Rare complication of treated immune thrombocytopenia
Authors: Radhika Gangaraju, Anton Rets, Kristi J. Smock, Nahla M. Heikal
Journal: American Journal of Hematology (2017)

A 58-year-old female presented to the emergency room with dark-colored stools, exertional shortness of breath and generalized weakness. Her past medical history included immune thrombocytopenic purpura (ITP) which was diagnosed several years prior to the current presentation (exact date unknown). Because of refractoriness to steroids, she was treated with rituximab and showed a good clinical response for a few years. She underwent splenectomy seven years ago due to relapsed ITP, which resulted in normalization of her platelet count. However, after 2 years, she developed recurrent thrombocytopenia with platelet count of 28 × 109/L and mild elevation in liver function tests. This prompted a computed tomographic (CT) scan of the abdomen which revealed an accessory spleen that was removed. She also received another treatment with rituximab at that time with improvement in platelet count. This produced a prolonged normalization of platelet counts and she did well until this acute presentation with gastrointestinal bleeding. Her physical examination was unremarkable except for pallor in mucous membranes. Her complete blood count (CBC) showed white blood cell (WBC) count of 8.6 × 109/L, hemoglobin (Hb) 7.6 g/dl and platelets of 326 × 109/L. Her prothrombin time (PT) and activated partial thromboplastin time (aPTT) were normal. Peripheral smear showed normocytic normochromic anemia, consistent with post-hemorrhagic anemia, borderline neutrophilia and normal platelet number with post-splenectomy changes, including moderate anisopoikilocytosis and Howell-Jolly bodies. The platelets showed preserved granularity and appropriate variability of size. Esophagogastroduodenoscopy showed erythema of the stomach with 8 mm bleeding polyp which was cauterized and removed. Though the patient had a history of ITP, due to normal platelet count and peripheral smear morphology on presentation, her bleeding was attributed to the structural lesion in the stomach and she was discharged home after stabilization of bleeding. She presented to her hematologist one month later, and complained of frequent nose bleeds and easy bruising. She was evaluated by an ENT physician who did not find any exposed vessel that would explain the epistaxis. This raised concern for a hematologic disorder that was causing epistaxis. CBC done in clinic showed a WBC count of 8.7 × 109/L, Hb of 12.5 g/dL, platelet count of 382 × 109/L and MCV 87.6 fL. The patient's samples were sent to our institution for diagnostic evaluation, including platelet aggregation studies. Since the CBC showed normal platelet count and peripheral smear was normal, we performed the coagulation screening tests (PT, aPTT) followed by a von Willebrand panel [von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and factor VIII activity (FVIII)]. The coagulation screening tests showed normal results with PT of 12.5 seconds (normal range 12.0–15.5 seconds) and aPTT of 31 seconds (normal 24–35 seconds). Von Willebrand panel indices were proportionately elevated: VWF:Ag of 256%, VWF:RCo of 259%, and FVIII of 349%, suggestive of an acute phase response. Since the initial tests were all normal, we suspected a platelet function defect. It should also be noted that fibrinogen and thrombin time measurements are often part of the initial work-up of a bleeding disorder, but due to the mucosal bleeding pattern in a patient with an ITP history and lack of clinical suspicion for fibrinogen disorders, these were not performed in this case. Light transmission platelet aggregometry (LTA) was performed on the patient's platelet-rich plasma using a PAP-8 Platelet Aggregation Profiler (Bio/Data Corporation, Paris, France). The following agonists were tested: collagen (5 µg/mL and 2 µg/mL), adenosine-diphosphate (ADP) (10 µM, 5 µM, and 3 µM), arachidonic acid (0.5 mg/mL), and ristocetin (1.5 mg/mL and 0.5 mg/mL). The platelet functional studies demonstrated markedly reduced aggregation to collagen, ADP, and arachidonic acid, as well as a borderline decrease of aggregation in response to high-dose ristocetin and normal response (absent aggregation) to low-dose ristocetin (Figure 1). Platelet aggregation studies in normal control donor (A) and the patient (B). Agonist concentrations and testing positions (1–8) are as above. Percent aggregation is depicted on the y-axis and time (minutes) is shown on the x-axis. The patient tracings demonstrate markedly decreased aggregation to all agonists except high dose