A career in solving clinical‐pathological conundrums: Heyde syndrome, anti‐platelet factor 4 disorders, and microvascular limb ischemic necrosis

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical‐pathological conundrums addressed during my 35‐year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high‐molecular‐weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin‐induced thrombocytopenia (HIT)—a highly prothrombotic, antibody‐mediated, anti‐platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin‐dependent platelet‐activating antibodies—permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin‐independent anti‐PF4 antibodies, now recognized as the explanation for vaccine‐induced immune thrombotic thrombocytopenia (VITT), as well as VITT‐like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep‐vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT‐ and cancer‐associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate “shock liver” (impaired hepatic synthesis of natural anticoagulants).

career at McMaster University.Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome.The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, antiplatelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis.Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection.Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/ microvascular thrombosis (venous limb gangrene).The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT-and cancerassociated venous limb gangrene provided insight into disturbed procoagulant/ anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

| HEYDE SYNDROME
Heyde syndrome is an eponymous term that indicates the association between aortic stenosis and gastrointestinal (GI) bleeding due to GI angiodysplasia. 1 This syndrome is named after Dr. Edward Heyde, internist, who noted in 1958 the concurrence of calcific aortic stenosis and idiopathic GI bleeding, for which the connection remained a mystery for over 30 years.

| History of recognition of Heyde syndrome
In 1986, as a hematology resident, I learned by chance of Heyde syndrome while preparing to give city-wide Hematology Rounds on the topic of acquired type 2A von Willebrand syndrome (vWS-2A) secondary to monoclonal gammopathy.I was reading all I could find about vWS-2A and learned that some affected patients had bleeding GI angiodysplasia; while reading about bleeding GI angiodysplasia I learned that the "idiopathic" bleeding first noted by Dr. Heyde was now recognized to represent GI angiodysplasia (an entity unknown in the 1950s).I was intrigued by two facts: first, surgical correction of aortic stenosis usually cured the GI bleeding; second, there was no known pathophysiological explanation for Heyde syndrome or for its surgical cure.
While preparing for my presentation, I encountered a key paper that immediately suggested to me the explanation for Heyde syndrome.This was the study 2 by Dr. Joan C. Gill, a Wisconsin hemophilia specialist who, while working in the laboratory of Dr. Robert Montgomery in Milwaukee, reported that "some patients with noncyanotic congenital heart disease may have an acquired abnormality of vWF [von Willebrand factor] that is normalized with correction of the abnormal hemodynamic state."It seemed not too much of a stretch for me to wonder if a subtle abnormality of vWF-namely the selective loss of high-molecular-weight (HMW) multimers-might underlie Heyde syndrome.Of course, young children with congenital heart disease do not have GI angiodysplasia (an acquired lesion of aging), so the significance of the Milwaukee paper in the context of Heyde syndrome was not readily apparent.This history of the discovery of the explanation for Heyde syndrome is presented elsewhere. 3

| Acquired von Willebrand syndrome type 2A
A few months into my appointment at the Hamilton General Hospital, I was referred a 65-year-old woman with severe recurrent episodes of GI hemorrhage secondary to jejunal angiodysplasia; being aware of Heyde syndrome, I recommended to proceed with aortic valve replacement, predicting that the bleeding state would disappear (writing one of my shortest consultation opinions: "fix the valve: the bleeding will stop").I ensured collection of serial plasma samples to investigate whether a deficiency in HMW vWF multimers was present, and whether such an abnormality would resolve postoperatively.Indeed, research technologist Jane C. Moore (McMaster University) demonstrated a vWS-2A laboratory profile (i.e., deficiency of the largest vWF multimers), which corrected post-surgery (Figure 1A).Along with the patient's gastroenterologist, Dr. David Morgan, we submitted to The Lancet our hypothesis that an acquired vWS type 2A was a plausible explanation for Heyde syndrome as well as its surgical cure.
The Editors accepted our paper, 4 but removed our case and relevant data.That gave us the opportunity to report our index patient case a decade later (with 10-year clinical and laboratory follow-up), and also to add a second case, thus showing that durable normalization of a subtle type 2A vWF deficiency, with long-term cure of GI angiodysplasia bleeding, occurs in Heyde syndrome patients following aortic valve replacement. 5

| Subtle VWF deficiencies and bleeding GI angiodysplasia
Jane Moore, David Morgan, and I subsequently reviewed the topic of Heyde syndrome, summarizing its pathogenesis (Figure 1B) 1 ; in our paper, we proposed that subtle type 2A deficiency of HMW vWF multimers might be relatively common in patients with stenosing cardiovascular disease, perhaps helping to explain why bleeding GI angiodysplasia is not uncommon in the elderly patient population with cardiovascular disease.

| HEPARIN-INDUCED THROMBOCYTOPENIA
When I first began my 3-year research fellowship in HIT, this entity had not yet entered the diagnostic mainstream; in fact, there remained some controversy as to whether a true immune-mediated heparin-induced prothrombotic disorder existed. 6,7Indeed, my own early experiences in the laboratory caused me some uncertainty (see Section 3.1).antibodies) by their ability to generate procoagulant platelet-derived microparticles. 9A later joint paper with Ms. Sheppard showed-from the perspective of generating procoagulant, platelet-derived microparticles-that HIT antibodies could be classified as a "strong" platelet agonist, indeed more capable of triggering a platelet procoagulant response than other strong platelet agonists, thrombin and collagen. 10These observations help to explain the remarkable prothrombotic nature of HIT.To date, Jo-Ann and I have co-authored 53 peer-review publications.Thirty years after she joined the McMaster Platelet Immunology Laboratory, Ms. Sheppard continues to work at a high level of efficiency and productivity.This longstanding partnership between a clinician-scientist and a multitalented technologist contributed fundamentally to my career success.

