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282 American Society of Hematology

Platelets: An Update on Diagnosis and Managementof Thrombocytopenic Disorders

Keith R. McCrae, James B. Bussel, Pier M. Mannucci, G. Remuzzi, and Douglas B. Cines

Thrombocytopenia in the pregnant patient mayresult from a number of causes, most of whichinvolve either immune-mediated platelet destruc-tion or platelet consumption. Many of these disor-ders share clinical and laboratory features, makingaccurate diagnosis difficult. Moreover, uterineevacuation is indicated in the therapy of somedisorders, while in others alternative interventionsmay allow the pregnancy to be carried to term.These and other issues are discussed as part of acomprehensive review of the differential diagnosisand management of thrombocytopenia in preg-nancy.

The term “refractory ITP” is used with referenceto two distinct groups of patients: 1) patients inwhom the platelet count cannot be easily in-creased, including those who are poorly responsiveto initial single agent treatment, and 2) those withpersistent thrombocytopenia despite the use ofconventional therapies. An approach to manage-ment of the former group will be presented, fol-lowed by a discussion of patients with chronicrefractory ITP. The latter will include presentation ofnew data on the role of Helicobacter pylori in ITPand whether its treatment ameliorates thrombocy-topenia, as well as the use of rituximab and othermodalities.

Thrombotic microangiopathies such as throm-botic thrombocytopenic purpura (TTP) are rare, butlife threatening causes of thrombocytopenia. Ultra-large multimers of von Willebrand factor (vWF)aggregate platelets intravascularly, and congenital

or immune-mediated deficiencies of a metallo-protease that cleaves these ultra-large multimersmay cause TTP. However, little information existsconcerning the behavior of this protease in otherphysiological and pathological conditions. Levelsof this protease have now been measured inhealthy individuals of different ages, full-termnewborns, pregnant women and a patients withvariety of pathologic conditions, and these data willbe reviewed herein.

Heparin-induced thrombocytopenia/thrombosis(HIT/T) remains the most common antibody-mediated, drug-induced thrombocytopenic disor-der, and a leading cause of morbidity and mortality.Based on clinical correlations and murine models,there is increasing evidence that antibodies tocomplexes between platelet factor 4 (PF4) andheparin cause HIT/T, and the molecular composi-tion of the relevant antigen has also become betterdefined. However, the introduction of sensitiveELISAs to measure anti-PF4/heparin antibodies hascomplicated diagnosis in some settings in whichthe incidence of such antibodies in unaffectedpatients exceeds the incidence of the disease. Inaddition, the FDA approval of Lepirudin andArgatroban has expanded the repertoire of agentsavailable for therapy of HIT/T and may change theapproach to management of asymptomatic patientswith thrombocytopenia. However, the optimal useof these drugs in commonly encountered settingsremains in evolution, and a need for alternativeapproaches to prevention and treatment is evident.

I. PREGNANCY-ASSOCIATED THROMBOCYTOPENIA:AN UPDATE

Keith R. McCrae, MD*

Thrombocytopenia affects up to 10% of all pregnancies.Though obstetricians manage most of these cases, morecomplex thrombocytopenic disorders are often referredfor hematologic consultation. Therefore, the consultinghematologist must have a working knowledge of the dif-ferential diagnosis and therapy of pregnancy-associated

thrombocytopenia. The goals of this section will be toreview the differential diagnosis, pathophysiology andmanagement of these disorders.

The Platelet Count in Normal PregnancyBefore considering pregnancy-associated thrombocy-topenia, it is useful to review the effect of normal preg-

* Department of Hematology/Oncology, Case Western ReserveUniversity, School of Medicine, BRB-3, 10900 Euclid Ave.,Cleveland OH 44107-4937

Hematology 2001 283

nancy on the platelet count. Though a number of smallstudies have failed to reach a consensus on this issue,recent studies that assessed platelet counts in more than4,000 pregnant patients each have demonstrated that themean and 2.5th percentile platelet count decreases byapproximately 10% in pregnant patients, and that thehistogram of platelet count distribution at term is nor-mally distributed but shifted to the left.1,2 In most cases,this “physiologic” decrease in platelets occurs in the thirdtrimester. These observations suggest that pregnancy isassociated with a mild, and generally unappreciated de-crease in the circulating platelet count.

Differential Diagnosis ofPregnancy-Associated Thrombocytopenia

Thrombocytopenia in pregnancy may occur secondaryto a variety of causes (Table 1), ranging from benigndisorders such as gestational thrombocytopenia to syn-dromes associated with significant morbidity.3-5 Most ofthese occur during specific periods of gestation, althoughthese periods may sometimes overlap. On occasion, pa-tients may present with a constellation of symptoms thatreflect characteristics of more than one disorder. How-ever, in most cases the underlying cause of thrombocy-topenia may be diagnosed when the time of onset, clini-

cal manifestations and laboratory studies are consideredtogether.

