PREDICTING THE SEVERITY OF NEONATAL ALLOIMMUNE THROMBOCYTOPENIA THROUGH RISK STRATIFCATION BY ASSESSING THE IgG SUBCLASSES INVOLVED

Abstract

Neonatal alloimmune thrombocytopenia (NAIT) is a potentially fatal condition that affects the fetus or newborn. Maternally derived IgG alloantibodies directed against paternally inherited specific platelet antigens cross the placenta and target fetal platelets. The most severe cases result in death due to intra-cranial haemorrhage (ICH) caused mostly by antibodies to human anti-platelet antigen (HPA)-1a (also known as glycoprotein IIb/IIIa or integrin αIIbβ3). Previous studies have tried, unsuccessfully, to predict which HPA-1a- positive mothers may be at risk of ICH and fetal death, focussing on antibody titre and /or IgG subclass. More recent evidence has implicated antibodies to integrin αvβ3, which shares a subunit with HPA-1a and is required for angiogenesis. Therefore, ICH could be due to vascular pathology caused by HPA-1a antibodies that cross-react with αvβ3. This study aimed to develop an assay that could detect antibodies to αvβ3 and to test if these were present in NAIT cases which had ICH. The second aim was to use LuminexTM technology, as previous studies used less sensitive methods, to investigate whether IgG subclass could be linked to NAIT severity. Human embryonic kidney (HEK) cells were transfected with expression vectors encoding the heterodimers αvβ3 or αIIbβ3. Protein expression was validated using monoclonal antibodies and known HPA positive sera by flow cytometry. Transfected cell strains were incubated with serum samples from NAIT referral cases testing positive for HPA-1a antibodies and were divided into ICH and non-ICH cohorts. Analysis using flow cytometry revealed that samples from both ICH and non-ICH when tested undiluted reacted with both HEK strains. A possible prozone effect was assessed by testing neat and 4-fold-diluted samples from the two cohorts but no such effect was observed. In order to detect αvβ3- specific antibodies, the αIIbβ3 antibody was absorbed out of the patient sera using a β3- coated resin. After absorption, three ICH samples were tested with αvβ3 transfected cells but did not have detectable antibody. The two cohorts (ICH v non-ICH) were tested at doubling dilutions from neat to 1/64 using the two different strains but no differences were observed between them. LuminexTM technology using an in-house multiplex bead assay with recombinant HPA-1a, HPA-1b and GPVI proteins was used to detect IgG antibodies and their subclass. Cut-offs were established using One Lambda’s normalised background ratio plus 3SDs from 22 female apheresis donors. Fifteen samples from the ICH cohort were analysed with 23 non- ICH, 7 post-transfusion purpura (PTP) and 15 platelet refractory patients. Differences were observed between non-ICH and platelet refractory samples for total IgG (p=0.027) and IgG1 (p-0.011), and with ICH, non-ICH, PTP versus platelet refractory for IgG2 subclass (p

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	John Aplin (Supervisor) & Melissa Westwood (Supervisor)