| Key experiment: Platelet donor heterogeneity
My research supervisor (and career mentor) Prof. Kelton developed the platelet serotonin-release assay (SRA; for review 11 ), a test to detect heparin-dependent platelet-activating antibodies.However, when I tested known HIT sera with the SRA, I sometimes found inconsistent results.I wondered whether differences in HIT serum reactivity might reflect "idiosyncratic" factors (e.g., perhaps certain platelet alloantigens or other donor-specific attributes) or whether perhaps there were "hierarchical" factors, for example, maybe some donor platelets were relatively unreactive, in general, to activation by HIT sera.
To address this issue, I tested 10 different HIT sera against platelets from 10 different donors, that is, 100 HIT sera-platelet reactions were evaluated.Then, these 10 sera and 10 platelet donors were ranked, from best to worst, based upon their overall mean reactivity.This simple experiment showed a hierarchical reaction profile, leading to my first original paper dealing with HIT. 12 These data had tremendous implications for diagnostic testing for HIT using platelet activation assays: (a) it is important to use optimal platelet donors ("pedigree" donors), (b) "weak-positive" HIT serum controls are needed to ensure any individual assay can successfully identify HIT sera (including weak ones). 11With such measures, our group has reported a 95% diagnostic sensitivity for the SRA in detecting HIT antibodies. 13

| Laboratory testing for HIT antibodies: PF4/ heparin and the iceberg model
In 1992, Jean Amiral and colleagues identified platelet factor 4 (PF4)/ heparin complexes as the antigen target for HIT antibodies and developed a solid-phase enzyme-linked immunosorbent assay (ELISA) to detect such antibodies. 14Several groups (including the McMaster Platelet Immunology Laboratory 15 ) quickly confirmed this key discovery (for review see Reference [7]).
Through numerous studies of blood samples obtained from heparin-exposed subjects, we determined many important aspects of the interrelationship between ELISA reactivity, SRA reactivity, and development of clinical HIT.For example, we found that many ELISApositive sera did not cause platelet activation (by SRA); further, the risk of developing HIT was essentially restricted to ELISA-positive/ SRA-positive subjects. 16We also identified that IgG class antibodies were the predominant immunoglobulin class that caused HIT, a finding consistent with the role of FcγIIA receptors (i.e., IgG platelet Fc receptors) in effecting platelet activation in HIT. 17 Another notable observation included the demonstration that the strength of immunoassay reactivity (e.g., higher optical density [OD] values by ELISA) were predictive of SRA-positive status. 18A corollary was the counterintuitive finding that a positive test for anti-PF4/heparin antibodies argued strongly against a diagnosis of HIT if the OD reading was only weakly positive (e.g., <1.0 OD units).This finding supported the growing concept that many patients were overdiagnosed as having HIT. 19ese various aspects of ELISA/SRA interrelationship could be expressed through the "iceberg" model of HIT, 20 namely that pathogenic HIT antibodies represented the "tip of the iceberg" (portion above the waterline) featuring IgG class antibodies of sufficient quantity and platelet-activating quality to cause HIT (Figure 2A).This concept evolved to the multiple iceberg model (Figure 2B), where icebergs of varying size (larger size indicating greater immunogenicity) and differing degrees of protrusion above the waterline (greater proportion of anti-PF4 immune response resulting in clinical HIT). 7udies of serial blood samples showed that the detection of HIT antibodies always preceded the occurrence of thrombocytopenia, 21 presenting a fundamental insight into HIT pathogenesis, namely that anti-PF4 antibodies must circulate in plasma in order to assemble the platelet-activating immune complexes in situ on platelet surfaces. 22e availability of well-characterized patient sera from heparinexposed patients has also proven invaluable in the assessment of novel laboratory assays for HIT, including ELISAs, 23 as well as more recent rapid assays to detect anti-PF4/heparin antibodies, [24][25][26] with enormous potential to influence evaluation of HIT in real time.The discovery that addition of PF4 enhances platelet activation by HIT antibodies 27 provided a tool to recognize the occasional patient with SRA-negative HIT, 28 further enhancing diagnostic sensitivity of the SRA.
Finally, my research associate Jo-Ann Sheppard was key to utilizing a fluid-phase ELISA 29 that was helpful in assessing cross-reactivity of HIT antibodies against different polyanions, such as fondaparinux.
Several years later, after the recognition of vaccine-induced immune thrombotic thrombocytopenia (VITT) as a consequence of the vaccine response to the Covid-19 pandemic led to a paradigm shift in the understanding of anti-PF4 disorders, Jo-Ann modified the fluid-phase ELISA and applied this assay to the investigation of anti-PF4 versus anti-PF4/heparin antibodies (discussed later in Section 4.4: VITT-like anti-PF4 disorders in non-vaccine, non-heparin settings). 30By avoiding the artifact of conformational modification of PF4 bound to solid-phase (in standard ELISAs), distinct reactivity profiles of anti-PF4 and anti-PF4/heparin antibodies could be discerned.

