2B or not 2B? A prothrombotic tendency masquerading as a bleeding disorder
Authors: Yvonne Brennan, Jennifer Curnow, Emmanuel J. Favaloro
Journal: American Journal of Hematology (2017)

A 76 year old male of Indian background was admitted with a 6 month history of severe lower back pain associated with weight loss. Background medical history consisted of type 2 diabetes mellitus, chronic kidney disease, severe aortic stenosis, ischemic heart disease with single vessel coronary artery disease, dyslipidemia and obstructive sleep apnoea. Magnetic resonance imaging scan revealed abnormal signal within the L3 vertebral body suspicious of marrow infiltration. The patient was initially reviewed by a clinical management team, at this stage excluding specialist hematology support, and thought to have vertebral tuberculosis (Pott?s disease) in view of his ethnicity, positive quantiFERON-TB gold assay and elevated C-reactive protein (CRP). An urgent computed tomography guided biopsy of the L3 vertebral body was therefore arranged, with routine “screening” blood tests ordered ahead of the biopsy. Somewhat unexpectedly to the clinical team, blood tests were suggestive of potential blood loss or active bleeding. Blood tests (summarized in Table 1) revealed chronic anemia with hemoglobin 86 g/L and low hematocrit 0.29. The anemia was normocytic with mean corpuscular volume 84fL (82-98fL). Platelet count was normal, as were prothrombin time (PT) and thrombin time (TT). However, activated partial thromboplastin time (APTT) was mildly prolonged at 44s (24–38 s), correcting on 1:1 mixing with normal plasma. Platelet Function Analyzer (PFA)-100/200 closure times (CTs) were maximally prolonged with both Collagen/Epinephrine (C/Epi) and Collagen/ADP (C/ADP). These results caused concern among treating clinical staff in view of the proposed biopsy, potentially excluding an inhibitor, but still conceivably consistent with a possible factor deficiency. Hematology input was subsequently requested to clarify the abnormal APTT and PFA results, as well as to potentially examine a possible bleeding disorder, given the anemia and low hematocrit. The hematology team identified that aspirin (100 mg daily, given for the patient's ischemic heart disease) had only been ceased on the day of testing. However, aspirin would be expected to prolong C/Epi only and could not explain the prolongation of both C/Epi and C/ADP. The low hematocrit established earlier, was therefore thought to be the main cause of the prolonged CTs, especially for C/ADP. Although we are unaware of any literature that indicates a specific cut-off for hematocrit that causes the PFA-100 CT to be prolonged/maximally prolonged, in our experience a hematocrit below around 0.35 may affect the CT, and a hematocrit below 0.3 (as for this patient) could easily yield a maximally prolonged CT, particularly if added to concomitant aspirin.1, 2 Nevertheless, now given the need to fully explain all abnormal test findings, the hematology team arranged additional tests: blood film review, fibrinogen, factor assays, lupus anticoagulant (LA), and von Willebrand factor (VWF) assays. Heparin contamination, also a possible cause of APTT prolongation, was thought to be unlikely in view of APTT correcting on 1:1 mix with normal plasma, as well as TT being in the normal range. Otherwise, an anti-FXa, or neutralisation testing using heparinase or polybrene could be used to rule out heparin or heparin contamination. Blood film review indicated normal platelet morphology. Laboratory findings revealed normal levels of factor VIII, IX, XI, and XII, and normal VWF antigen and activity (Table 1). Fibrinogen level was elevated and LA was not detected (although LA clotting assays were mildly prolonged, both screen and confirm assays were similarly prolonged, mixing tests failed to identify an inhibitor, and the normalized ratio (1.10) was well below the assay cut-off value of 1.20 for a positive LA). Although these results provided some reassurance, with hypofibrinogenemia and LA excluded, and hemophilia and von Willebrand disease (VWD) seemingly unlikely, the prolonged APTT remained unexplained and a potential cause of continued concern to the clinical team. Due