Autoimmune Disease Testing

Autoimmune Disease TestingGroup 9Fatima Tu ZahraKomal Binte Ajmal Maham NadeemMuhammad AhmedSaifullah khanSumayya IrfanAutoimmune DiseasesAhmedAutoimmunityImmune System of bodySelf antigensLoss of immune homeostasisAbnormal response against its own tissues

Hallmark of AutoimmunitySelf reactive T-cellsAutoantibodiesInflammation

Laboratory DiagnosisCant be done by a single testSeveral tests are needed to be doneComplete blood countComprehensive metabolic panelImmunological studiesSerologies Flow cytometryCytokine analysisHLA typingDisease activityErythrocyte sedimentation rate (ESR)Initial Laboratory Evaluation

Blood analysisNormochromic anemiaPlatelet countWBC countLeukopenia and Thrombocytopenia common in SLE patients

SerologyAbnormalities in serum level of specific enzymesAbnormalities in metabolic processesE.g. Autoimmune Hepatitis: Elevated TransaminaseElevated BilirubinElevated serum proteinsNon-specific markers : Same in drug toxicity

Coagulation StudiesAntiphospholipid SyndromeProlongation of Activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT)Inhibition of Clotting factorSarcoidosisHypercalcemiaAutoimmune Inflammatory Myopathies(Dermatomyositis, Polymyositis and Inclusion body myositis)Elevated creatinine kinaseElevated ALTElevated AST

UrinalysisGlomerulonephritis and Interstitial nephritisProteinuriaHamaturiaActive sediment of RBCs and WBCs

All These TestsNon-specificBut initial alarm for autoimmune diseaseFerritinKomal Binte AjmalFerittin

Iron binding proteinIt sequesters iron in a non toxic and readily available formCritical component of iron homeostasis

Hyperferitinemia

Hyperferitinemia is associated with inflammation due to infection , malignancies or due to auto immune diseasesThough elevated levels are less often observed in cases of auto immune diseases

Auto immune diseases

Rheumatoid arthritisHigh concentrations of Ferritin are found in synovial fluid and synovial cells or RA patientsSLE Ferritin levels were high in the urine of patients with nephritis , pleural fluid and in the CSF of patients suffering from meningitisContTHYRODITISHormonal pathways are involved in the regulation of FerritinElevated Ferritin levels POLYMYOSTITIS /DERMATOMYOSTITISInflammatory auto immune myopathy characterized by muscle weakness and visceral movementHigh Ferritin level

C reactive protein Pentamic protein found in blood plasma Synthesized by liver Also an inflammatory marker Detect inflammation which can be ether due to infection or auto immune diseases

Significance Of CRPThe level of CRP in the blood is normally low.A high or increasing amount of CRP in the blood suggests the presence of inflammationbut will not identify its location or the cause. In individuals suspected of having a seriousbacterialinfection, a high CRP can be confirmatory. In people withchronicinflammatory conditions, high levels of CRP suggest a flare-up or that treatment has not been effective.If the CRP level is initially elevated and drops, it means that the inflammation or infection is subsiding and/or responding to treatment.

DISEASESRASLEVasculitis

LIMITATIONSNon specific It only detects the presence of inflammation but do not diagnose the cause ESR Commonly known as erythrocyte sedimentation rate Simple , inexpensive but non-specific test This test is used to measure the inflammation in body which can either be a result of 1)infection2)cancer 3)auto immune disorders

How the test is performed

The test measures how fast the RBCs fall to the bottom of a thin long tubeThe speed at which the RBCs fall to the bottom In case of Inflammation, abnormal proteins appear in blood which cause RBCs to clump together and make them fall quickly

SIGNIFICANCE OF TEST IN AUTO-IMMUNE DISORDERSElevated levels are shown which indicate inflammationDISEASES rheumatoid arthritis , SLE are most common

LimitationsLimitations of this test It is a non specific test , it just indicates the presence of inflammation but do not tell the exact location , cause and diagnosis of disease

