In Focus
Overview of fetal and neonatal alloimmune thrombocytopenia (FNAIT)
A quest for predictive parameters
Gerald Bertrand
HLA-HPA laboratory at the Blood Center of Rennes, France Read bio>
Gail Pahn
Platelet and Neutrophil Reference Laboratory at Australian Red Cross Lifeblood Brisbane, Australia Read bio >
Mathieu Roussy
HLA and HPA reference laboratory at Hema-Quebec, Canada. Read bio>
William Lemieux
HLA and HPA reference laboratory at Hema-Quebec, Canada. Read bio>
Lucie Richard
HLA and HPA reference laboratory at Héma-Québec, Canada. Read bio>
Lilach Bonstein
Blood Bank Laboratory and the National Platelet & Neutrophil Immunology Laboratory, Rambam Health Care Campus, Haifa, Israel
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating event occurring in 1/1000-2000 neonates, resulting from the destruction of fetal/newborn platelets by maternal alloantibodies (Figure 1).
These alloantibodies are directed against the immunologic determinant of platelet glycoproteins (GP), inherited from the father by the fetus, known as human platelet antigens (HPA).
Currently there are 41 documented HPAs1. Platelet alloimmunization may arise subsequent to feto-maternal exchange, and in contrast to hemolytic disease of the newborn (HDN) can occur during the first pregnancy (Press here to find out). Recent findings have shown that fetal syncytiotrophoblasts microparticles expressing GP can spread in maternal blood and may also be one of the immunizing events during pregnancy.
Figure 1. Physiopathology of Fetal/Neonatal Alloimmune Thrombocytopenia
Workshop
One of the major goals for the Platelet Immunology Working Party (PIWP) from ISBT, through the International Platelet Immunology Workshop, is to address the analytical approach to FNAIT and give platelet immunology reference laboratories more tools to elucidate complex cases.
When to suspect FNAIT?
An FNAIT diagnosis is suspected when a neonate presents with early onset isolated severe thrombocytopenia (less than 24-48 hours of age). It is a diagnosis of exclusion after considering other causes of fetal/neonatal thrombocytopenia: sepsis, disseminated intravascular coagulation and congenital conditions associated with thrombocytopenia. Association of FNAIT with other causes, especially maternal autoimmune thrombocytopenia, should also be considered. The diagnosis is all-important for the management of the index case and subsequent pregnancies3 (Figure 2).
Figure 2. Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence‐based practice, an international approach.
Laboratory diagnosis
Clinical presentation of FNAIT must be confirmed by investigation from reference laboratories specialized in platelet immunology, such as members of the International Platelet Immunology Working Party (PIWP). Laboratory diagnosis relies on detection and identification of maternal HPA alloantibodies, and identification of the causative paternal antigen. This is possible through performing internationally recognized and validated assays. During the International Platelet Workshop, participants are invited to perform serologic and genotyping techniques to resolve the cases submitted by the organizing committee. Sensitive and specific PIFT (flow cytometry method) and MAIPA (ELISA method) serological assays are designated to detect and identify maternal anti-platelet antibodies and are among the most widely utilized techniques. PIFT (Figure 3) detects antibodies bound to the surface of platelets, while MAIPA (Figure 4) identifies specific anti-HPA maternal antibodies by isolation of the platelet glycoproteins using monoclonal antibodies. Other commercial techniques can be useful, such as the Luminex based PakLx, the Capture-P (SPRCA) and the apDia MAIPA. Some laboratories have developed their own “in house” techniques (PABA, MPHA, FAS, SPRCA). Many reference laboratories will use a combination of two techniques for antibody identification. In addition to the serology, genotyping of the major HPA genes can be very useful to confirm or discard a doubtful reactivity. Genotyping can be obtained through SSP, RFLP, SSO, Bead-array, RT-PCR (Figure 5), Sanger or NGS. Each of these techniques have their advantages and limitations.
Variation in HPA alloimmunization
In individuals of European descent, HPA-1a alloimmunization is reported to be involved in 75% of cases, followed by HPA-5b and -3a alloimmunizations2. These proportions may however vary in different populations according to antigen prevalence. Alloantibodies against HPA-4 and isoantibodies against GPIV (CD36) (link to CD36 article) are most common in Asian and African communities. Although HLA typing is not usually required, there is evidence that most cases of severe HPA-1a alloimmunization can be associated to the presence in the mother of the DRB3*01:01 allele4,5.
