human platelet antigen.ppt

Shamee Girish

Introduction 2hr old infant with petechial rash and purpura soon after delivery No hepatosplenomegaly No evidence of infection Platelet count of 11000 Cerebral ultrasound intracranial haemorrhage Maternal platelet count normal no history of AI disease

1:1500 pregnancies Occurs in first pregnancy in 50% cases 80% HPA-1a incompatibility 10-15% HPA-5b incompatibility (Caucasians ) 3% of all thrombocytopenia 27% of severe cases

Antibody studies on maternal serum Antibody detection and identification Paternal platelet cross-matchesAntigen studies Phenotyping of parents (+/- neonate) Genotyping of parents (+/- neonate) Genotyping of fetus (in subsequent pregnancies)NAITP

Classification - platelet antigens

Classification Depending on biochemical natureCarbohydrate antigenA, B, H, P etc2. Protein antigenHLA class I A, B, CGPII b / III a GPI b / IX / V3. Haptensquinine heparin quinidine antibiotics

HLA antigens Megakaryocyte derivedAdsorbed from plasmaPlt m RNA produced Class I HLA HLA genes short arm chromosome 6 Integral membrane glycoprotein MW: 56000 dalton NO. of HLA molecules : 15000-120000 / platelet Antigenic strucure:2 chains heavy chain ,light chain 2- microglobulin epitope private epitope, public epitopecross reactive groups (CREGS)

ABH Blood group antigensIntegral membrane proteins ,Expressed on GPGPIa / IIa most of ABH per molecule of GPGP IIb , PECAM-I - majority of ABH per pltsAmount of Antigen variesFrom person to personFrom platelet to plateletA antigen 100-16,000 molecules / pltB antigen of A antigenHIGH EXPRESSOR : elevated A1 activitytype Itype II3 times the normal7 timesheterogenous distributionhomogenous

Other Antigens Ii, Lewis, P antigensCD 36: ( GP IV ) GPI anchored to plt membrane receptor for thrombospondin GPIV null develop anti Naka, clinically significant deficiency associated with cardiomyopathy, MI CD 109: 175 kd glycoproteinFound in activated T cellsGOV antigens < 2000 molecules / pltsGOV a, GOV b Antigens labile in storage

Platelet specific antigensNomenclatureHistorical system:named by authors first reporting the systemsname of the patient in which Ab was detectedmost of these first discovered during the invest of NAITlead to confusionISBT nomenclature 1990numerical arrangement - date of publicationalphabetically frequency in populationw for workshop, antibody against only one alleleAlleles arise as a result of single amino acid Substitutions at different positions

Platelet specific antigensMisnomer have broader tissue distributionare expressed on receptor molecules involved in cell-matrix interactions (found in endothelial cells )Well characterized biallelic alloantigens + rare antigensAutosomal Codominant inheritanceTheir phenotype frequency varies in different racial populationsHPAs are localized on four platelet membrane glyco-proteins (GP) GPIIb, GPIIIa, GPIb and GPIa.

HPA systemAntigens%GPHPA 11a1b98%27%IIIaChr 17HPA 44a4b99.9%

HPA systemAntigens%GPHPA 33a3b85%63%IIbChr 17HPA 99bw0.6%HPA 55a99%Ia chr 5HPA 22a2b99%15%IbChr 17HPA 1212bw0.3%Ib chr 22HPA 1515a15b80%60%CD109Chr 6

Gene frequencies in Indian populationMohanty et alTrans med, 2005

HPA systemAntigens%HPA 11a/a1b/b1a/b87.95 (98)0.65 (27)11.38HPA 22a/a2b/b2a/b99.34 (99)0 (15)0.65HPA 33a/a3b/b3a/b0.13 (85)99.86 (63)0HPA 44a/a4b/b4a/b99.86 (99.9)0 (

