The Glanzmann Thrombasthenia (GT) is a rare inherited disorder of platelet function causing quantitative or qualitative defects of the platelet membrane glycoprotein (GP) IIb-IIIa (αIIbβ3) complex.1,2 The incidence of the disease is approximately 1 per million, but this increases to 1 in 200,000 in areas of high consanguinity.3 The GT may be sub-classified using laboratory criteria into type 1 variants (< 5% GPIIb-IIIa expression), which is associated with severe bleeding and type 2 (5–20% expression of the GPIIb-IIIa) or type 3 qualitative GT variants, in which the bleeding phenotype may be less severe.1

The Bernard-Soulier Syndrome (BSS) is usually associated with an autosomal recessive inheritance that causes a defect of the platelet membrane glycoprotein (GP) Ib-IX-V complex. The incidence is similar to the GT incidence, as well as the relation to consanguinity.3 The main characteristic of the disease is macrothrombocytopenia with giant platelets often leading to misdiagnosis.

The treatment for both conditions is based on platelet transfusions, which can cause adverse events, such as allergic reactions, volume overload, transfusion-related acute lung injury and transfusion-transmitted infections. Alloimmunization against human platelet antigen (HPA) or human leucocyte antigen (HLA) epitopes is also common4 for patients requiring frequent transfusions and may be associated with the platelet refractoriness and therapeutic failure of platelet transfusion.

It is also not uncommon that GT and BSS patients develop antibodies against the portions of normal glycoproteins that are deficient in the disorders, which lead to difficulty in the transfusion support.1 The risk of antibody formation may be different, depending on the subtype of the GT, considering that patients with homozygous or compound heterozygous mutations totally lacking αIIbβ3 are more prone to develop antibodies than others with some αIIbβ3 expression.5 One small study showed the presence of anti-HPA antibodies in 81% of patients with no αIIbβ3 expression, versus 25% with less severe mutations.6 The literature is yet incomplete in reference to the risk of antibody formation in BSS, but it is probably less frequent than in the GT.7

The presence of platelet alloantibodies may cause immune platelet refractoriness, worsening the transfusion management of patients with rare inherited bleeding disorders. Recombinant activated factor VII (rFVIIa) can be used for the treatment and prevention of bleeding episodes and as prophylaxis for invasive procedures in patients with GT and alloantibodies, as well as in patients with past or present immune platelet refractoriness. Nevertheless, off-label use of rFVIIa in patients with GT who have frequent bleeding episodes is increasing, mainly to avoid alloimmunization to platelets.8

The methods to detect the platelet alloantibodies may include techniques, such as the platelet immunofluorescence test (PIFT),9 which is a screening assay for the detection of antibodies in the serum, frequently used for cross-matching the patient serum with the platelets to be transfused. It is a sensitive test, but it does not allow the identification of the specificity of the antibody. Other tests, such as the MAIPA (using ELISA technique)10 and the bead-based Luminex Platelet Assay, combine a good sensitivity and specificity,11,12 however they are more laborious and costly.

In this study, we implemented a practical and efficient strategy with the combination of methods to detect the presence of alloantibodies in the GT and BSS patients, aiming to describe their frequency in this population. We also suggest an algorithm for alloantibody detection in the GT and BSS patients.

Inherited platelet disorders, such as the Glanzmann Thrombasthenia and Bernard-Soulier Syndrome, are rare hemorrhagic disorders with a frequent need for platelet transfusion and, consequently, the formation of anti-HLA and anti-HPA antibodies in a varying proportion of patients, which can result in platelet refractoriness, directly impacting the clinical management. By implementing three different techniques, we developed an algorithm (Figure 1) for evaluating the presence of alloantibodies in a population of patients with GT and BSS from a reference center in southeastern Brazil and we found a frequency of platelet alloantibodies of 18.1% in the GT patients (n = 2/11 patients - 1 patient with anti-HLA and 1 patient with anti-HPA) and 25% in the BSS patients (n = 1/4 patients - anti-HLA).