Figure 3. Indirect Platelet Immunofluorescence test
Is maternal alloantibody titer a predictive parameter?
Some studies demonstrate that maternal alloantibody concentration or titer is a predictive parameter for severe FNAIT/intra cranial hemorrhage (ICH) when the dosage is performed early in pregnancy6, however a clear consensus is not achieved in some countries. This is the kind of controversy that the PIWP can address for discussion and assays can be designed in the biannual Workshop. Despite this, an increase in antibody concentration during the course of pregnancy is a good indication of immune stimulation. Follow-up of the antibody concentration during IVIG-treated pregnancies appears to be helpful for identifying therapy failure and managing delivery.
Figure 4. Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) assay
Platelet transfusion for FNAIT affected newborns
Although, the severely affected newborn should be promptly transfused with platelets without waiting for laboratory results, the ideal platelets for transfusion are those that will not be destroyed by maternal alloantibodies present in the newborn. Many countries have developed national registries to quickly identify unrelated compatible HPA-typed donors and, as in France, cryopreserved platelets may be readily available for transfusion or, as in Quebec (Canada) where the platelet inventory always contains two or more HPA-1a negative platelet units ready for distribution. As for emergency cases, random platelet transfusion is an appropriate strategy pending the availability of compatible units. Addition of IVIG can also be considered according to the gravity and evolution of the case. Normal platelet counts are usually obtained with/without treatment in 8-10 days, and, in the absence of ICH, the outcome is favorable.
Antenatal therapy
Antenatal therapy is proposed for women with a history of an affected infant and for those presenting antibodies directed against HPAs (Figure 2b)3. The pregnant women should be followed in referral centers offering genetic counseling. In the case of paternal HPA heterozygosity for the culprit antigen as for cases with an unidentified father, fetal HPA genotyping is necessary. Non-invasive procedures using cell-free fetal DNA isolated from maternal plasma are now available for the most immunogenic HPA systems. There is a consensus on maternal therapy with IVIG with/without steroids as a first line treatment. Dosing and timing of maternal therapy depends on severity of previously affected sibling (with/without ICH), and the strategy in use locally. Fetal blood sampling is the only way to determine the fetal platelet count. However, this invasive procedure, responsible for 1 to 2% of fetal loss, is no longer recommended. Elective C-section can be proposed as a safe way of delivery in some countries.
Figure 5. SNP Genotyping of HPA5 by TaqMan Real Time - PCR
References
- https://versiti.org/products-services/human-platelet-antigen-hpa-database/hpa-gene-database
- de Vos TW, Porcelijn L, Hofstede‐van Egmond S, Pajkrt E, Oepkes D, Lopriore E, et al. Clinical characteristics of human platelet antigen (HPA)‐1a and HPA‐5b alloimmunised pregnancies and the association between platelet HPA‐5b antibodies and symptomatic fetal neonatal alloimmune thrombocytopenia. Br J Haematol. Nov 2021;195(4):595‑603.
- Lieberman L, Greinacher A, Murphy MF, Bussel J, Bakchoul T, Corke S, et al. Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach. Br J Haematol. may 2019;185(3):549-562.
- Kjeldsen-Kragh J, Titze TL, Lie BA, Vaage JT, Kjær M. HLA-DRB3*01:01 exhibits a dose-dependent impact on HPA-1a antibody levels in HPA-1a–immunized women. Blood Adv. 9 avr 2019;3(7):945‑51.
- Wienzek‐Lischka S, König IR, Papenkort E, Hackstein H, Santoso S, Sachs UJ, et al. HLA‐DRB3*01:01 is a predictor of immunization against human platelet antigen‐1a but not of the severity of fetal and neonatal alloimmune thrombocytopenia. Transfusion (Paris). mars 2017;57(3):533‑40.
- Kjær M, Bertrand G, Bakchoul T, Massey E, Baker JM, Lieberman L, et al. Maternal HPA-1a antibody level and its role in predicting the severity of Fetal/Neonatal Alloimmune Thrombocytopenia: a systematic review. Vox Sangunis. january 2019;114(1):79-94.