GP complexes & function

GPIIb/IIIa HPA 3HPA 9

Hetero dimeric noncovalent complexes genes are present on chromosome 17 Resting platelets ~ 50,000 copies / platelets Unique to the megakaryocytic lineage complexes assemble in endoplasmic reticulum GPIIb-IIIa association prerequisite for transport to surface membranePlatelet activation agonistsGPIIb-IIIa Conformational change Binds to fibrinogen, related adhesive proteinsRGD sequence(Arg-Gly-Asp)Mediate binding Fibrinogen vWF, fibronectin vitronectinPolymorphism influence activation, aggregation

GPIa-IIaGPIIaGPIaI domainHPA 5HPA 13Type 1

GPIa-IIa very late antigen 2 found on activated T lymphocytes 800 2800 copies per platelet I-domain carrying integrin Binds to Mg2+ in its Metal ion dependent adhesion site (MIDAS) major platelet collagen receptor expression state impact on platelet adhesion HPA 5 system

GPIb-IX-VHPA 2HPA 12

GPIb/IX/V ~25,000 copies per resting platelet Involved in initial stages of plt adhesion to damaged vessel wall mediates adhesion of unstimulated platelets to vWF in subendothelium receptor for: (facilitates thrombin induced plat activation)thrombin coagulation factors XI, P-selectinF XII cold storage of platelet clustering of GPIb/IX/V binding of macrophage increased rapid clearance from circulation

Severe decrease GPIb/IXBSS

Cell Adhesion zipper GpIb projecting from the cell membrane - interact with another GpIb receptor through an intervening thrombin molecule GPIb - thrombin arranged as an adhesive ribbon structureThis linear zipper configuration resembles the "cell-adhesion zipper"

Vessel injurysubendothelial vWF exposureGPIb/IX/VGPIa/IIaIntraplatelet signalingConformational changes in GPIIb/IIIaBinds to fibrinogen with high affinityPlatelet activationRelease of granular contentsGrowing aggregation

Technique for platelet antigen typingSerological typing Genotyping Antisera against inquired Ag is usedAssay using whole plateletSera must be free of HLA antibodiesMethods:PIFTELISAPlt agglutination techMAIPAMACEDone with genomic DNAMolecular basis of 15 Ags resolved14 are SNPsMethods: PCR- SSP RFLP ASO OLA SSCP

Genotyping TechniquesFirst generationAllele-specific oligo hybridizationSecond generationRestriction fragment length polymorphismSequence-specific PCRThird generationFluorescence-based technique (Light Cycler, TaqMan) - real time PCRFourth generationMicroarray-based technologyHigh-throughput multiplex single-nucleotide polymorphisms

PCR-ASO/SSO hybridization

ABABABRFLP

ABPCR-Sequence specific primersHomozygous B

Single strand conformation polymorphism

fluorescent dyequencher dyeR5RfluorescenceReal time PCRRTaq polymerase displaces the annealed probe

Platelet antibody detectionDifficulty in measuring platelet associated IgG10100100010000100000307603,50020,000Red cell IgGplatelet IgGAIHAAITP Background noise on control platelet high positive result obtained with 2 to 5 fold increase in IgG per platelet( RBC- 10 to 20 times)

Platelet antibody assaysPhase I assaysPlatelet aggregationInhibition of platelet aggregationInhibition if clot retractionInhibition of platelet migrationComplement fixationPlatelet factor 3 release51Chromium release14C- Serotonin release

Phase I assays Indirect assay patients serum mixed with control platelets measure platelet dependent end points- platelet aggregation, lysis, release- migration inhibition- procoagulants formation Disadvantages:- many antibodies do not cause plt activation- low sensitivity- low specificity no longer used

Phase II assaysDetection of Platelet surfaceassociated immunoglobulinsPlatelet suspension immunofluorescence test (PSIFT)Flow cytometryRadioimmunoassay Antiglobulin consumptionSolid phase red cell adherence (SPRCA)Total platelet associated immunoglobulinsNephalometryElectroimmunoassayRadial immunodiffusion

Phase II assays Introduced in early 1970 qualifies and quantifies the plt antibody measures the amount of antibodies on the surface or within the platelet they use anti immunoglobulin probe ( polyclonal/mono/staphy A) Two step assaytest platelets + known amount of Anti human IgGquantify residual unbound IgGIncubate

