PROGRESS AND DEVELOPMENT

OF PLATELET ANTIBODY DETECTION

L.Porcelijn, E. Huiskes, M. de Haas

INTRODUCTION

• Human platelet antibody (HPA) detection is necessary for the diagnosis and therapeutic decisions for refractoriness to platelet transfusions, post transfusion purpura and fetal and neonatal alloimmune thrombocytopenia.

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• In the last four to five decades many new developments, both in knowledge and methods, have increased the quality of platelet serology.

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• However, the quest for the optimal antibody detection method(s) encountered, sometimes unexpected, difficulties.

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• In this review the various aspects concerning platelet antibody test methods and detection of platelet antibodies both for the diagnostic and screening setting are discussed.

PHASE 1�

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🡺Early methods for platelet antibody detection

a) platelet aggregation tests

b) serotonin release

c) complement fixation techniques

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🡺The first platelet specific antibody, anti-Zw (a) (later named HPA-1a) was demonstrated in the platelet aggregation test .

THE HISTORY OF PLATELET ANTIBODY DETECTION

PHASE 2��

🡺In the 1970s- the availability of radiolabelled or immune fluorescence labelled anti-human imunoglobulins of the IgG, IgM or IgA class more sensitive and specific methods e.g.

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• Radio Immune Assay (RIA)
• Platelet Immunofluorescene Test (PIFT)
• Mixed Passive haemagglutination Assay (MPHA)

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🡺Limitation

the co-expression of human leucocyte antigen (HLA) class-I antigens and Fc-gamma-receptor (FcγR)IIa on platelets causing difficulties to distinguish HLA antibodies from human platelet antigen (HPA) antibodies and non-specific binding of IgG via the Fc-part to the Fc-receptor.

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🡺Chloroquine -

removes the HLA antigens from the platelet surface and prevents HLA antibodies from binding while the human platelet antigens (HPA) carrying glycoproteins remain intact

PHASE 3�

🡪Isolated Glycoproteins (GPs)

a) First assays to detect antibodies

b) bypassing the HLA and Fc-receptor ‘problem’, applied sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

c) Unreliable test – heating up to 95 °C causes denaturation of proteins

PHASE 3�

• 1970’s 🡪 solubilization of the platelet membrane and extraction of the membrane proteins, retaining their antigenicity, with non-ionic detergents was describe.

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• Together with the availability of Gp-specific monoclonal antibodies (moab) platelet antibody detection methods became possible.

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✌ Woods & Mcmillan 🡪 1st GP-specific immunoassay for detection of

platelet antibodies against GP llb/llla and GP lb/IX

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• Furihata et. Al and Kiefel et. al 🡪 introduced the antigen-capture ELISA (ACE) and monoclonal antibody immobilization of platelet antigens (MAIPA, see Fig. 1)

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✌1989-Menitove et al 🡪 introduce modified antigen-capture ELISA ( MACE)

MAIPA ASSAY

• 1990s,🡪 beads coated with sheep or goat-anti-mouse moab serving as solid phase target for the specific mouse-anti-human-platelet glycoprotein complexes were used in modified MAIPA assays.

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• Advantage 🡪 of these beads assays was shorten the test time of approximately 3.5 h compared to eight hours for the MAIPA or later 5 h for the modified rapid MAIPA

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→ Joutsi et. Al 1997

✶ Introduce detection of antibodies on different Gps

✶ Use different fluorescent labels for the GP-specific moab in MAIPA ; later

✶ Use polystyrene beads with varying fluorescence intensity

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→ Nguyen et al 2004

✶ A ‘beads MAIPA’ called SASPA, with different IgG subtype specificities to

prevent cross-capture of the GP-specific moab’s,

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→ Fujiwara et al 2009

✶ Introduce a method with mouse-anti-GP beads, called ICFA

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METHODS FOR SIMULTINEOUS DETECTION OF MULTIPLE HPA ANTIBODIES

→ Metzner et al 2017

✶ An equivalent of the MACE on Luminex beads called platelet antibody bead array (PABA)

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→ Meyer 2006, Bakchoul 2008

✶ an HPA antibody detection method using gel ID cards (gel antigen specific assay, GASA)

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However, because the (modified) MAIPA assay is time-consuming and technically demanding, many (non-reference) laboratories use more convenient solid-phase ELISA techniques with GP’s from HPA typed donor coated on microtiterplates or Luminex beads (Fig. 2), which became commercially available in the decades after the introduction of the MAIPA

�Luminex beads based HPA-antibody�detection assay

Fig. 2. Luminex beads based HPA-antibody

detection assay.