ristocetin, which demonstrates a borderline decreased aggregation pattern [Color figure can be viewed at wileyonlinelibrary.com] The pattern of platelet dysfunction seen in this patient is consistent with Glanzmann's thrombasthenia (GT), which in the inherited form is a rare autosomal recessive disorder caused by mutations in the genes encoding platelet surface glycoprotein receptors GPIIb (CD41) and/or IIIa (CD61).1, 2 This leads to absent or decreased expression of the GPIIb/IIIa receptor for fibrinogen binding on the surface of platelets or less commonly, to receptor dysfunction. These patients have a variable bleeding diathesis; though most of them present in early childhood with moderate to severe bleeding. Since our patient did not have any bleeding complications until later in her life, congenital GT was not suspected. In contrast, acquired GT (AGT) is a condition resulting from functional defects of GPIIb/IIIa due to blocking autoantibodies directed against this receptor without increased clearance. The prevalence of AGT is also low with less than 30 cases reported in the literature. To further investigate the cause of her platelet functional defect, we performed flow cytometric analysis of platelet glycoprotein expression on a BD FACS Canto II flow cytometer instrument (BD Biosciences, San Jose, CA), and included detection of CD41 (CD41*APC, BioLegend), CD42b (CD42b*PE, BioLegend), and CD61 (CD61*PerCP, Becton Dickinson). CD41 and CD61 are epitopes on the GPIIb/IIIa complex and CD42b is an epitope expressed on the GPIb/IX/V complex. The flow cytometric evaluation of CD41, CD42b, and CD61 expression was within normal limits (Figure 2). Flow cytometric analysis for platelet glycoprotein expression showing normal expression of CD41, CD42b, and CD61 [Color figure can be viewed at wileyonlinelibrary.com] With the flow cytometry results we were able to rule out decreased expression of platelet surface glycoproteins and the two classic congenital forms of platelet function defects, Bernard—Soulier (not suggested by the functional studies) and GT. However, this did not explain the cause of her bleeding, and dysfunction of GPIIb/IIIa was not yet excluded. We further proceeded with platelet antibody testing. The platelet-associated antibodies test (direct platelet antibody) that detects autoantibodies attached to patient's platelets, was performed by flow cytometry using the following antibodies: rabbit F(ab′)2*FITC control reagent (Dako), polyclonal rabbit F(ab′)2 anti-human IgG*FITC (Dako), polyclonal rabbit F(ab′)2 anti-human IgM*FITC (Dako), and mouse anti-human CD42b*PE (Immunotec, Beckman-Coulter). CD42b was used for platelet gating. IgG antibody attached to the platelet surface, but not IgM, was detected (Figure 3). The indirect platelet antibody identification test detects free antibodies in patient's serum without differentiating between allo- and auto-antibodies.3 The test was performed by a qualitative solid phase ELISA assay (PAK PLUS, Immucor), designed to detect antibodies to HLA class I antigens and to antigenic epitopes on the major platelet glycoproteins Ib/IX, Ia/IIa, IIb/IIIa and IV. The indirect antibody panel showed the presence of antibodies broadly reactive against GPIIb/IIIa and GPIa/IIa. CD42b-gated platelets from the patient demonstrating presence of IgG, but not IgM, attached to the platelet surface [Color figure can be viewed at wileyonlinelibrary.com] The pattern of serum free antibody reactivity to more than one platelet antigen in the indirect platelet antibody test is suggestive of autoimmune rather than alloimmune antibodies, and is concordant with the presence of IgG attached to the patient's platelets in the direct platelet antibody test. The platelet dysfunction pattern is consistent with Glanzmann's thrombasthenia with normal expression of GPIIb/IIIa, confirming the diagnosis of AGT. The patient continued to have intermittent epistaxis needing embolization and easy bruising, but these bleeding episodes have not been severe enough to warrant any systemic therapy. With the knowledge that she has a functional platelet defect, this patient should be closely monitored for any signs of bleeding especially during surgical procedures. AGT is a rare hematological disorder in which patients present with a bleeding diathesis later in life without history of bleeding in childhood. It occurs due to development of autoantibodies or a plasma protein inhibitor which interfere with the function of platelet glycoprotein receptor GPIIb and/or IIIa.4, 5 GPIIb/IIIa is the most densely expressed glycoprotein on the platelet surface and serves as the receptor for