to the urgent need for the vertebral body biopsy, more detailed hemostatic tests could not be performed in time. Although it was felt by the hematology team that a bleeding disorder was unlikely, especially given isolated abnormal APTTs are not good predictors of bleeding, the patient proceeded to have the biopsy under some prohemostatic cover at the insistence of the proceduralist and treating team. Thus, 4 units of fresh frozen plasma (∼14 mL/Kg) and 1 unit of pooled platelet transfusion (∼284 × 109 platelets) were provided. The biopsy was uncomplicated. Although the Hematology team provided advice that fresh frozen plasma administration was not necessary as levels of factors associated with bleeding had been demonstrated to be normal, the fresh frozen plasma was requested with the aim to “correct” the prolonged APTT, and potentially cover any unrecognized factor deficiency, due to strong concern about the risk of spinal hematoma associated with the procedure. Platelets were transfused in the context of recent aspirin ingestion, again acknowledging that data establishing efficacy for this approach is lacking. Biopsy histopathology revealed large cell carcinoma that was diffusely positivity for AE1/AE3, CD10, CD20, positive nuclear staining with PAX8 and focal positive cytoplasmic staining with RCC antigen, consistent with metastatic clear cell renal cell carcinoma. With the diagnosis of spinal metastases, the patient was planned to have an L3 vertebrectomy. PFA-100/200 were repeated prior to the surgery. Despite being 43 days after aspirin was ceased, CTs remained prolonged, albeit less prolonged than initial (aspirin influenced) values. In view of pending surgery, chronic anemia, low hemoglobin, low hematocrit, prolonged PFA CTs, and prolonged APTT, the clinical hematology team liaised with the specialized hemostasis laboratory in an attempt to unravel this case. Repeat factor assays, repeat VWF tests, and comprehensive testing of platelet function by light transmission aggregometry (LTA) were undertaken. Part of the focus of the investigation related to VWF testing was to exclude the remote possibility of an acquired von Willebrand syndrome (AVWS), due to the patient's severe aortic stenosis (aortic velocity 4.40 m/s, mean pressure gradient 51 mmHg, aortic valve area 0.71 cm2), and which required surgery; however, the proposed aortic valve replacement was cancelled in view of his newly diagnosed metastatic cancer. Factor assays were again normal, as were all VWF test parameters. Indeed, VWF levels were noted to be high (Table 1). LTA showed normal responses to arachidonic acid (aggregation at 1.6 uM), epinephrine (10 uM), and collagen (1 and 2 ug/mL). The patient's platelets, however, agglutinated at 0.5 mg/mL of ristocetin, which was therefore consistent with a positive low dose ristocetin-induced platelet agglutination assay (LD-RIPA; Figure 1). Interestingly, LTA also revealed responses to low levels of ADP (secondary at <0.2 uM ADP). Representative sample results for LTA. Panel A: Patient Ristocetin Induced Platelet Aggregation (RIPA) threshold = 0.5 mg/mL. Panel B: Control RIPA threshold = 1.0 mg/mL. Panel C: Patient ADP secondary aggregation threshold <0.2 uM. Panel D: Control ADP secondary aggregation threshold 2.5 uM. Panel E: Patient collagen threshold <0.25 ug/mL. Panel F: Control collagen threshold 0.5 ug/mL. [Color figure can be viewed at wileyonlinelibrary.com] VWF test results did not support AVWS, as there was neither evidence of reduced VWF, nor a low ratio of activity/antigen, which could otherwise be consistent with aortic stenosis induced loss of high molecular weight VWF. The positive LD-RIPA was an additional unexpected finding, and is otherwise a hall-mark diagnostic feature of a type 2B or platelet type (PT-) VWD.3, 4 However, VWF test results were not consistent with this possibility either. It was decided to repeat the LTA for confirmation and also perform RIPA mixing studies. Similar findings were observed on repeat LTA testing (Table 1). Thus, LD-RIPA was again positive. However, LTA also again revealed responses to low levels of ADP (secondary at <0.2 uM ADP). RIPA mixing studies were inconclusive, neither suggesting either 2B VWD