Previous historical reviews have described a prevalence of platelet alloantibodies varying from 25% to 49% among GT patients,4,13 which may be anti-HLA or anti-HPA.1 In the international GT registry data, 65/218 (30%) of patients treated with different hemostatic agents developed platelet alloantibodies, of which 47 were against the GPIIb-IIIa and 21 towards the HLA.14,15 The only patient from our cohort who presented an anti-HPA antibody did not have clinical evidence of platelet refractoriness. The presence of the alloantibody in the absence of clinical significance may be explained by previous studies that had already suggested that platelets may be effective, even in patients with platelet alloantibodies, because of long time intervals between exposure to platelets in the GT and perhaps also due to the transient nature of the alloantibodies.16 Another possibility already mentioned in other studies is the presence of “natural” antibodies to HLA antigens, even without transfusion, just like the anti-A and anti-B.17 In our study, the early initiation of the use of platelet transfusion does not seem to have interfered with the formation of alloantibodies, as none of the patients with detected alloantibodies received transfusion before the age of 5.

In the context of the clinical management of bleeding or prophylaxis for invasive procedures in patients with these disorders, the identification of platelet alloantibodies is extremely important, as an important therapeutic strategy for patients with GT is the use of rFVIIa, which is a high-cost medication, with an approved indication only for patients with a previous or current history of platelet refractoriness and identified anti-HPA and / or anti-HLA antibodies in Brazil. We identified 2 patients with anti- HLA antibodies showing reactivity greater than 97% in the PRA-HLA testing. This positivity rate is critical, as it will make it extremely difficult to access a negative crossmatch apheresis platelet, requiring an extensive HLA-typed donor bank to find a compatible donor. In this scenario, the rFVIIa becomes a fundamental therapeutic tool. Since 2014, the rFVIIa had been approved by the FDA for use in the US in patients with GT with refractoriness to platelet transfusions, with or without antibodies for platelets. Similarly, the European label for the rFVIIa has been extended recently to include patients with GT with past or present refractoriness to platelet transfusions, or where platelets are not readily available.18 However, in many other countries, such as Brazil, currently the detection of platelet alloantibodies is still essential for these patients to have access to this therapeutic option.

In order to facilitate, systematize and reduce the cost of identifying these antibodies, through this study we developed the algorithm in Figure 1. From the results of this study, we suggest the application of the PIFT as an initial screening test for the detection of platelet alloantibodies in all patients with GT or BSS, even if they have never been previously transfused. Furthermore, it is of great value in patient management, since in case of a positive result, it is even possible to select a platelet unit to be transfused to the patient if necessary, as at least 4 different platelet samples are used in the methodology of the test. Although the PIFT has a good sensitivity, it does not discriminate between anti-HLA and anti-HPA antibodies.19 After an initial positive result, it is necessary to confirm it using a test of higher specificity, such as the MAIPA or PAKLx, that also allow us to identify which alloantibody is present. In our study, we found a 100% agreement between the results of both techniques, similar to previous studies,11 suggesting that any of them can be used individually. The two methodologies use more laborious techniques and are more expensive, which is why they could be reserved only for initially positive patients in the PIFT test.20

To our knowledge, this is the first description of frequency of alloimmunization in inherited platelet disorders from Latin America. Patients included in the study have been followed in the same outpatient clinic for a long time and by the same medical team. All patient samples were tested in the same reference immunohematology laboratory, using the same techniques, which ensures the reproducibility of the results. The access to different laboratory techniques for the detection of alloantibodies allowed us to evaluate the results looking for the best cost benefit in the process of identifying these antibodies.

As limitations, data on clinical characteristics and bleeding manifestations were collected retrospectively, which limits the availability of detailed data. The time of blood collection for the antibody testing was not related to the moment of the transfusion and the test was performed only once for each patient, without a follow-up. Our study was not able to evaluate the hypothesis of fewer antibodies in patients with type 2 GT caused by the protective effect of some amount of antibody because none of our patients had the type 2. Another major limitation is the small number of patients included, which is justified by the rarity of these disorders.

In conclusion, our findings describe a relatively low frequency of alloimmunization in our population of patients with hereditary platelet disorders. However, we emphasize the extreme importance of identifying these antibodies in the clinical management of these patients, to enable access to the rFVIIa. Following the algorithm we developed, it is possible to perform the detection in steps, maximizing the use of resources and making the best use of each technique and the results they provide. Further studies in a larger number of patients are necessary for a better understanding of antibody frequency and factors that lead to the formation, as well as to define the clinical significance of these alloantibodies.