| HIT features moderate severity of thrombocytopenia
An intriguing clinical observation was that many HIT patients have only moderate degrees of thrombocytopenia.Unlike classic D-ITP caused by quinine, sulfa drugs, and vancomycin, where the platelet count is less than 20 Â 10 9 /L in most cases, in HIT, the opposite is true: most ($90%) HIT patients have platelet count nadir values that are greater than 20 Â 10 9 /L. 31This reflects the fundamentally different pathogenesis of D-ITP (platelet clearance via drug-dependent anti-glycoprotein [GP] IIb/IIIa or anti-GP Ib/IX IgG) versus HIT (FcγIIA-mediated platelet activation). 32e platelet-activating nature of HIT has important implications for diagnosis in the setting of thrombocytosis, for example, patients with postoperative thrombocytosis 33 or in patients with myeloproliferative neoplasm. 34In 2003, we showed that a relative drop in platelet count-for example, 50% or greater-from the postoperative peak platelet count (that immediately precedes the putative HIT-associated platelet count fall), provides a more sensitive and specific definition of thrombocytopenia, in comparison to using an arbitrary threshold such as 100 or 150 Â 10 9 /L. 33

| HIT frequency: Unfractionated versus lowmolecular-weight heparin
Early in my career, I had the opportunity to test systematically serial plasma samples obtained from a clinical trial of unfractionated heparin (UFH) versus low-molecular-weight heparin (LMWH) thromboprophylaxis in hip replacement surgery.Using the standard definition of thrombocytopenia (platelet count fall to <150 Â 10 9 /L), we found a higher frequency of HIT with UFH vs LMWH (9/332 vs. 0/333; F I G U R E 2 Iceberg model of HIT.(A) Structure of the HIT iceberg.The size of the iceberg reflects the totality of the anti-PF4 immune response, as indicated by a positive polyspecific anti-PF4/heparin ELISA that detects antibodies of IgG, IgA, and/or IgM classes.A subset of such patients will have detectable IgG class antibodies; a subset of those patients will have platelet-activating antibodies, as indicated by a positive serotonin-release assay.The portion of the iceberg above the waterline represents clinically-evident HIT, comprised of patients with HITassociated thrombosis as well as other patients without thrombosis ("isolated HIT").(B) Multiple iceberg model.HIT icebergs can be depicted in a variety of ways to indicate differences in frequency of detecting anti-PF4/heparin antibodies, as well as differences in frequency of clinically evident HIT.For example, anti-PF4/heparin antibodies are frequently detected after cardiac surgery (likely due to immunization triggered by UFH given at time of cardiac surgery), but with a somewhat greater frequency of HIT (area above the waterline) with UFH versus LMWH, reflecting a greater breakthrough of HIT with UFH.In general, there is a lower frequency of anti-PF4/heparin antibody formation, and a corresponding lower frequency of clinically evident HIT, with LMWH (versus UFH), for both surgical and medical patient populations.ELISA, enzyme-linked immunosorbent assay; HIT, heparin-induced thrombocytopenia; ICU, intensive care unit; LMWH, low-molecular-weight heparin; PF4, platelet factor 4; UFH, unfractionated heparin.p = 0.0018). 35Subsequent studies 36,37 corroborated a higher risk of HIT with UFH vs LMWH.

| HIT is a venous prothrombotic disorder
When I first studied HIT, the prevailing dogma was that HIT was associated with arterial, but not venous, thrombosis; indeed, HIT was initially known as the "white clot syndrome" based upon the characteristic platelet-rich thrombi extracted from arteries by vascular surgeons. 6Yet, in my experience, the majority of HIT-associated thrombotic complications were venous, not arterial (4:1 venous: arterial ratio) 38 ; indeed, when I submitted the corresponding manuscript 38 for publication, it was rejected on the basis of being a retrospective study, with the venous thrombosis predominance potentially confounded through recognition bias; the reviewers recommended that only a prospective study could establish a venous thrombosis association with HIT.Fortunately, our paper on HIT frequency with UFH versus LMWH had just been published, 35 and in that clinical trial (prospective study), we found that 7 of the HIT patients had venous thrombosis, versus only 1 who had an arterial thrombosis.When we pointed this out to the Editors, our submission to American Journal of Medicine was then accepted.This marked venous thrombosis predominance in HIT has since been corroborated on numerous occasions; this history of this evolution in thinking of HIT from (only) arterial to a venous-predominant prothrombotic disorder is recounted elsewhere. 39

| HIT features unusual thrombotic events
The availability of a high-quality test for HIT antibodies-the SRA [11][12][13] -along with supportive ELISA data-permitted identification of numerous HIT presentations in diverse clinical settings.We were able to show that HIT was associated with: deep-vein thrombosis (DVT-including bilateral DVT) 33,35 ; upper-extremity DVT (almost invariably intravascular catheter-associated) 40 ; post-intravenous bolus heparin acute anaphylactoid reactions (also called "acute systemic reactions") 34,37,41 ; necrotizing skin lesions (usually at heparin injection sites, 42 but occasionally distinct from heparin injections 43 ); unilateral or bilateral adrenal hemorrhagic necrosis 42,44 ; cerebral venous sinus thrombosis, 45 and so forth.A truism of HIT: the more unusual the thrombotic event is, the more likely it is that HIT is the underlying explanation. 46

