Application of Flow Cytometry to Diagnose Platelet Dysfunction

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2 Application of Flow Cytometry to Diagnose Platelet Dysfunction THANZ Workshop 28 th October 2017 Elaine Uhr, Institute of Haematology, Royal Prince Alfred Hospital, Sydney, Australia

3 Overview Platelet flow cytometry Flow cytometry identifies the platelet disorder at receptor level and forms a link between screening optical aggregation and molecular characterisation of the exact mutation Platelet flow requires - optics suitable for small particle analysis - a pure platelet population for single cell analysis and for quantitation of low density receptors Examples of special applications possible when the flow assays are fully optimised - inherited disorders, platelet antibodies and receptor blocking studies Platelets - numerous membrane receptors present - abundant granules

4 Platelet Granules - Flow Cytometry Granule numbers Detection post activation Lysosomes ~ 13 acid hydrolases Lysosome protein CD63 α granules ~ 80 proteins α granule protein CD62P Dense granules 3-6 chemicals Dense granule mepacrine Holmsen H, 1994 Skaer (EM + stains) - mepacrine is specific for dense granules Skaer R, B.J.H. 1981;49:435

5 Platelet Membrane Receptors - Flow Cytometry Detection Receptor numbers GPIIb/ ~50,000 GPIIIa Detection CD41 CD61 GPIb/V/IX ~25,000 CD42b (CD42a) GPIa/IIa ~ 2,000 CD49b GPIV ~20,000 CD36 5HT 2A variable Atabrine binding IgG 200,000 x normal in ITP Goat anti human IgG

6 Anticoagulant Effect on Platelets Electron Micrography: Citrate pseudopodia (Ca ++ dependent functional assays e.g. platelet activation) 1µm K 2 EDTA preserves the membrane for 24 hr Platelet antibodies Full blood count and blood film are both required prior to all platelet investigations Platelets, Ulutin (ed) Exerpta Medica 1975

7 Platelet Morphology Platelet size is an important diagnostic feature of all platelet disorders MagnificationX1000 Thrombocytopenia Reduced BM production Large 1-2% Normal distribution Large 5-10% ITP Immune destruction Large 10-25% Inherited Macrothrombocytopenia Giant > 30%

8 FBC and Morphology - Diagnostic for Inherited May-Hegglin May-Hegglin - a platelet disorder where the FBC analyser and morphology provide the diagnosis Large platelets Reduced platelet count Dӧhle bodies in neutrophils Toxic granulation May-Hegglin Prominent dark staining Dӧhle Bodies Pale homogeneous staining Dӧhle Bodies 7yr old boy with May-Hegglin disorder - autosomal dominant MYH-9 gene mutation MYH-9 =non muscle myosin Macrothrombocytopenia ISPUB ISSN

9 CD62P - α Granule Protein Present After Secretion CD62P copies of α granule protein detected after granule secretion Structure - long, thin sticky mucin repeats rapidly shed from platelet membrane Function - short term bridging between Platelets/Monocytes and Platelets/Neutrophils Flow - platelet activation assay for the absence of CD62P post agonist = Inherited Grey Platelet Syndrome - platelet - leucocyte assay for acquired in-vivo platelet activation CD41 -PE Platelet-monocytes = 33% of total monocytes Platelet - monocyte aggregation assays performed to monitor double hip replacement/ haemophiliac/ VIII inhibitor/ Novoseven Concern that high dose Novoseven may trigger in vivo platelet activation = No One only intra operation sample positive for platelet/monocyte aggregation at 33% CD14 FITC (monocytes) McEver Thromb. Haemostasis 1991; 66:806:80

10 Receptor GPIIb/GPIIIa: Detected as CD41 and CD61 CD41 / CD61-50,000 receptors per platelet Absence of CD41 and/or CD61 Alloimmune thrombocytopenia = inherited Glanzmann Thrombasthenia may present post platelet transfusion or via placental transfer = FMAIT (anti HPA1a) CD41/CD61 Fibrinogen binding site GPIIIa (CD61) Structure - β integrin complex Function - after activation fibrinogen binding site is exposed and produces platelet/platelet close contact to initiate calcium dependent aggregation FMAIT = feto maternal alloimmune thrombocytopenia HPA = Human platelet antigen