fibrinogen, making it one of the key players in platelet aggregation.1, 6 Autoantibodies directed against GPIIb/IIIa in ITP patients were first described by van Leeuwen et al., and the first case of AGT was described by Greaves et al.7, 8 Patients with AGT can have bleeding despite normal platelet counts or may present with bleeding which is disproportionate to the platelet count.6 It is seen in association with autoimmune disorders, lymphoproliferative disorders or use of certain medications.5, 9, 10 It is characterized by the typical GT pattern of decreased or absent platelet aggregation to collagen, ADP, epinephrine (not tested in this case), and arachidonic acid while response to ristocetin is normal or mildly decreased, similar to the congenital form. However, expression of platelet glycoprotein receptors on the surface of platelets is normal.11 The clinical bleeding history, age at presentation, and presence of platelet autoantibodies to GPIIb/IIIa are helpful to differentiate it from inherited forms of GT, with receptor dysfunction rather than deficiency. The reason for decreased ristocetin-induced platelet aggregation in some subjects with GT is currently unknown since ristocetin mediates aggregation through the GPIb receptor rather than IIb/IIIa, but this phenomenon has been reported in the medical literature.12 There is paucity of literature estimating the risk of bleeding with the degree of thrombocytopenia in ITP patients, and platelet count is often used as surrogate marker for risk of bleeding in clinical trials of ITP. In general, platelet counts above 30 × 109/L do not correlate with bleeding and counts below 10 × 109/L have some correlation; however, the details of this relationship have not been well characterized.13 In patients with AGT due to ITP, platelet counts are not a useful predictor of bleeding risk due to the presence of platelet dysfunction. In our patient, evaluation of platelet function was done using LTA. Giannini et al., performed a different platelet function assay, PFA-100, in a case of AGT.14 PFA-100 is performed as a point-of-care method in some centers for rapid detection of impaired primary hemostasis, but its diagnostic utility is limited. Although this method does identify severe platelet function disorders and would have been expected to be abnormal in our case, we do not routinely offer this testing as it does not have sufficient sensitivity or specificity to be used as a screening tool for platelet disorders, in particular mild platelet dysfunction and presence of anti-platelet medications such as aspirin.15 Although platelet-associated IgG can sometimes be observed in normal individuals, patients with ITP have significantly greater amounts of IgG on platelets than normal people16 and flow cytometry is a sensitive test to detect these platelet associated antibodies.17 We were also able to find platelet-specific antibodies free in the serum using the Pakplus indirect immunoassay, indicating that the patient still has ITP-type antibodies despite normalization of her platelet count after splenectomy. The abnormal aggregation study, which is a functional test, with normal expression of GPIIb/IIIa by flow cytometry; is an indication of a functional defect, though it would not differentiate between an inherited versus an acquired functional defect due to antibodies. Another way to confirm the functional blocking effect of antibodies to GPIIB/IIIa is to perform mixing studies. In aggregation, this is done by mixing patient serum or plasma with normal platelets and showing inhibited aggregation in a similar abnormal pattern to the patient.18 Mixing studies have been described using aggregometry, a platelet adhesion assay, and flow cytometry to diagnose AGT in a 27 year old male with non-Hodgkin lymphoma.8 These were performed by adding serum of the patient, or of a healthy control (1:1), to gel-filtered control platelets. Aggregometric mixing tests showed that the patient's serum, but not control serum, inhibited ADP induced platelet aggregation of control platelets. In mixing tests using a platelet adhesion assay, plasma of the patient or controls were incubated with whole blood from healthy donors, and patient's serum reduced platelet adhesion to a collagen-coated surface at high shear stress. Flow cytometric mixing tests revealed that both GPIIb/IIIa expression, and function were inhibited by patient's serum.14 Although mixing studies have potential utility in the work-up, these tests are difficult to standardize and validate. They are not performed at our institution, and to the best of our knowledge, this test is not offered as a clinical test. In