| HIT features unusual timing characteristics
Methodic evaluation of serial blood samples obtained from heparinexposed patients showed that the onset of detectability of the pathogenic IgG class antibodies is relatively speedy in HIT, beginning only 6 days (median) from the start of heparin treatment, followed soon after by the fall in platelet count and thrombotic complications (Figure 3A). 21Moreover, this timeframe did not differ whether or not the subjects had a previous history of remote heparin exposure.How could one reconcile these observations with the recognized phenomenon of "rapid-onset" HIT, where the platelet count falls abruptly when heparin is given to a patient known to have had a previous history of heparin exposure?
In a painstaking analysis of 243 patients with HIT, including analysis of the timing of onset of thrombocytopenia in relation to concurrent and previous heparin exposures, John Kelton and I made the surprising finding that rapid-onset HIT was invariably associated with recent prior heparin exposure, that is, within the past 90 days (and especially within the past 30 days); we showed that rapid-onset HIT was explained by already circulating HIT antibodies (triggered by the recent heparin exposure) that caused an abrupt platelet count decline when heparin was restarted. 47In the same paper, we showed that HIT antibodies were invariably transient (Figure 3B), which helped to explain why there was such a close relationship between rapid-onset HIT and recent heparin exposure; in essence, if the prior heparin exposure was more than several months ago, there would be no residual HIT antibodies, and thus no possibility of rapid HIT occurrence.A corollary of these observations is that there is a minimum period of time-at least 5 days-for a patient to develop clinically-significant levels of HIT antibodies sufficient to cause thrombocytopenia, irrespective of the patient's history of previous heparin exposure, including even whether they had had a previous history of HIT itself.if there had been another recent prior heparin exposure that had triggered immunization; in other words, HIT never begins 2, 3 or 4 days after any heparin exposure.
These observations also indicated that heparin can safely be readministered to a patient with a previous history of HIT, provided that at least a few weeks or months had elapsed, and HIT antibodies are no longer detectable.In this way, heparin can be given for important indications-such as for intraoperative anticoagulation during cardiac or vascular surgery-despite a history of previous HIT (with a nonheparin anticoagulant given for postoperative anticoagulation, if required).Subsequent studies supported the notion that this approach is safe; moreover, recurrence of HIT five or more days postreexposure itself is uncommon (<3% frequency). 48metimes, to solve clinical problems I encountered when performing hematology consultations, I utilized undiscarded plasma/ serum samples remaining within the various clinical laboratories, in order to address certain problems in the research laboratory, for example, to prove presence of HIT antibodies prior to an episode of rapid-onset HIT, or to ascertain the timing of HIT antibody seroconversion when a patient had multiple different exposures to heparin; an example of this latter type of study was summarized in a paper, called "CSI Hematology" (Clinical Sample Investigation-Hematology). 49 In our medical community, plasma samples referred to the regional coagulation laboratory are retained for 6 weeks, thus affording the opportunity to systematically address various laboratory issues, in complex coagulopathies, in a post-hoc manner.

| 4Ts scoring system
The key research findings regarding thrombocytopenia severity (including relative platelet count declines from pre-HIT "baseline"), timing of platelet count fall, various clinical manifestations including the types of thrombosis, as well as absence of other explanations for thrombocytopenia, were combined into a scoring system, named the 4Ts, for "Thrombocytopenia", "Timing", "Thrombosis", and "oTher". 50,51The 4Ts is widely used, and is recommended by HIT diagnosis and treatment guidelines. 52The development of this scoring system was an example of a tremendous collaboration with Greinacher's group, as the scoring system (developed in Hamilton 50 ) was then evaluated both at our center and in Germany. 51

| Novel anticoagulant and adjunct HIT treatment approaches
HIT is a dramatic disorder which during the earliest and middle stages of my career had no established treatment.My approach was to evaluate carefully those HIT patients who were treated with a variety of anticoagulant approaches.In this fashion, I recognized that some treatments were ineffective (warfarin, ancrod), 53 and that others appeared effective (danaparoid sodium, 53 fondaparinux, 54 rivaroxaban 55 ).Working at a center (McMaster University) noted for its methodological expertise, reliance on retrospective patient cohorts may seem counterintuitive, but for a relatively uncommon disorder with (historically) delayed laboratory diagnosis, and ensuing difficulties in performing prospective research studies, this approach helped identify effective treatments.4][55] Indeed, my personal view is that factor Xa inhibitory treatment has important advantages over direct thrombin inhibitors (DTIs) for management of HIT. 13 Similarly, the adjunct treatment, high-dose intravenous immunoglobulin (IVIG), has been used by myself and others to treat HIT.In 2017, I reviewed the literature on IVIG for treating HIT, and concluded that this adjunct therapy was likely to be valuable for patients with atypical presentations of HIT (discussed in the next section). 56day, high-dose IVIG is considered an important treatment adjunct for platelet-activating anti-PF4 disorders that feature heparinindependent platelet-activating properties.

| HEPARIN-INDEPENDENT PLATELET-ACTIVATING ANTIBODIES
A central paradigm of HIT is that it represents a heparin-dependent, platelet-activating disorder.Thus, for a patient who forms HIT antibodies, thrombocytopenia should only occur if the patient continues to receive heparin, and should resolve quickly upon stopping heparin.
However, over the past two decades, it became increasingly apparent that there is a spectrum of anti-PF4 disorders-including atypical forms of HIT-that feature antibodies with heparin-independent platelet-activating properties, including some patients who never were exposed to proximate heparin.Figure 4 illustrates five different anti-PF4 disorders, classic HIT (as already discussed), but 4 other entities briefly discussed here.
F I G U R E 3 Temporal features of heparin-induced thrombocytopenia (HIT).(A) Clinical events in relation to anti-PF4/heparin antibody formation following hip replacement surgery with heparin thromboprophylaxis.The enzyme-linked immunosorbent assay (ELISA) used was a commercial, polyspecific anti-PF4/polyanion ELISA that detects antibodies of IgG, IgA, and/or IgM classes.The figure compares the optical density (OD) values for patients with HIT (n = 12), defined as >50% platelet count fall, versus seroconverting, non-HIT patients (n = 36).The data show the characteristic timeline for HIT-related events: anti-PF4/heparin seroconversion (median, 4 days after start of heparin), followed by onset of the platelet count fall (median, 6 days after start of heparin), followed by at least a 50% platelet count fall from the postoperative peak value (median, 8 days after start of heparin) to clinical evidence of thrombosis (median, 10 days after start of heparin

