroid therapy, but the titer of ANA is not affected by steroids. A dropping titer indicates true clinical recovery.

INDIRECT IMMUNOFLORESCENT ANA

ANTI-NUCLEAR ANTIBODIES
ANA - abs directed against nuclear antigens. most frequent and highest in titer in SLE. Positive in 98% of SLE patients.

ANA testing (QUALITATIVE)

ANAs are detected by indirect immunofluorescence assays Substrate: - Cryopreserved tissue such as rat liver, mouse kidney etc. - Tissue culture cell lines: HEp-2 .

Frion in 1957 introduced the indirect immunoflourescence technique. PRINCIPLE The indirect immuno florescence antibody test is based on the development of nuclear florescence when antinuclear antibody is fixed to nuclear material which then reacts with FITC conjugated antihuman gamma globulin. This antibody reacts with the nuclei of a variety of cells like normal human leucocytes, cells from chronic myelocytic leukemia, leukocytes from various mammalian species. Normal rat liver is the most commonly used substrate. REAGENTS 1. Phosphate buffered saline pH 7.4. 2. Reagent grade acetone. 3. F.I.T.C conjugated antihuman fraction II.

INDIRECT IMMUNOFLORESCENT ANA TECHNIQUE

4. Sodium meta silicate 5. Mounting medium SAMPLE Patient serum OTHER REQUIREMENTS Rat liver cryostat sections. PROCEDURE 1. Prepare cryostat cut rat liver section 2. Sections are mounted on sodium meta silicate coated slides (-20c). 3. Just before use the slides are fixed in acetone at room temp. for 10 mins and then air dried (one slide should be used for one test)

4. Approx 0.1 ml of the test serum is layered over tissue section with a pipette and incubated at room temp. for 30 mins in a moist chamber. 5. Known +ve and ve controls should be tested with each group of slides. 6. Excess serum is removed initially by directing a gentle stream of phosphate buffer saline across the slide and then by washing the tissue by giving 2 changes of phosphate buffer saline at 5 mins interval on oscillating shaker at 45 oscillation speed/min. 7. F.I.T.C conjugated polyvalent antihuman immunoglobin is layered over the tissue and incubated for 30 mins at room temp. 8. The slides are washed in phosphate buffer saline and then mounted with a coverslip in the barbital buffered glycerol solution. 9. The slides are then examined with a florescence microscope and the pattern is noted.

Patterns of IF staining
Four patterns of staining are seen: 1. Homogenous (diffuse) antibodies to dsDNA, histone strongly associated with SLE, drug induced SLE, RA 2. Speckled antibodies against non histone proteins Characteristic of SLE, Sjogren, Systemic Sclerosis 3. Nucleolar antibodies against ribosomal precursor of RNP seen in SLE,Systemic Sclerosis, Sjogren 4. Rim (peripheral) antibodies to dsDNA and soluble nucleoproteins characteristic of SLE

Immunofluorescent staining of ANAs

Anti DNA antibodies

Anti ss- DNA: nonspecific and not in clinical use Anti-ds DNA: specific for SLE Antibodies that react to both ss-DNA and ds-DNA

DETECTION OF ds DNA ANTIBODIES BY ENZYME IMMUNOASSAY

PRINCIPLE Var ELISA and ds DNA antibodies is an indirect non competitive enzyme immunoassay for the quantitative and qualitative determination of ds DNA antibodies in serum. The wells of micro plate are coated with recombinant plasmid ds DNA. Antibodies specific for ds DNA present in patient sample bind to the antigen. In the IInd step, the enzyme labeled 2nd antibody(conjugate) binds to ve antigen-antibody complex. The enzyme labeled Ag-Ab complex reacts with the added substrate to form a colored solution.

REAGENTS Microplate strips, Calibrators, +ve control, Wash buffer concentrate , Sample diluent, IgG HRP conjugate, Substrate TMB (3,3',5,5' Tetra methyl Benzidine, stop solution (0.5 MH2.SO4). PROCEDURE 1. Dilute serum/ plasma (1;101)with 1000l buffer +10 l serum. 2. Remove strips from pouch and put firmly into strip holder. 3. Wash wells one time with wash buffer immediately prior to use. Fill wells with 300 l wash buffer, soak for 20 sec in well and remove. 4. Dispense 100 l of calibrators, controls and diluted patient sample into approx wells. 5. Incubate at 37C for 30 mins. 6. Aspirate fluid from wells and wash well 3 times with wash buffer. 7. Dispense 100 l of conjugate into all wells and incubate for 30 mins.

Aspirate fluid from wells and wash 3 times with wash buffer. Dispense 100 l of substrate TMB into all wells and incubate for 10 mins in dark. 10. Dispense 50 l of stop solution into all wells. Read absorbance at 450 nm, max 30 mins after adding stop solution

8. 9.

INTERPRETATION
ASSESMENT -ve Equivalent +ve SEMI QUANTITATIVE EVALUATION <351u/ml 35-55 1u/ml >551 u/ml QUANTITATIVE EVALUATION Ratio<1.0 Ratio 1.0-1.4 Ratio>1.4

REFERENCES
Practical Hematology By John V.Dacie Laboratory Medicine Hematology By Miale John B-5th edition Clinical Aspects of Immunology By P.G.H.Gell ,R R A Coombs-3rd edition STITES Clinical Hematology By Wintrobe-9th edition Encyclopedia of Immunology By Roitt-Vol I,II,III Manual of Clinical Laboratory Immunology By Noel.R.Rose www.Google.com