Inflammatory MarkersMaham NadeemCeruloplasminGlycoproteinProduced in liverCarries or transports more than 95% of copper in blood plasmaPlays a role iniron metabolism

MechanismBehaves as an enzyme (ferroxidase) Catalyzes oxidation of Fe2+(ferrous iron) into Fe3+(ferric iron) Assists in transport in the plasma in association with transferrinEssential for normal movement of iron from cells to plasma

PeriodontitisA set of inflammatorydiseasesaffecting the tissues that surround and support theteethRisk identified and quantified by biomarkersCreates area of local inflammation indicated by elevated serum levels of various pro-inflammatory markers specially ceruloplasmin.

Increased levels in:Inflammatory statesPregnancyLymphomaRheumatoid arthritisAlzheimer's disease

HaptoglobinProteinEncoded by theHPgene.Produced in response to tissue injury

MechanismInintravascular hemolysis, free haemoglobin(Hb) released fromerythrocytesFree Hb bound by haptoglobinHaptoglobin-hemoglobincomplex formedPhagocytosed by macrophagesLevels decline

In extravasclar hemolysis:Macrophages phagocytose erythrocytesHb not released into circulationNo Hb-haptoglobin complex formedSerum Haptoglobin levels riseIncreased levels in:InflammationMalignancySurgeryTraumaPeptic ulcer diseaseUlcerative colitis

AlbuminSerum proteinSynthesized by the liver Main function is to regulate thecolloidal osmostic pressureof bloodInflammation results in a lower synthesis rate Decreased serum concentrations (negative acute phase protein

MechanismDistributed between the vascular and interstitial spacesDuring inflammation, IL6 increasesResults in albumin being pulled from the intravascular spaces and circulating to the liver until theinflammatory diseaseresolvesLiver starts producing other proteins needed to fight the inflammation.Inflammation simultaneously decreases albumin synthesis and increases albumin catabolism.

Decreased levels in:Autoimmune diseasesInfectionsMalignanciesTraumaSurgery, burn injuriesCrohns disease

Auto-antibodiesSaifAuto-antibodyAuto anti-bodies are the proteins that are directed against bodys own cells, tissues and organ causing inflammation and damage.Unknown causes of productionsGenetic predisposition and environmental triggersExposure to virus and toxinsHormonal causes ( women are more susceptible)

Types of auto-antibodies

Rheumatoid Factor (RF)

RF binds to Fc portion of polyclonal IgG.Helpful in evaluation of patients with Rheumatoid Arthritis.Specificity=70% sensitivity=70%Progressive joint morbidity and rheumatoid nodules.Sjogren's syndrome, SLE, Cryoglobulinemia, interstitial fibrosis and silicosis.Diagnosis with ELISA.Anti-citrullinated peptide antibody.Inflammation in RA.Activation of peptidylarginine deiminase which incorporates citrulline into certain proteins.Antibody against CP.Specificity= 95% sensitivity= 70%.

RF/ACP antibody testBoth of these tests are primarily used as diagnostic tool for Rheumatoid arthritis.Procedure is same as that of ELISA.Commercially available kits

Anti Nuclear Anti-bodiesDiverse group that react against nuclear, nucleolar and perinuclear antigensHallmarks the serologic diagnosis of SLE.Also present in some non-rheumatic disorders like Hashimoto and Graves disease.ELISA method.Subtypes: anti-Ro antibodies,anti-La antibodies,anti-Sm antibodiesanti-nRNP antibodiesanti-Scl-70 antibodies, anti-histone antibodies, antibodies to nuclear pore complexes, anti-centromere antibodies,andanti-sp100 antibodies.Anti Nuclear Anti-bodiesDetection methods include ELISA and Indirect immunofluorescence.Indirect immunofluorescencePrimary antibody attaches to antigenSecondary antibody with fluorophore binds to primary antibodyFluorophore emits light when excited.Amplification can be done using multiple secondary antibodies.