| Autoimmune HIT (aHIT)
Autoimmune HIT, or aHIT, sometimes known as atypical HIT, features HIT patients where the thrombocytopenia begins or worsens after stopping heparin ("delayed-onset HIT"), 57 or where the thrombocytopenia persists for a week or more despite stopping heparin ("persisting" or "refractory HIT") (for review 58,59 ).Another aHIT entity includes patients only exposed to small amounts of heparin (heparin "flush" HIT). 60Also, most patients with fondaparinux-induced HIT have the serological features of aHIT.When the McMaster laboratory performs the SRA using aHIT sera, we find substantial heparinindependent serotonin-release (HISR), usually >80% release, at 0 U/ mL heparin (buffer control).However, in some patients the degree of HISR is less (only 30%-50%), but whether this reflects testing against platelets from donors that do not support well the phenomenon of HISR, or other features, is unclear.A recent study by a French group 61 using a 30% threshold for HISR concluded that the corresponding HIT patients tend to have atypical clinical features of HIT.The importance of identifying patients with aHIT is that they often fail conventional treatment approaches, and may benefit from adjunct high-dose IVIG to deescalate their HIT hypercoagulability state.

| Spontaneous heparin-induced thrombocytopenia (SpHIT)
In 2008, my coauthors and I reported that a prothrombotic HITmimicking disorder with HIT antibodies could occur in patients without proximate heparin exposure, a disorder called "spontaneous HIT" (SpHIT). 62Interestingly, the cases do not really arise spontaneously, but rather occur after certain triggers, such as following knee replacement surgery, or after infection. 63Perhaps the use of a tourniquet during knee replacement surgery, with release of cellular debris (including polyanions) somehow triggers an anti-PF4 immune response.Recent observations support an adjunctive role for highdose IVIG in treating this rare disorder (for review see Reference [63]).

| Vaccine-induced immune thrombotic thrombocytopenia (VITT)
The Covid-19 pandemic, and the ensuing vaccine campaign, led to the unexpected outcome of an ultrarare adverse event of two adenovirus vector vaccines, ChAdOx19 and Ad26.COV2.S, characterized by thrombocytopenia, thrombosis, elevated D-dimers, and frequent hypofibrinogenemia. 64,65Patient serum contains anti-PF4 antibodies directed against the heparin-binding site on PF4, a target site that is distinct from the heparin-dependent antibodies generated in classic HIT.Remarkably, 50% of VITT patients evince cerebral venous sinus thrombosis; a high proportion of patients (approximately one-third) develop splanchnic vein thrombosis.We and others have shown that high-dose IVIG inhibits VITT antibodyinduced platelet activation, usually accompanied by abrupt platelet count recovery, although IVIG-refractory patients may need therapeutic plasma exchange. 66I G U R E 4 Five platelet-activating anti-PF4 disorders.Shown for each of the five anti-PF4 disorders are the corresponding year for first reports, as well as abbreviations (in red text).At the top of the figure, platelet factor 4 (PF4) is conceptualized as a "globe" with heparindependent antigen sites formed at the north and south "poles" when heparin binds to the heparin-binding site ("equator"); in contrast, nonheparin-dependent antigen sites exist at the "equator" (recognized by heparin-independent antibodies as seen in aHIT, SpHIT, SpVITT, and VITT).Greinacher, 67 has led to identification of VITT-like antibodies in diverse prothrombotic clinical settings.For example, my collaborators and I reported that adenovirus infection itself can trigger a lifethreatening VITT-mimicking disorder.67,68 VITT-like platelet-activating antibodies have also been implicated in chronic prothrombotic disorders characterized by thrombocytopenia, 67,69 including with monoclonal gammopathy of clinical significance.70 These events are the most recent example of an insight into anti-PF4 pathophysiology developing more or less simultaneously in the McMaster and Greifswald laboratories.

| ACRAL MICROVASCULAR LIMB ISCHEMIC NECROSIS SYNDROMES
HIT has a remarkably high frequency ($5%) of ischemic limb injury. 39e of the conundrums of HIT was how patients could develop ischemic limb losses even when arterial pulses were present, a disorder known as venous limb gangrene (DVT with associated acral tissue necrosis).As discussed in this section, elucidating the explanation for venous limb gangrene has helped provide insight into other disorders of acral limb ischemia.

| Venous limb gangrene complicating HIT
We performed a retrospective case-series of patients with ischemic limb losses in the setting of DVT. 71Pathology studies showed microvascular thrombosis in the setting of DVT to account for ischemic limb losses.We also had serial plasma samples available on several of the patients, which provided the opportunity to examine various potential explanations to explain the microthrombosis observed.
The most surprising finding was that warfarin treatment was implicated in the pathogenesis of venous limb gangrene.Affected patients had a characteristic supratherapeutic international normalized ratio (INR), typically >4.0, which was significantly greater than in control patients; affected patients also had greatly elevated thrombinantithrombin (TAT) complexes, supporting a role for ongoing severe thrombin generation in these patients with HIT.
A key diagnostic clue was the occurrence of non-acral skin necrosis in one of the 10 patients we reported, a finding which suggested to one of the coauthors (Prof.John Kelton) that warfarin could be playing a causal role not only in the non-acral skin necrosis, but perhaps also in explaining the acral necrosis seen in all 10 patients.
Indeed, this proved to be the case.