Phase II assays Direct binding assaylabeled ligand (I125 ,HRP, fluorescein ) + platelets Quantify the amount of ligands bound Total platelet associated IgG detergent lysis of platelet membrane

Release of IgG into the solution

Quantification (immunoprecipitation, Nephalometry, radial immunodiffusion)

Phase II assaysAdvantages Measures the immunoglobulin bound to plateletgreater specificity Disadvantages Measures all Ig bound pathological or non specific immunoglobulin granule IgG obtained by fluid phase endocytosisincreased in thrombopoietic stimulus

Phase III assaysMonoclonal antibody immobilization of platelet antigens (MAIPA)Antigen capture ELISA (ACE)Modified antigen capture ELISA (MACE)Immunobead assayImmunoblotting

Phase III assays First suggested by Van Leeuwen Measures the binding of antibody to a specific platelet glycoprotein

Advantages - No nonspecific binding- Precise identification of Ab bound to plt GP Disadvantages- costly - Steric hindrance auto/allo Abs share antigenic epitope with monoclonal antibodies

Solid phase red cell adherence assaySimple to perform once establishedDirect assay: measures antibody on plateletsIndirect assay: measures antibody in the serumDisadvantage: fails to distinguish platelet specific, non-specific antibodies Reagents:Rabbit antithrombocyte globulinPolyspecific AHGR1r red cells sensitized with anti-DPlatelets blood donors, platelet panels family members ABO compatible

Equipment

Method of solid phase assay Platelets to be typed for HPA are added to a U-shaped microplate coated with rabbit anti-human thrombocyte antibody, spun and incubated.

Excess platelets are removed by washing a platelet monolayer is left.

The monolayer is incubated with typing anti serum.

Platelet antibody will bind to platelet antigen.

microplate is washed.

Anti-human IgG + C3d reagent and Rh(D) sensitised red cells are added and the plate is spun.

HPA typing - SPRCA Donor platelets Vs typing anti sera IgG coated indicator red cellspositivecontrolnegativecontrol

Modifies SPRCATarget platelet + chloroquine or acid

Disrupts the class I HLA heavy chain peptide 2 microglobulin trimolecular complex

Modifies the antigenic epitope

Reduces binding of HLA specific antibodies

Flow cytometrySensitization of platelets with patients serumPolyclonal or monoclonal antibodies specific for human immunoglobulin, labelled with fluorescent dye (FITC)Results : MCF normal platelet + patient serum MCF normal platelet + normal serum

Advantages Very sensitive Antibodies specific for labile epitope can be detected ( unlike phase III assay )Disadvantages Does not differentiate between platelet specific and nonspecific antibodies

MAIPAMonoclonal antibody specific immobilization of platelet antigenDirect or indirect assayDonor pltDonor pltTarget plt simultaneously sensitized with pt serum and murine MoAbRecognize the desired target moleculeWashed and solubilized in nonionic detergentCentrifuge remove cytoskeletal fragmentsAntigen of interest

Wells coated with goat anti mouse AbsSupernatant lysate added to the wellsEEnzyme labelled goat anti human Ig probe

Advantages SimpleHighly specificPreservation of antigenic glycoprotein. Sensitization before solubilizatonLess nonspecific binding of IgG to the plastic surface less background noise

Disadvantages CostThey detect only antibodies that bind to plt proteins captured with monoclonal antibodiesLarge number of monoclonal antibody reagents neededFalse positive result:some normal subjects have anti-mouse antibodies

Immunobead assayDirect or indirectPlatelet glycoprotein is captured onto the solid phase by the murine monoclonal antibody directed against the glycoprotein of interestELabelled anti human antibodyAuto/allo antibody complexed with glycoprotein Anti-platelet monoclonal antibody attached to bead

Clinical significance NAIP

PTP

Refractoriness to platelet transfusion

Hpa 3b most common allele of hpa 3 thrombin and GpIb-IX complexes in the region between platelets GpIb and thrombin molecules are arranged as an adhesive ribbon structure,