11 Optimal Times After Blood Collection for Platelet Function Assays Platelets in citrated whole blood with no tourniquet, at room temperature and no added agonist Fibrinogen binding % pos <15%, <<15% 30 minutes optimal Platelet Activation assay = platelets not yet activated optimal 4 hours LTA PRP (400x10 9 /L + 2µM ADP) = maximum aggregation responses for shape change/ primary wave/ plateau/ secondary wave

12 Inherited CD41/CD61 Disorder - Glanzmann Thrombasthenia Father - normal platelets (transport control) Fibrinogen binding = normal CD41 and CD61 = normal 100% 100% CD41 PE % pos Fibrinogen FITC % pos Fibrinogen binding <1% because CD61 = absent CD41 PE %pos 26% CD42b PE %pos Baby - Glanzmann thrombasthenia fibrinogen <1% CD42b PE %pos CD41 FITC %pos CD41 0% CD42b PE %pos CD42b PE %pos CD61 FITC %pos CD41 and CD61 = absent CD61 1% Fibrinogen FITC %pos CD41 FITC %pos CD61 FITC %pos Kaluza v1.0

13 Platelet Alloantibody: Anti HPA1a Seeped Out of the Donor Liver, Obliterated Recipient Platelets and was Detected in Recipient Serum Patient platelets 14% 5µL patient serum incubated with recovered Patient HPA1a pos platelets Antibody in the serum resulted in 67% platelets with attached antibody 5µL neg control serum 67% 47% 5µL weak pos control matched to WHO std 89% 2µL anti HPA1a pos control serum Assay- optimised neg/pos cut off ~50% matched to anti HPA1a WHO Std

14 Platelet Autoantibody: Platelet Specific α Granule IgG Platelet autoantibody (polyclonal) IgG present is specific to patient s own platelets detected on the platelet membrane after washing every technique to detect platelet antibodies requires a pre analytical wash step to remove the plasma protein halo surrounding each platelet EM of unfixed, unstained platelet rich plasma Note: during washing platelet specific α granule IgG is secreted onto the platelet surface membrane and is different from IgG present (due to another disease process) that may passively coat the platelet membrane Hughes et al Sem in Hematol, 2000: p222

15 Platelet Autoantibody : Platelet Associated IgG (PAIgG) Platelet Coated with High Titre ANA IgG - False Positive PAIgG Example : Patient with ANA titre >>2560 = anti centromere and platelet count 50 x 10 9 /L Centrifugal wash anti centromere Dialysis wash 70% 8% CD41 PE %pos CD41 PE %pos HEp-2 pattern SaH IgG FITC %pos SaH IgG FITC %pos IgG present in a patient with high titre ANA antibodies passively coated the platelet membrane was reduced by dialysis washing to normal platelet IgG = 8% pos IgG in the plasma coated, but was not bound to the platelet surface membrane and so was easily removed Current clinical practice - screen for and exclude collagenous disorders prior to diagnosis of ITP Bradwell A, Hughes R. Atlas of HEp-2 patterns 2007, 3 rd ed Birmingham UK

16 Platelet Autoantibody: CD36 is Target Antigen for Antibody to ADAMTS13 in TTP CD36-20,000 receptors per platelet and is also present on monocytes and endothelium Structure: large extracellular loop with many sticky mucin repeats for adhesion Function : scavenger receptor for antigens/pathological ligands CD36 Schultz by immunoblot identified CD36 as target antigen for antibody to ADAMTS13 - he demonstrated TTP antibody attached to CD36 and initiated - thrombotic complications via CD36 on platelets and endothelium TTP antibody at high titre is attached to and blocks the expression of platelet receptor CD36 TTP = thrombotic thrombocytopenia purpura Platelet CD36 has a similar role in Plasmodium falciparum infection J Clin Invest 2001;108:785 Wikipedia Schultz, BJH 1998;103:849

17 Platelet Autoantibody : SLE Related TTP Blocking of CD36 Receptor Expression by TTP antibody TTP patient treated with Plasma exchange + anti CD20 (Rituximab) Patient platelets demonstrated a reduction in TTP antibody titre on day dialysis wash removed blocking of the TTP antibody on CD36 2. CD36 expression was 100% positive (normal) for patient s platelets fresh to the circulation Centrifugal wash Dialysis wash 60% 90% 100% TTP = thrombotic thrombocytopenia purpura Note: CD36 conjugated antibody - IgG only (not IgM) Kaluza V3.0