Anti-dsDNA antibody.Diagnosis of Lupus nephritis, viral disease like parvo B19 and BK virus. Previously measured using radioimmunoassay (particularly the Farr assay)Nowadays measured using ELISA.Immunofluorescence assaytarget antigenCrithidia luciliae, a flagellated protozoa containing a dsDNA-containing small organelle called a kinetoplastThe antibodies to dsDNA are detected semi-quantitatively by demonstrating IgG bound to the kinetoplast.Extractable Nuclear AntigensAnti-ENA testing

SumayyaWhat are these?Soluble Cytoplasmic and Nuclear AntigensMain antigens used in the laboratory for disease detection are:RoLaSmRNPScl-70Jo1

Production of auto-antibodies to ENA are indicators of specific diseased conditions associated with many systemic Rheumatic diseases (SRD), including Systemic Lupus Erythmatosus (SLE), Rheumatoid Arthritis ( RA), Sjrgrens Syndrome, Sclerosis and polymyositis. 47How are they measured?Indirect ImmunoflourescenceElevated titers and staining pattern indicates important results

Attention: The test lacks specificity and elevated titers in individuals who do not have the disease might be detected leading to false positive results and referral to the rheumatologists

What do the results indicate?High enough titer will be indicative of SRDPresence of proteins in the situation when immune system is unable to distinguish self from foreign. Patients with positive ANA and symptoms of SRD are recommended Anti-ENA testsThe signs and symptoms include: Inflammation of organs, Muscle pain, Join pain and Fatigue

Anti-ENA Test: What does it Measure?Detects Auto-antibodies which specifically attack proteins in the nucleusThe following auto-antibodies are detected:Anti-Ro (for Sjrgrens Syndrome) Anti-La (for Sjrgrens Syndrome) Anti-Sm (for SLE)Anti-RNP (for MCTD)Anti-Scl-70 (for Scelrosis)Anti-Jo1 (for Polymyostitis)

Ouchterlony double immunodiffusionAgar gel immunodiffusion or passive double immunodiffusionImmunological technique Involved in Identification, Detection and Quantification

The Technique

Indirect Solid Phase Enzyme Immunometric AssayAnti-ENA Screen Kit

Principal: Anti-ENA testing is based on the binding of the antibodies from the patient to the antigens coated on a microplate

ResultsRefference: ENA Panel, Accessed at: http://labtestsonline.org/understanding/analytes/ena-panel/tab/test/

Signal Recognition ParticleAnti-Signal Recognition Particle Auto-antibodiesWhat is it?AbundantCytosolicUniversally conserved ribonucleoproteinThey target and bind to specific proteins in the EPR

Anti-SRP auto-antibodiesPresence is associated with severe acquired necrotizing myopathiesCharacterized with elevated serum Creatine Kinase levels, severe muscle weakness and poor response to traditional immunoresponsive therapyDiagnosis: 3-4 monthsWhy is it highly specific for myositis?It is extremely rare to find these antibodies for other conditions of autoimmunity or muscle disease5-8%of the patients with inflammatory myopathy have anti-SRP antibodiesStrong evidence of the presence of the diseaseAlso indicate certain clinical patternsThe Anti-SRP antibodiesDirected against the SRP complexSix subunits in the family associated with the a small RNA moleculeThe patients anti-SRP sera may recognize one the six subunits or 7S RNASRP54- The Main TragetAnti-SRP antibodies interfere the invitr translocation of the secretory proteins in to the endoplasmic reticulumCK levels are also elevated so their elevation is an important indicator of Anti-SRP antibodiesDetection MethodsIndirect Immunoflourescence test (Not Specific)Needs to be confirmed by an immunodot assay or protein immunoprecipitation (Not Quantitative)Immunodot AssayEnzyme Immuno AssayThe antigens are spread as discrete dots on to a solid membraneSpecimen is added to a reaction vesselThe strip is inserted The patients antibodies are allowed to bind to the antigens on the solid membraneThe non-specifically bound material is removedAlkaline phosphatase anti-Human antibodies are allowed to bind to the patients antibodiesThe strip is transferred to enzyme substrate reagentReaction with bound Alkaline phosphataseObservance of a distinct dot RNA Immunoprecipitation Assay