| Venous limb gangrene complicating cancerassociated DIC
Adenocarcinoma-associated DIC can be a HIT-mimicking disorder, including its association with acral limb ischemic necrosis.We performed a study 72 to evaluate patients who developed venous limb ischemic necrosis in the setting of heparin treatment in which HIT was excluded serologically.We identified warfarin as a key contributor to venous limb ischemia in the setting of uncontrolled cancer-associated DIC.As in warfarin-associated venous limb ischemic necrosis complicating HIT, a supratherapeutic INR was demonstrated to be a marker of this disorder; moreover, the explanation proved to be a surrogate marker for severe protein C depletion via parallel depletion in procoagulant factor VII. 73 Figure 5 shows a plot of thrombin-antithrombin (TAT) complex versus protein C (PC) activity both for patients with cancer-associated venous limb ischemia and a control patient population with supratherapeutic INR secondary to excessive warfarin anticoagulation.The remarkable finding was that PC activity levels were actually lower in patients with excessive warfarin; yet microvascular thrombosis occurred in the cancer patients, suggesting that the finding of markedly elevated TAT in the cancer patients-but not in the overanticoagulated warfarin patients-is the key pathophysiological difference.In essence, it is the failure of PC anticoagulant activity in the setting of extreme cancer-associated hypercoagulability that poses risk for microvascular thrombosis.
Another relevant observation was the lack of non-acral tissue necrosis in this patient population.This suggests that acral (distal extremity) limb necrosis in other clinical settings might also be associated with natural anticoagulant depletion.This provides a segue into the next section dealing with ischemic limb injury in non-HIT/noncancer settings, such as septic or cardiogenic shock.    1 presents a "Top Ten" list of clinical conundrums addressed over the past three decades.

| Symmetrical peripheral gangrene in critical illness
disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), Heyde syndrome, platelet factor 4 (PF4), symmetrical peripheral gangrene (SPG), vaccine-induced immune thrombotic thrombocytopenia (VITT), von Willebrand factor (vWF) multimers, warfarininduced venous limb gangrene 1 | INTRODUCTION I became intrigued by hematology in first-year medical school, when taught how blood film and bone marrow aspirate morphology could diagnose many disorders.Laboratory importance was further emphasized at the McMaster hematology residency program, prompting a heparin-induced thrombocytopenia (HIT) laboratory research fellowship under Professor John Kelton.In July 1990, I began my career as a clinical and laboratory hematologist at Hamilton General Hospital with an academic appointment at McMaster University, with Heart and Stroke Foundation of Ontario research funding to study HIT.This dual clinical-laboratory role suited me perfectly; many oftentimes vexing problems-conundrumsencountered on the ward or in the clinic could be solved in the laboratory.
But as techniques to detect HIT antibodies improved, the delineation between HIT and mimicking non-HIT thrombocytopenic disorders became easier to discern.And as my worldview of HIT evolved, I found a HIT aficionado doppelgänger in Germany-Andreas Greinacher-who had also developed a platelet activation assay for HIT antibodies.When meeting with Andreas (later, Professor Andreas Greinacher, Head of Transfusion Medicine at University-Greifswald) at international conferences, particularly the American Society of Hematology (ASH) meeting in Anaheim in December, 1992, we realized that our experiences with HIT mirrored one another's, and it made sense for us to work collaboratively.One consequence was our coauthoring (along with Prof. Beng Chong, University of New South Wales, Australia) a consensus review of HIT.8

F I G U R E 1
Heyde syndrome: clinical picture and pathogenesis.(A) Long-term correction of vWS-2A laboratory profile following aortic valve replacement surgery.This patient, who required 22 units of red cell concentrates to manage 9 episodes of GI bleeding secondary to jejunal angiodysplasia, had long-term (>12 years) resolution of bleeding following aortic valve replacement with bioprosthesis.(B) Proposed pathogenesis of Heyde syndrome.AVR, aortic valve replacement; GI, gastrointestinal; HMW, highmolecular-weight; U, units; vWF, von Willebrand factor.Source: Figures (modified) are reprinted with permission, from: Warkentin TE, Moore JC, Anand SS, Lonn EM, Morgan DG.Gastrointestinal bleeding, angiodysplasia, cardiovascular disease, and acquired von Willebrand syndrome.Transfus Med Rev. 2003;17 (4):272-286.Copyright: Elsevier.Another important professional development was my longstanding collaboration with my key research technologist, Jo-Ann I. Sheppard, B.Sc., beginning in October 1993.She immediately contributed to a key paper showing that HIT antibodies are distinguishable (versus classic drug-induced immune thrombocytopenia [D-ITP]

Figure
Figure 3C illustrates an informative patient case exhibiting both typical-onset and rapid-onset HIT.Although these features of HIT are now widely accepted, they were controversial 20 years ago.HIT, therefore, was shown to have dichotomous temporal features: following any given heparin treatment exposure, unexpected onset of thrombocytopenia either begins at least 5 days later (reflecting a new heparin-induced immunization event) or begins abruptly (within 24 h) IgG, immunoglobulin G; LMWH, low-molecular-weight heparin; MGCS, monoclonal gammopathy of clinical significance.Source: Reprinted, with permission from: Warkentin TE.Autoimmune heparin-induced thrombocytopenia.J Clin Med.2023; 12 (21): 6921.Copyright: Theodore E. Warkentin.