Luminex beads with different colors carrying different HPA typed GP structures, comparable with a platelet antibody identification panel, are used. In this figure the human serum containing anti-HPA-1a antibodies is added to a mixture of beads. The HPA-1a antibodies will bind to the HPA-1a positive beads. After incubation with a fluorescence labelled anti human IgG, lasers measuring the bead color

and the presence of fluorescence (mean flouresence intensity) are used for the identification of the antibody

SURFACE PLASMA RESONANCE

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• Not available in all platelet serology laboratories
• The SPR method for the detection of HPA antibodies seems promising.

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🡺Bakchoul et al. (2011)

• successfully tested Isolated IgG fractions from maternal sera in flow cells with purified HPA-1 typed GPIIb/IIIa from platelet donors

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• MAIPA - 68 detected HPA1a
• SPR - 75 detected HPA1a

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🡺Peterson et al. (2012)

• showed a higher sensitivity using SPR for 61 suspected FNAIT cases with HPA-1a negative mothers

83 suspected FNAIT case with HPA1a negative

HPA-typed cell lines and glycoprotein contstructs

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• The above-mentioned test methods are all based on the use of HPA typed donor platelets.

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• The availability of certain typed donors can be a problem as the frequency of some HPA’s is extremely low.

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• To overcome this problem, Hayashi et al. established an HPA typed cell line panel as an alternative source of platelet antigens

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• In addition to the low availability of certain typed donors, conformational changes of GPs after isolation and incomplete solubilisation can cause incorrect results.

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• To prevent these problems the use of recombinant glycoprotein constructs, as described by Stafford et al. in 2008 and validated by Chong et al. in 2015 can be a solution

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🡺Until now 41 human platelet antigens are known, of which 12 are part of bi-allelic systems, with varying allele frequencies in different populations (references http://www.versiti.org/HPA).

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🡺To value the antibody detection results it is necessary to consider the clinical symptoms, the ethnic origin of the patient and the HPA pheno- or genotyping results.

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🡺 The intact platelet antibody detection assays are generally less sensitive and specific than glycoprotein due to ;

✶ low and/or varying expression of certain GP(e.g. GPIa/IIa carrying

HPA-5 and CD109 carrying HPA15)

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✶ the expression of HLA-class-I making it difficult to distinguish anti-

HLA from anti-HPA

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✶ the presence of FcγRIIa on platelets causing non-specific binding of the Fc-tail of antibodies

General introduction HPA antibody detection

On the other hand, disadvantages of GP specific assays are :

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a) GP isolation prior to incubation with the patient’s serum can result in antigen loss and non-specific binding of antibodies due to damage or conformational change non-specific binding of antibodies to the microtiterplate wells or beads can cause false positive results

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b) Only test for antibodies of the IgG class

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c) The mouse-anti-human moab can block human antibody binding

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d) Only antibodies directed against antigens located on the investigated glycoproteins will be detected

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● Most commercial assays test for the a and b antigens within the HPA-1, -3 and -4 systems (GPIIb/ IIIa), as well as the HPA-2 (GPIb/IX) and HPA-5 (GPIa/IIa) systems.

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● The commercial Luminex beads assay PAKLx also includes a GPIV specific bead.

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• However, none of them includes CD109 (HPA-15), which might be due to technical difficulties in coating this glycosylphosphatidylinositol (GPI)-linked cell surface antigen

• Most platelet serology reference laboratories nowadays use one or several glycoprotein-specific antibody detection methods

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• 95% use MAIPA whether or not in combination with commercial or in-house modified ELISA assays or Luminex based beads assays

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HPA antibody detection

🡺ANTI-HPA 1

• consists of the antigens HPA-1a and -1b

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• among Caucasians these antigens are expressed by 97.6 % and 28 %, allele frequencies 0.846 and 0.154, respectively)

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• can cause refractoriness for platelet transfusions (PTR), FNAIT and post-transfusion purpura (PTP)

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🡺Anti HPA-2a and -2b

- very rarely involved in FNAIT

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- HPA-2b antibodies regularly seen in PTR ; and

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- Often a combination of antibodies of the IgG and IgM class.