or PT-VWD, and instead that both (rather than one or other of) the patient's plasma and platelets contributed to the increased RIPA responsiveness. Typically, LD-RIPA positivity is a defining feature of type 2B VWD or PT-VWD,3, 4 caused by gain-of-function mutations in the VWF gene or glycoprotein Ib platelet alpha subunit (GP1BA) gene, and resulting in increased affinity between VWF and the platelet GPIbα receptor. Ristocetin promotes this binding, which is enhanced in 2B and PT-VWD due to defective VWF or platelets, respectively. Although LD-RIPA is defined as positive when patient platelet rich plasma aggregates at ≤0.6 mg/mL of ristocetin rather than the normal threshold of 0.8–1.2 mg/mL, we felt it unlikely that this patient had either a type 2B or PT-VWD, or even AVWS. At our institution, all LD-RIPA results are investigated further by RIPA mixing studies to determine if the response lies with the patient's plasma (indicating type 2B VWD) or platelets (indicating PT-VWD).4 Our patient's LTA and RIPA mixing studies were not typical of either 2B or PT-VWD. What we saw was a slightly enhanced RIPA responsiveness in the mixed samples containing patient platelets/normal plasma and patient plasma/normal platelets, compared to normal platelets/normal plasma, but neither of which reached the level of hyper-responsive RIPA seen with the original patient platelets/patient plasma (Table 2). Also, our patient showed generalized hyperaggregability with LTA, with responses to low levels of all agonists that we tested for threshold events. Probable exclusion of 2B or PT-VWD was also suggested by the patient's lack of past abnormal or excessive bleeding history, and also inconsistent with other phenotypic tests (VWF tests, normal platelet count). The patient had undergone appendectomy at age 26, abdominal laparotomy for partial bowel obstruction at age 45, and 15 teeth extractions, all without any bleeding complications. His International Society on Thrombosis and Haemostasis Bleeding Assessment Tool score was 0. He had 6 brothers, 2 sisters, 3 daughters, and 5 grandsons, none of whom had an abnormal bleeding history. In addition, the patient was not on any medication known to affect platelet function testing, consisting of paracetamol, hydromorphone, methadone, pregabalin, insulin, valsartan, pantoprazole, docusate and sennosides, and darbepoetin alfa. The patient was cleared by the hematology team and underwent angiogram and embolization of the L3 lesion followed by L3 vertebrectomy and L2-L5 spinal fusion with pedicle screw fixation without any prohemostatic support. The procedure was notably uneventful. Venous thromboembolism (VTE) prophylaxis with unfractionated heparin 5000units subcutaneously twice a day was commenced on day 1 postvertebrectomy. Despite chemical VTE prophylaxis, he developed a symptomatic left soleal vein deep vein thrombosis (DVT) on day 11 post-vertebrectomy, and low molecular weight heparin 1mg/kg twice a day was commenced. Sometime after stabilisation of the patient, the hematology team felt that some clarification of previous unexpected results should ensue to provide some closure for the patient and the medical team. Repeat LTA RIPA mixing study was performed, together with VWF Multimeric profiling and genetic testing of the VWF and GPIb genes. LTA again showed generalized elevated aggregation, including LD-RIPA, ADP, and also collagen (Figure 1), and RIPA-mixing studies were again nonspecific, suggesting a contribution of both platelets and plasma to LD-RIPA responsiveness (Tables 1 and 2). As expected, VWF multimeric analysis returned normal results (Figure 2). Sequencing of exon 28 of VWF and GP1BA did not identify any mutations associated with 2B and PT-VWD respectively. However, the patient was found to be heterozygous for the B allele (3 tandem repeats) and C allele (2 repeats) in exon 2 of GP1BA. Genotype BC of the variable number of tandem repeats (VNTR) polymorphism of GP1BA has been associated with recurrent ischemic stroke in patients who fail aspirin treatment.5 Von Willebrand factor multimer analysis confirming that the patient had the full range of VWF multimers. A: 1. Normal control. 2. Our patient. B: 1. Normal control. 