4. 4 |
VITT-like anti-PF4 disorders in non-vaccine, non-heparin settings Contrary to the expectation that the HIT-mimicking entity, VITT, would vanish following switch from adenoviral vector Covid-19 vaccines to mRNA-based Covid-19 vaccines (the latter not associated with VITT 65 ), in reality, patients with VITT-mimicking prothrombotic disorders have increasingly been recognized.The development of novel laboratory methods to detect VITT antibodies-the aforementioned McMaster fluid-phase ELISA 30 and a rapid anti-PF4 chemiluminescence immunoassay (CLIA) developed in the laboratory of Prof.

I# 5
sometimes have been asked to review complex clinical cases with adverse outcomes in medical-legal settings.In so doing I encountered several cases of ischemic limb losses in critical illness in which HIT was a diagnostic consideration.However, in some cases, HIT was not present, yet dramatic usually bilateral lower-limb (and sometimes upper-limb) ischemic limb injuries occurred despite detectable arterial pulses.The question arose: what was the explanation for microvascular thrombosis in such patients with symmetrical peripheral gangrene (SPG)?I viewed this problem from the perspective of a profound disturbance in procoagulant/anticoagulant balance, given my experience in F I G U R E 5 Warfarin-associated microvascular limb ischemic necrosis syndromes: procoagulant/anticoagulant imbalance.(A) Clinical picture of warfarin-associated venous limb gangrene complicating HIT.Shown is a schematic representation of typical clinical and laboratory findings of warfarin-associated venous limb gangrene complicating HIT.The representative scenario is that of cardiac surgery for mechanical heart valve; hence, besides intraoperative heparin (not shown), the patient receives postoperative heparin with overlapping warfarin anticoagulation.Clinical and laboratory features include: thrombocytopenia (onset day 7), deep-vein thrombosis (DVT), supratherapeutic international normalized ratio (INR), and venous limb gangrene developing in the same limb as with the DVT.Reprinted, with modifications, with permission from: Warkentin TE.Ischemic limb gangrene with pulses.N Engl J Med. 2015; 373 (7): 642-655.Copyright: Massachusetts Medical Society.(B) Procoagulant-anticoagulant imbalance in patients with venous limb gangrene.Shown are thrombin-antithrombin (TAT) complex levels versus Protein C activity levels in patients with HIT and cancer.Green-colored symbols (n = 8) are from 4 patients (2 blood samples per patient) with HIT who developed warfarin-associated venous limb gangrene; red-colored symbols are from 4 patients (7 samples from the 4 patients) with cancer who developed warfarin-associated venous limb gangrene.Open circles (n = 67) represent plasma samples obtained from 12 warfarin-treated HIT patients (9 with DVT) who did not develop venous limb ischemia/gangrene.Reprinted, with modifications, with permission from: Warkentin TE, Cook RJ, Sarode R, Sloane DA, Crowther MA.Warfarin-induced venous limb ischemia/gangrene complicating cancer: a novel and clinically distinct syndrome.Blood.2015; 126 (4): 486-493.Copyright: American Society of Hematology.(C) Clinical picture of warfarin-associated venous limb gangrene complicating cancer.Shown is a schematic representation of typical clinical and laboratory findings of warfarin-associated venous limb gangrene complicating cancer.The representative scenario is that of initial "idiopathic" deep-vein thrombosis (DVT) in a patient with unrecognized metastatic adenocarcinoma.There is an initial increase in the platelet count during therapeutic-dose heparin (reflecting inhibition of thrombin-induced platelet activation), followed by rapid onset of thrombocytopenia when heparin is stopped following achievement of a therapeutic INR on warfarin.Over the next few days, in association with further rise in INR to supratherapeutic levels, venous limb gangrene occurs in the same limb as with the DVT.HIT antibody test is usually negative or weakly positive, and the platelet count typically rises with resumption of heparin treatment.Reprinted, with modifications, with permission from: Warkentin TE.Ischemic limb gangrene with pulses.N Engl J Med. 2015; 373 (7): 642-655.Copyright: Massachusetts Medical Society.(D) Procoagulant-anticoagulant imbalance in cancer patients with venous limb gangrene.Left panel: Thrombin-antithrombin (TAT) levels are shown for cancer-associated venous limb gangrene patients versus warfarin-treated non-cancer control patients with supratherapeutic INR due to warfarin overdose.Right panel: Protein C activity levels are shown for cancer-associated venous limb gangrene patients versus warfarin-treated non-cancer control patients with supratherapeutic INR due to warfarin overdose.Reprinted, with modifications, with permission from: Warkentin TE, Cook RJ, Sarode R, Sloane DA, Crowther MA.Warfarin-induced venous limb ischemia/gangrene complicating cancer: a novel and clinically distinct syndrome.Blood.2015; 126 (4): 486-493.Copyright: American Society of Hematology.HIT-and cancer-associated hypercoagulability where absence of nonacral necrosis did not necessarily mean that natural anticoagulant depletion was not playing a key pathophysiological role.Indeed, based upon my review of the literature, I found that natural anticoagulant depletion was commonly suspected and investigated when patients had purpura fulminans (i.e., combined non-acral and acral tissue necrosis); however, natural anticoagulation depletion was usually not considered when the clinical picture was predominant or exclusive SPG.Moreover, I observed in medical-legal cases that proximate acute ischemic hepatitis ("shock liver") was a common proximate event in patients with SPG, thus presenting a plausible explanation for profound natural anticoagulant depletion (as the liver synthesizes both PC and antithrombin [AT]).Thus, I began to investigate in my own clinical practice patients who developed SPG.In the first such reported case, Deborah Siegal, Richard Cook, and I showed profound procoagulant/anticoagulant imbalance in a patient who developed SPG in the setting of cardiogenic shock with DIC and proximate shock liver (which we called "acute hepatic necrosis")74 ; profound depletion of both AT and PC, along with extreme hypercoagulability, was documented in this patient.Three years later, in a paper published in Seminars in Thrombosis and Hemostasis, I summarized my experience in the evaluation of 15 patients with SPG complicating critical illness.75The key findings included: (a) marked DIC with usually profound thrombocytopenia and greatly elevated D-dimer levels; (b) high frequency (>90%) of proximate shock liver; (c) greatly reduced PC and AT activity levels (usually <20% and <40%, respectively), and (d) a characteristic temporal relationship between the onset of shock/shock liver and subsequent onset of ischemic limb injury (median, 3 days [range 1.5-5 days]).The concepts (with case examples) have also been presented in several articles.[76][77][78][79][80]These studies show the potential benefits of researching an adverse drug effect-HIT-which can provide unexpected benefits in terms of thinking about other mimicking clinical entities, in this case, the evaluation of microvascular acral limb ischemia in non-HIT clinical settings.T A B L E 1 Top 10 Clinical Conundrums Addressed by the Author.Clinical problem Explanation #1 Explanations for microvascular limb ischemic syndromes • HIT-associated venous limb gangrene • Cancer-associated venous limb gangrene • Symmetrical peripheral gangrene in critical illness Profound disturbance in procoagulant-anticoagulant balance: (a) DIC state (e.g., HIT, adenocarcinoma, septic or cardiogenic shock) with (b) natural anticoagulant depletion (warfarin (HIT, adenocarcinoma), "shock liver" (critical illness) #2 Anti-PF4 disorders beyond classic HIT: identification of aHIT, SpHIT, VITT, and SpVITT Role of heparin-independent platelet-activating antibodies as unifying theme for aHIT, SpHIT, VITT, and SpVITT #3 Identifying off-label treatment options for anti-PF4 disorders Observational data in well-characterized HIT patients: danaparoid sodium, fondaparinux, factor Xa-inhibiting DOACs, high-dose IVIG #4 Scoring system to assist clinicians in identifying patients who might have HIT 4Ts scoring system (based on elucidating clinical features of HIT) Unusual temporal features of HIT ("rapid" vs "typical" HIT, with implications for heparin re-exposure in selected clinical settings (e.g., cardiac surgery) Rapid (minimum, $5 day) IgG immune response (without IgM precedence) and antibody transience #6 HIT as the great masquerader: what is the clinical spectrum of HIT? Helping to characterize a wide spectrum of clinical sequelae of HIT