our case, we used flow cytometry for the detection of platelet associated (direct) autoantibodies. Direct autoantibodies can also be eluted from the platelets by low PH buffer and their specificity can be tested using an ELISA plate coated with GPIIb/IIIa, GPIb/IX, and GPIa/IIa to confirm the pathophysiology of the disease.19 This test is not offered in our institution. The direct antibody test that we performed may be more sensitive because it detects any antibodies associated with platelets but obviously less specific than the eluate test which would be recommended in cases of AGT since it has the ability to identify the specificity of the autoantibodies. Several treatment strategies have been used for treatment of bleeding in AGT patients including platelet transfusions. Although routinely used for treatment of GT, transfusion may not be useful in AGT as they are rapidly inactivated or cleared by the antibodies. Successful therapy with corticosteroids,20 intravenous immunoglobulin,21 rituximab,9, 22 and plasma exchange23 have been reported in literature. In patients with AGT in association with lymphoproliferative disorders, treatment of the underlying malignancy with chemotherapy can correct the AGT. Cases of AGT in association with lymphoma, in which the bleeding diathesis resolved with chemotherapy or surgical removal of the tumor without the need for immunosuppression have also been reported.4, 24 Rituximab has been used in several patients with success.25 Recombinant FVIIa (rFVIIa) is approved for use in patients with congenital GT with platelet alloimmunization and refractoriness to platelet transfusions.26 In cases of life threatening bleeding or emergent procedures in AGT, rFVIIa has been used with anti-fibrinolytic agents to achieve hemostasis.27 The literature on treatment of acquired Glanzmann's thrombasthenia is mostly limited to case reports and case series, and there are no studies comparing the efficacy of different treatment regimens considering the rarity of the condition. In our patient, splenectomy and subsequent removal of an accessory spleen normalized the platelet count, and antibody-bound platelets were in circulation for a longer time, unmasking the antibody-mediated platelet function defect. These platelets would have otherwise been cleared from the circulation as described in a case of Evan's syndrome by Fuse et al., where the patient developed a severe bleeding tendency due to AGT after normalization of platelet count after splenectomy.21 Similar findings were noted in other cases of ITP that developed AGT after splenectomy.8, 20, 28, 29 In the mouse model of ITP, the main accumulation site of platelets was translocated to the liver after splenectomy, suggesting that reticuloendothelial systems (liver, bone marrow, lymph nodes) other than the spleen are also responsible for the destruction of platelets.30 One may also hypothesize that the antibody production from reticuloendothelial sites other than the spleen may increase due to platelet recovery after splenectomy. Porcelijn et al., described six cases of AGT where the lack of platelet destruction can be explained, either by the subclass of the autoantibodies (i.e., IgG2 or IgG4 as their Fc receptor binding capacity is poor) or by the arrested platelet destruction by IgG1 or IgG3 autoantibodies after splenectomy.9 Unfortunately, IgG subclasses were not evaluated in this case. In summary, although splenectomy is a beneficial treatment for ITP, overproduction of antiplatelet antibodies triggered by platelet recovery should be taken into account, especially when patients subsequently manifest a bleeding diathesis with normal platelet count. In conclusion, AGT is a rare cause of bleeding diathesis, mostly seen in patients with lymphoproliferative disorders or autoimmune disorders such as ITP postsplenectomy. A systematic approach to investigate platelet function disorders (including platelet function mixing studies if available), GP expression by flow cytometry, and detection of GPIIb/IIIa autoantibodies will lead to the diagnosis as in our patient and other cases described in the literature. These patients need to be diagnosed and followed up closely for any signs of bleeding, with caution during surgical procedures since they may have normal platelet count and their risk of bleeding may be overlooked. There are no uniform guidelines for the diagnosis or management of these patients and treatment should be individualized depending on the severity of bleeding and underlying medical conditions. The authors declare no competing financial interests. Radhika Gangaraju http://orcid.org/0000-0002-5542-4754