18 Mepacrine Assay - Dense Granule Storage Pool Disease (SPD) Platelet are highly auto-fluorescent Normal blood smears showed an intense yellow fluorescence due to serotonin in platelets Serotonin Nieuwenhuis - tested 106 confirmed SPD and only 33% were abnormal by optical aggregation So we set up and optimised the flow cytometric mepacrine assay to measure quantitative uptake and secretion of fluorescent mepacrine (mimics serotonin) in order to more accurately detect mild inherited and mild acquired SPD Nieuwenhuis et al Blood 1987;70: Holmsen H. J Lab Clin Med 1970;75:

19 Anti-Malarials: Mepacrine and Atabrine as Flow Reagents Mepacrine sold in Europe, UK and Australia. In Australia mepacrine supply ceased, replaced with atabrine sold as mepacrine Both reagents are similar - mepacrine has an additional 2x (CH 2 Cl) We needed to re optimise the mepacrine assay to use atabrine as substitute reagent Mepacrine - synthetic antimalarial developed Germany/UK 1930s Atabrine - developed by US army 1942 Quinacrine N mustard dihydrochloride Sigma Q2876 MW 541 Relatively insoluble Quinacrine dihydrochloride Sigma Q3251 MW 472 Highly soluble Belson R Biotechnic Histochemistry 1973; 48: 311

20 Assay Requirements for Mepacrine and Atabrine Atabrine binding % pos 30 Mepacrine assay 1. - optimal incubation time 30 min 2. - mepacrine uptake continued after 30 min Atabrine assay 1. - optimal incubation time 20 min and assay required completion within <30 minutes including thrombin addition and flow cytometer analysis 2. - Atabrine detached from the platelet in a time dependent manner during incubation 3. - to achieve acceptable assay increments atabrine also required a further 1/10 dilution

21 Uptake of Mepacrine and Atabrine Into Red Cells Mepacrine uptake into red cells - 34% RBC positive for mepacrine Atabrine uptake into red cells 3.6% Absence of atabrine uptake into red cells explained the requirement for a further 1/10 dilution of atabrine to achieve suitable result increments in the atabrine assay Anti-malarial action of mepacrine - Mepacrine enters RBC and destroys gametocytes via DNA damage Wikipedia

22 Mepacrine and Atabrine Replace Serotonin as Ligand on Serotonin Receptors 5HT 3A and 5HT 2A By two different mechanisms the most likely explanation:- Mepacrine assay measures uptake into and secretion from platelet dense granules via serotonin receptor 5HT 3A a ligand-gated ion channel receptor Atabrine assay measures binding to and dissociation from the serotonin receptor 5HT 2A a G protein coupled receptor For further confirmation we carried out blocking studies Serotonin receptors: Currently there are 14 receptors in 7 families. All are G protein coupled receptors except 5HT 3A a ligand - gated ion channel receptor for uptake of ions and small particles e.g. serotonin Wikipedia

23 In Vivo Blocking Effect on Mepacrine and Atabrine Assays Post SSRI Overdose Selective serotonin reuptake inhibitor (SSRI) overdose - patient in Emergency Mepacrine µm Mepacrine Assay Uptake Platelets % positive Atabrine µm Atabrine Assay Binding Platelets % positive (5-15) (30 50) (15-30) (45 75) (30-60) (60 80) (55-90) (70 85) Parentheses denote reference ranges Mepacrine assay - normal uptake into dense granules after SSRI ingestion Atabrine assay - absent binding to the 5HT 2A SSRI - occupied the serotonin (atabrine) binding site and blocked binding of atabrine to 5HT 2A Application for Atabrine assay- to identify post partum haemorrhage due to SSRI ingestion near/at delivery (but not throughout pregnancy) as literature states SSRI ingestion is often not disclosed Heller et al, BMC Pregnancy Childbirth 2017;17:166

24 In Vitro Blocking Effect on Mepacrine and Atabrine Assays by Granisetron an Ion Channel Blocker Mepacrine assay Atabrine assay - Granisetron blocking of 5HT 3A receptor prevented mepacrine uptake into platelets - little or no blocking by Granisetron compared with blocking in the mepacrine assay Granisetron blocking - via the 5HT 3A receptor for mepacrine uptake and not via the atabrine receptor The routine investigation of a new reagent resulted in a separate useful clinical diagnostic assay Drug blocking efficiency was calculated as the % reduction in mepacrine % positive platelets (or atabrine) after incubation with each drug addition compared with % positive platelets incubated with buffer only. Granisetron was tested in vitro at therapeutic concentrations