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| FINAL THOUGHTS Solving the individual patient case, and particularly case-series with common themes (e.g., outcomes of heparin reexposure in patients with known previous HIT; microvascular limb ischemia complicating HIT, cancer, or critical illness), has been immensely rewarding, and represents perhaps an underappreciated scientific approach unknown to many clinician-scientists who focus on large clinical trials.A dual clinical-laboratory career in hematology provided me a fantastic opportunity to explore in the laboratory vexing problems encountered during clinical practice; and the vibrant McMaster research community provided the ideal venue to pursue and to elucidate many intriguing clinical-pathological conundrums.Reflecting on my career, Table ).The horizontal bars indicate the corresponding data ranges for the four aforementioned serological and clinical events, summarized as median (small black squares within rectangles), IQR (open rectangles), and range (ends of thin black lines).From postoperative day 6 onwards, there is a significant difference in strength of ELISA reactivity (per OD values) between seroconverting patients who developed HIT vs. non-HIT controls.Reprinted, with modifications, with permission from: Warkentin TE, Sheppard JI, Moore JC, Cook RJ, Kelton JG.Studies of the immune response in heparininduced thrombocytopenia.Blood.2009;113(20):4963-4969.Copyright: American Society of Hematology.(B) Transience of HIT antibodies (Kaplan-Meier analysis).The time to a negative HIT test is shown, both for an antigen assay (commercial polyspecific anti-PF4/polyanion ELISA that detects IgG, IgA, and/or IgM class antibodies) and a platelet activation assay, the serotonin-release assay.The antigen assay tended to become negative more slowly than did the activation assay (P = 0.007 by the sign test).Reprinted, with modifications, with permission from: Warkentin TE, Kelton JG.Temporal aspects of heparin-induced thrombocytopenia.N Engl J Med. 2001; 344(17): 720-728.Copyright: Massachusetts Medical Society.(C)Representativecase of typical-onset HIT followed by an episode of rapid-onset HIT.The patient initially received a 10-day course of heparin (10 000 U per day by subcutaneous injection).Typical-onset HIT developed on day 6, according to a positive test for HIT antibodies.On day 30, the patient received heparin again (5000 U as a bolus injection followed by intravenous infusion), and rapidonset thrombocytopenia developed.Reprinted, with modifications, with permission from: Warkentin TE, Kelton JG.Temporal aspects of heparininduced thrombocytopenia.N Engl J Med. 2001; 344(17): 720-728.Copyright: Massachusetts Medical Society.