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• Anti HPA3

-HPA3a and 3b, can be involved in FNAIT, PTR and PTP.

- HPA3 antibodies can be difficult to detect with GP-specific antibody detection assays

-Alloantibodies against HPA-3b are very rare.

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HPA-4

• located on β3 in the α2β3 complex
• In the Asian population, immunization against HPA-4b is regularly seen in Japan.

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🡪HPA-5

-located on GPIa of the GPIa/IIa complex

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Anti-HPA 15

- located on CD109

• Antibodies against HPA-15a or -15b are associated with cases of FNAIT and PTP, but are more regularly detected in patients suffering from PTR

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Anti-NaK^a (CD 36)

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✹ CD36 is known as platelet GPIV (NAKa ) on platelets.

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✹ A lack of GPIV on platelets (known as type II CD36 deficiency) is seen in approximately 0.1 % of Caucasians, 1.3 % in Chinese, 2.4 % African-Americans, 4.8 % Afro-Caribbean’s, 8 % sub-Saharan Africans and 5–10 % Japanese .

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✹ Immunization against GPIV, also known as NAKa can occur in a small percentage of these individuals (approximately 0.5 % of Asians and Africans) who also lack CD36 on other cell types (type I CD36 deficiency), causing FNAIT or PTR.

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✹ Anti-NAKa can best be detected with GPspecific antibody detection assays, but false negative results can occur because of GP-specific mouse-anti-human moab’s blocking the human antibody binding

conclusion

• The availability of labeled anti-human-Ig antibodies, platelet membrane solubilization, GP-specific moab’s, Luminex beads and recombinant GP constructs in combination with HPA (geno)typing possibilities have changed platelet serology.

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• However, there still is not one method which can detect all possible clinical important antibodies.

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• It remains clear that antibody detection should be done in laboratories with sufficient knowledge and experience and that also for those (reference) laboratories standardization of test methods is necessary.

HISTORY AND DEVELOPMENT

OF PLATELET IMMUNOLOGY

IN PDN

⇒ Training on platelet workshop at Pusat Perkhidmatan Darah Kuala Lumpur.

⇒Train on method Solid Phase Red Adherence Cell (SPRAC)

⇒Early stage using Capture-P (commercial kit)

• MAIPA is a gold standard method for all countries including Malaysia.
• Training provided for each development.

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● 1995 - platelet test mainly using Capture-P

- tested in batch once a month

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● 1998 - training in Australia SPRCA Assay together with MAIPA Assay

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● 2001 - Development platelet antibody testing using panel provided by

Australia Red Cross

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● 2005 - Retraining on MAIPA Assay in PDN

- Trained by Australia Scientist

- currently using MAIPA assay with developement method

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● 2008 - training in Australia for platelet Immunology including molecular

● 2014 - Rapid Maipa training in Guangzhou 2014

● 2018 - Modified Rapid Maipa training in Japan 2018

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Test offered in Platelet Immunology in PDN

METHOD USE IN PLATELET IMMUNOLOGY, PDN

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• A simple method
• Able to detect platelet reactive antibodies attached to the surface of the platelet membrane.
• Includes : ABH antibodies, HLA Class1 antibodies, HPA antibodies, auto antibodies, non-specific platelet reactive antibodies
• Capability : High sensitivity but low in specificity
• Mixed antibodies often mask and complicate the identification of antibodies toward HPA

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• A complex method
• Detection is depending on the specific monoclonal antibody against the investigated glycoprotein
• Includes : Antibody identification for HPA antibodies, antibody screening for HLA antibodies
• Capability : Good sensitivity but high specificity
• Can separate and identify specific antibody
• Resolution for weak and mix antibodies

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SPRCA

(Solid Phase Red Cell Adherence Assay)

Whole / intact platelet assay

MAIPA

(Monoclonal Antibody Immubilisation of Platelet)

Antigen Glycoprotein capture assay

�HPA Antibodies specific detected in PDN�2011-2020�

THANK YOU