25 Platelet Flow Cytometry Summary / Conclusion CD42b/42a ~25,000 fragile, ph sensitive 5HT3A Conc/time dependent uptake CD41/61 ~50,000 triggers aggregation time dependent CD62P ~1000 transient bridging 5HT2A serotonin binding rapid dissociation CD36 ~20,000 scavenger prothrombotic complications CD49b ~2000 adhesion, low density CD63 kill pathogens Indicator for activation due to artefact Conclusion - Receptor function determines the receptor structure - platelets have a collection of receptors all of which have special needs - platelet flow assays have been optimised to address the individual receptor differences

26 More Troops Died from Malaria in WWII than from Battle Wounds The British Experience Field Marshal Right Hon The Viscount Slim 13 th Governor General of Australia The American way Outbreak of Plasmodium falciparum 10% only had protective serum Mepacrine levels Good doctors are of no use without good discipline. It is they who see the daily dose of mepacrine is taken American Journal of Tropical Medicine & Hygiene If mepacrine was not taken, I sacked the commander. I only had to sack three; by then the rest had got my meaning. - Lieutenant General William Slim ( ), Burma Campaign, 1943

27 Platelet Activation Assay - to detect receptors and proteins from secreted granules Label and prepare tubes before blood collection Add buffer, antibodies specific for receptors/granule proteins, dilute agonist where required, diluted whole blood Incubate exactly 10 minutes Add Analyse Result very dilute fixative to temporarily arrest activation on the flow cytometer as % platelets positive for receptors and secreted granule protein markers

28 Method: Flow Platelet Antibody Assays Autoantibody Assay PAIgG - 50µL whole blood per tube is washed in acid buffer (neg/pos/patient), - platelets harvested - incubated with GaH IgG F(ab ) 2 - FITC in neutral buffer with dilute BSA - washed to remove excess anti IgG and anticd41-pe added - Results are expressed as % platelets positive for IgG and % large platelets Alloantibody Assay - washed platelets are pre-incubated with neg/pos/patient sera - washed - auto antibody assay followed as above without added BSA

29 Mepacrine Assay - Method Add anti CD41-PECy5 as platelet marker mepacrine dilutions whole blood diluted 1/40 Incubate exactly 30 min at 37⁰C Remove aliquots add thrombin add buffer to all tubes Analyse on the flow cytometer Results uptake (incubation tube) secretion (aliquot tube with added thrombin) Calculate release % reduction in mepacrine staining

30 Mepacrine Assay Technical Investigations Summary Physical Influences - whole blood - dil 1/40(20µL), no centrifugation (mechanical activation) - platelet count - adjust dilution when count > 400 x 10 9 /L - platelet size - large - uptake, secretion at higher conc mepacrine Technical Influences - buffer selection - HEPES Mg ++ free inert to platelet membrane - incubation time - 30 min at 37⁰C, time = cytoplasmic staining NS binding - mepacrine concentrations - 2.5,5,10,20µM(final conc) = cytoplasmic staining) - thrombin concentration U (final conc) - reduce for low platelet count (0.01U) - result expression - %pos for improved sensitivity - robust reference ranges - separate RR for children is not required for WB assay

31 FBC Analyser - Platelet Satellitism CD62P Bridges between platelets and neutrophils Blood collection 1 hour post 2 hours post 16 hours post Platelet count x 10 9 /L Platelet-neutrophil aggregates are a transient effect (post anti epileptic drug carbamazapine)

32 Platelet Allo-antibody Assay Anti HPA1a - FMAIT Platelet allo antibody - single point mutation on CD61 - anti HPA1a when baby platelets are HPA1a pos Mother 7% 5µL antihpa1a incubated with maternal platelets Mother s platelet phenotype HPA1a negative CD41-PE SaH IgG -FITC HPA1a pos platelets 10µL maternal serum incubated with HPA1a positive platelets Mother s serum potent allo-antibody anti HPA1a destroyed baby platelets CD41-PE 94% SaH IgG -FITC Baby Washed baby platelets at birth coated with anti HPA1a (IgG) antibody that crossed the placenta Baby platelets at birth are HPA1a positive, coated with anti HPA1a antibody CD41-PE 89% FMAIT = feto- maternal alloimmune thrombocytopenia SaH IgG -FITC