Anti-Neutrophil Cytoplasmic AntibodyA group of auto-antibodiesMainly, IgG typeThey are agianst cytoplams of neutrophils and monocytesDetected in a number of auto-immune disordersParticularly associated with vaculitisImmunoflourescence and ANCADone on ethanol fixed neutrophilsFormalin fixed may also be usedHelp differentiate ANCA patterns

Difference between P-ANCA and C-ANCA

ANCA Antigensc-ANCA Protinase 3P-ANCA MyeloperoxidaseIn addition p-ANCA has its antibodies against Bacterial permeability increasing Factor (BPI), Cathepsin G, Elastase, Lactoferrin and lysozyme. Others: HMG1, HMG2, alpha enolase, catalase and actin e.tc. ELISA is also used for antibody detectionThe Complement SystemIt is a part of the immune system that is involved in complementing the antibodies and the phagocytic cells to clear the pathogens.Has several triggers, when it is stimulated it directs proteases that cleave specific proteins and release cytokines that start an amplifying cascade.Over 30 proteins and fragments make up this systemIts role in DiseasesIt is involved in a number of autoimmune disorders including, Arthritis, autoimmune heart disease, multiple sclerosisDeficiency in the terminal pathways contribute to these diseasesImmunoglobulinsThe body may start mistaking self antigens for foreign So immunoglobulin test help detect autoimmune diseasesIgA, IgG and IgM are often measured togetherCan be used to detect Juvenile idiopathic arthritis, lupus and celiac diseases

CryoglobulinFlow cytometry cytokine studies MHC studies

Fatima Tuz Zahra

Cryoglobulin

Abnormal blood proteinGamma globulins with a molecular weight of approximately 200,000.Precipitates blood serum when it is chilledBinds with complement proteins and other peptides to form immune complexes Cause tissue damage

Types of CryoglobulinType I Monoclonal immunoglobulins IgM isotypeMultiple myelomaWaldenstrom's macroglobulinemiaLymphoproliferative disordersTypes of CryoglobulinType II Mixture of polyclonal igG and monoclonal igm Type III Combination of polyclonal igG and polyclonal igM.Hepatitis CSmall vessel vasculitisHow the Test is PerformedBlood is typically drawn from a vein, usually from the inside of the elbow or the back of the handCryoglobulins are very hard to detect accurately Temperature sensitiveDetection MethodsIf the cryoglobulin test is positive :Protein electrophoresisImmunofixation electrophoresis (IFE)MHC/HLA

MHC class I and II genes Genetic determinants of many autoimmune diseasesMHC class I molecules include HLA-A, -B, and C. MHC class II molecules include HLA-DR, HLA-DQ, and HLA-DP.HLA class I and II have been associated with rheumatic disordersHLA studiesHLA-B27 is present in approximately 90% - 95% of white patients with ankylosing HLA-DR1 and HLA-DR4 increase the risk of polyarticular juvenile idiopathic arthritis (JIA) in many populations. HLA-DR3 and HLA-DR2 are associated with lupus in Caucasian populationsDetection of HLAElisaGel electrophoresisPolymerase chair reaction (PCR)

Cytokine studiesCell signaling molecules that aid cell to cell communication in immune responses Stimulate the movement of cells towards sites of inflammation, infection and trauma.Cytokines that influence inflammation include il-1, il-6 and tnf-alpha.DetectionDNA microarray studiesCytokines Human Autoimmune DiseasesRheumatoid ArthritisSystemic Lupus ErythematosusMultiple SclerosisPsoriasisSystemic Sclerosis

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