2. Patient with known type 2B VWD showing predominant lack of large VWF multimers. [Color figure can be viewed at wileyonlinelibrary.com] VWD is the most common inherited bleeding disorder, and caused by quantitative (types 1 and 3) and/or qualitative (type 2) defects in VWF.3 Type 2B VWD is rare, representing <5% of all VWD cases,6 and involves gain-of-function missense mutations within or near the A1 domain of the VWF gene, resulting in increased affinity of VWF to the platelet glycoprotein (GP) Ibα receptor.7 This increased binding leads to clearance of large VWF multimers and platelet sequestration. PT-VWD is even rarer than 2B VWD, and instead occurs when the gain-of-function mutation lies with the GP1BA gene.4, 8 PT-VWD is often misidentified, either as the “more common” 2B VWD, or more usually as ITP.4, 8 Apart from positive LD-RIPA, both 2B and PT-VWD are usually characterized by reduced VWF levels that are typically functionally discordant, variable thrombocytopenia, predominant loss of large VWF multimers on multimeric analysis, and respective presence of mutations either in exon 28 of VWF (where the A1 domain is encoded) or GP1BA.3, 4, 8-10 Although RIPA hyperresponsiveness is a defining feature of 2B or PT-VWD, samples will infrequently provide false positive results,11 as in our patient, or false negative results. RIPA is practically challenging to perform, requiring rapid testing of freshly collected blood, and establishment of local normal and “pathological” ranges, with potentially frequent changes in ristocetin batches.11 Nevertheless, a false positive LD-RIPA at the level expressed by this case (0.5 mg/mL), and consistently reproducible (three occasions in our case), is a rare event. Indeed, we recall seeing such a low level “false-positive” (0.5 mg/mL) LD-RIPA on only one other occasion in our experience, with this not being observed on repeat testing (i.e., was not reproducible) in that patient. However, ‘false positive’ events with responsiveness seen at 0.7, or less often at 0.6, mg/mL have occasionally been seen by us (as previously reported).11 Alternatively, some patients with 2B-VWD may not show evidence of enhanced RIPA3 or may only show RIPA positivity at concentrations higher than 0.5 mg/mL.9, 10 There are ongoing efforts to develop alternatives to RIPA such as an ELISA utilizing a gain-of-function GPIbα mutant as the capture for VWF.12 Sequencing of GP1BA or exon 28 of VWF is considered the most accurate test to diagnose PT- or 2B VWD; however, cost, availability and turnaround times are issues that prohibit this from routine availability.4, 10 Severe aortic stenosis, which our patient had, has been associated with AVWS mimicking type 2A VWD. Here, proteolysis of large VWF multimers may occur as it passes through the stenotic valve under high shear forces.13 Thus, AVWS was initially considered a possible differential diagnosis in our patient, but was also discounted, being characterized by acquired history of bleeding, low VWF levels or at least low VWF activity/antigen ratios consistent with loss of large VWF multimers,14, 15 none of which our patient had. AVWS has also been associated with numerous medical disorders including cancer, lymphoproliferative and myeloproliferative disorders, immunologic diseases, and other cardiovascular diseases.14 AVWS mimicking 2B VWD is extremely rare; the few published case reports having all been associated with monoclonal gammopathy of undetermined significance.16-18 Overall, our patient showed several unexpected findings on pre-biopsy blood tests including abnormal APTT and prolonged PFA CTs, which raised concerns with the managing clinical team over bleeding risk, particularly in the context of anemia and low hematocrit, forcing the hematology team to provide some prohemostatic support for the initial vertebral biopsy. Once an abnormality is detected, even if it may not be the cause of the bleeding, there is a tendency to investigate causes. The elevated APTT was not due to factor deficiency or LA. Nevertheless, the patient's APTT was frequently mildly prolonged during the course of his hospitalization, albeit with some fluctuation. Although no definitive cause for the prolonged APTT could be identified, we