33 Receptor GPIb/GPV/GPIX : Detected as CD42b (and CD42a) CD42b - 25,000 receptors per platelet (vwf receptor complex) Absence of CD42b = Inherited Bernard Soulier Syndrome, Heterozygote detection requires - well defined reference ranges for CD42b and CD42a CD42b = GP1b CD42d = GPV Many mucin and leucine repeats for rolling and adhesion CD42a = GPIX Numerous cytoplasmic tails for rapid signalling Structure = sticky mucin and leucine repeats and numerous tails Function = rolling and adhesion at high shear rates to vwf on damaged endothelium = signalling

34 Platelet Alloantibody: Anti HPA5b (FMAIT) - CD49b CD49b (GPIa/IIa) - 2,000 receptors per platelet FMAITis rare with reduced severity 74% CD41 PE %pos IgG - FITC %pos 20ul anti HPA5b required for incubation step with HPA5b positive platelets Function: adhesion to exposed collagen Structure: numerous mucin repeats long cytoplasmic tail for signalling Technical: all reagents (e.g. antisera) require titration to obtain optimal quantitative response

35 Mepacrine Assay - Effect of Mepacrine Concentration at higher mepacrine concentrations % release is reduced and results are invalid due to mepacrine present in the cytoplasm The flow cytometer cannot discriminate between mepacrine present in the dense granules and mepacrine in the cytoplasm 2.5µM mepacrine 20µM mepacrine Confocal microscopy 5µM 2.5µM mepacrine is present in granules and is secreted after thrombin is added Fiona Kupresanin and Phillip Fromm, Anzac Research Institute, Concord 20µM mepacrine is present in the platelet cytoplasm and remains post thrombin induced granule secretion only mepacrine in granules is secreted

36 Mepacrine Assay - Effect of Mepacrine Concentration 20µM mepacrine is present in granules and in the platelet cytoplasm after thrombin induced granule secretion. This produces invalid results for mepacrine release at mepacrine concentrations >10µM 2.5µM mepacrine 20µM mepacrine Confocal microscopy 5µM 2.5µM mepacrine is present in granules only and is secreted after thrombin is added 20µM mepacrine is present in granules and in the platelet cytoplasm The flow cytometer cannot discriminate between mepacrine present in the dense granules and mepacrine in the cytoplasm Fiona Kupresanin and Phillip Fromm, Anzac Research Institute, Concord

37 Glanzmann Platelets - Normal Mepacrine Assay Results Mepacrine assay is independent of fibrinogen binding to GPIIIa (unlike optical aggregation) Mepacrine % pos platelets Mepacrine % pos platelets post secretion CD41 PECy5 CD41 PECy5 22% 8% 5uM mepacrine 5uM mepacrine + thrombin Mepacrine assay with CD41 absent - normal mepacrine uptake and normal thrombin induced secretion Release = % difference between % uptake and % post secretion 5uM mepacrine = 63% (NR 47-70%) Mepacrine release = % reduction in mepacrine staining post secretion

38 Atabrine Assay Scattergram Platelets Atabrine binding 54% platelets pos Atabrine assay flow cytometric histograms 0.5µM atabrine bound to normal platelets = 54% platelets were positive for atabrine binding Thrombin triggered atabrine release resulted in reduced % platelets positive for atabrine binding = 14% Atabrine staining of RBC = 0.3% red cells positive positive RBCs were back-gated into the flow scattergram

39 Effect of Palonosetron Blocking on Mepacrine and Atabrine Assays Palonosetron claimed high potency, specific 5HT 3A blocker? Mepacrine assay - Palonosetron demonstrated high potency blocking with prozone effect. High efficiency blocking in the atabrine assay suggested Palonestron blocking action is not via 5HT 3A alone. Prozone effect is absent in the atabrine assay Drug blocking efficiency was calculated as the % reduction in mepacrine positive platelets (or atabrine) after incubation with each drug dose compared with % positive platelets incubated with buffer only

40 FBC Analyser Mean Platelet Volume and Morphology Platelet size is an important diagnostic feature of all platelet disorders Whole blood collected into K 2 EDTA, platelet histograms - ABBOTT Sapphire MPV = <5 fl MPV = 6-9 fl MPV = fl MPV = >16 fl Normal (2-4µM) X1000 Thrombocytopenia Reduced BM production Large 1-2% Normal distribution ITP Immune destruction Large 10-25% Inherited Macrothrombocytopenia Giant > 30% present outside the platelet gate