suspect this was due to the patient's elevated CRP interfering with the APTT assay. CRP is known to bind negatively charged phospholipids, and phospholipids are used as a catalytic surface in APTT assays. Prolongation of APTT has been shown to increase proportionally with CRP concentrations.19, 20 Of interest, LA was not detected, although the clotting tests used (including those based on dilute Russell Viper Venom time) were also mildly prolonged. The prolonged PFA CTs were most likely due to the concomitant low hematocrit, initially compounded with aspirin therapy.1, 2 That this then led to LTA, with additional unexpected findings, potentially suggestive of 2B or PT VWD, caused additional surprise, but perhaps was not totally unexpected given the circumstances. RIPA mixing studies suggested that both the patients’ platelets and plasma contributed to the heightened response (Table 2). The patient's platelets were generally highly reactive, also responding to low dose ADP and collagen, which presumably drove some of the LD-RIPA responsiveness. It is noteworthy that the patient had high levels of VWF, perhaps a feature of his illness, and this possibly drove the plasma contribution to the LD-RIPA responsiveness. In summary, given the high VWF levels and nonspecific RIPA mixing results, together with generalized elevated LTA responsiveness (ristocetin, ADP, collagen), the positive LD-RIPA results were concluded to represent non-specific hyperaggregability, likely secondary to underlying malignancy, and not 2B/PT-VWD or even AVWS. Additional evidence for this conclusion were lack of clinical or other laboratory evidence of VWD, absence of bleeding history, normal VWF multimeric analysis as well as genetic testing. More interesting was that genetic testing not only failed to identify any 2B/PT-VWD mutation, but that sequencing of GP1BA identified a prothrombotic polymorphism. Contrary to having a bleeding phenotype, and needing treatment with prohemostatic support, this case reflected a prohemostatic environment, with high levels of VWF and generalized “platelet hyperreactivity.” Clinically, the patient developed a DVT despite VTE prophylaxis. Although we suspect this prohemostatic environment was a result of his underlying malignancy, it is possible that his severe obstructive sleep apnoea (OSA) requiring nocturnal continuous positive airway pressure therapy also contributed. Patients with severe OSA have been shown to be hypercoagulable on the basis of thromboelastography.21 The mechanism may be ADP-induced platelet hyperaggregability as shown by flow cytometry22 and optical aggregometry.23 Importantly, had the patient been inappropriately identified as having a bleeding disorder based on LD-RIPA, and treatment been undertaken for a type 2B VWD, potentially involving prohemostatic support with VWF concentrate, then treatment could have plausibly promoted even more serious thrombotic events, potentially including stroke, although this is mere supposition. In conclusion, we report a patient with false positive LD-RIPA who was instead found to have a prothrombotic tendency (cancer; postoperative state; potential genetic risk factor), highlighting the importance of considering a patient's clinical history as well as assessing all supporting laboratory evidence in making a diagnosis of hemostasis abnormality prior to major surgeries, in particular 2B or PT-VWD in this case. The patient eventually underwent a course of radiotherapy to the lumbar spine. Staging tests for the renal cell carcinoma did not identify any other malignant foci. He completed 6 months of therapeutic dose low molecular weight heparin. He declined any further treatment and succumbed to his cancer approximately one year after initial diagnosis. The authors would like to thank staff within the Department of Laboratory Haematology for performing some of the tests reported in this article. Nothing to report. Y. Brennan managed the patient after hematology support was requested, and wrote the initial manuscript. J. Curnow also managed the patient, and critically revised the manuscript. E. Favaloro managed the process of laboratory testing, arranged genetic analysis, and critically revised the manuscript.