Causes of Pregnancy-Associated Thrombocytopenia

Gestational (incidental) thrombocytopeniaGestational, or incidental, thrombocytopenia is the mostcommon cause of thrombocytopenia in pregnancy. Thisdisorder affects approximately 5% of all pregnantwomen, and accounts for > 75% of all cases of preg-nancy-associated thrombocytopenia.3,5-7 The pathogen-esis of gestational thrombocytopenia is not understood,but may reflect the effects of hemodilution or acceler-ated platelet clearance through immune or non-immunemechanisms.3, 6 Though the majority of patients withgestational thrombocytopenia maintain platelet countsin the range of 110,000-150,000/µL, most authoritiesconsider a platelet count of greater than 70,000/µL in anotherwise healthy pregnant woman with no prior his-tory of immune-mediated thrombocytopenia to be con-sistent with this disorder. The likelihood of another, moresignificant cause of thrombocytopenia increases signifi-cantly in patients with platelet counts below this value.

Gestational thrombocytopenia is a benign disorder,likely a more pronounced variant of the “physiologic”thrombocytopenia that accompanies pregnancy. Patientswith gestational thrombocytopenia do not have an in-creased incidence of bleeding, nor are they at increasedrisk for delivery of thrombocytopenic offspring.3,5-8

Hence, the medical evaluation of an otherwise healthypregnant woman with a platelet count greater than70,000/µL and no prior history of thrombocytopenia maybe limited to a general physical examination with care-ful blood pressure measurement and thorough examina-tion of the peripheral blood film.9,10 As it is inevitablethat a small number of women meeting these criteriawill have mild immune thrombocytopenia purpura (ITP),a thorough review of pre-gestational platelet counts isindicated.

Immune thrombocytopenia purpura. ITP, the mostcommon cause of significant thrombocytopenia in thefirst trimester, accounts for approximately 1 case ofthrombocytopenia per 1,000 pregnancies and 5% of allcases of pregnancy-associated thrombocytopenia.3,5,11,12

The pathogenesis of ITP in pregnant, as in non-pregnantpatients, involves the activity of antibodies directedagainst platelet glycoproteins, primarily GP IIb/IIIa andGP Ib/IX, and clearance of these IgG-coated plateletsby the reticuloendothelial system.13

The diagnosis of ITP in pregnancy is not difficultwhen patients have a prior history of thrombocytopeniaor in the setting of moderate to severe thrombocytope-nia (i.e. < 50,000/µL). However, it may not be possibleto distinguish ITP from gestational thrombocytopenia

Table 1. Causes of thrombocytopenia in pregnancy.

Isolated thrombocytopenia

Gestational

Immune (ITP)

Drug-induced

Type IIb von Willebrand disease

Congenital

Thrombocytopenia associated with systemic disorders

Pregnancy-specific

Preeclampsia

HELLP (hemolysis, elevated liver function tests, lowplatelets syndrome)

Acute fatty liver

Not pregnancy specific

Thrombotic microangiopathies

Thrombotic thrombocytopenic purpura

Hemolytic uremic syndrome

Systemic lupus erythematosus

Antiphospholipid antibodies

Disseminated intravascular coagulation

Viral infection (human immunodeficiency virus [HIV],Epstein-Barr virus [EBV], cytomegalovirus [CMV])

Bone marrow dysfunction (primary or secondary)

Nutritional deficiency

Hypersplenism


in a mildly thrombocytopenic patient with no prior his-tory of thrombocytopenia. Levels of platelet-associatedIgG are elevated in both groups.14 The utility of MAIPA(monoclonal antibody immobilization of platelet anti-gens) assays, which are designed to specifically mea-sure antibodies reactive with platelet glycoproteins, isalso uncertain in this setting.15 Practically, in the absenceof a pre-gestational platelet count, the onset of signifi-cant (< 100,000/µL) thrombocytopenia early in gesta-tion, with declining platelet counts as gestationprogresses, is most consistent with ITP, while gestationalthrombocytopenia appears to develop more commonlyin the second or third trimester.11

Though decisions concerning management of ITPduring pregnancy affect both mother and fetus, therapyis focused primarily on amelioration of thrombocytope-nia in the mother. The need for therapy is based on theseverity of thrombocytopenia and whether there is ac-tive bleeding. Patients with a platelet count greater thanapproximately > 30,000/µL and no bleeding generallydo not require treatment. However, when the severity ofthrombocytopenia increases or bleeding develops,therapy is indicated.9,11,16 Moreover, as pregnancy ap-proaches term, more aggressive measures to raise theplatelet count to a safe level (> 50,000/µL) to minimizethe hemorrhagic stresses of delivery and allow the ad-ministration of epidural anesthesia may be re-quired.9,11,16,17

Management of a pregnant patient with ITP is simi-lar to that of non-pregnant individuals. Due to their effi-cacy and low cost, many consider corticosteroids to bethe first line of therapy.3,9,10,16 However, in addition totheir usual side effects such as osteoporosis and weightgain, corticosteroids increase the incidence of pregnancy-induced hypertension and exacerbate gestational diabe-tes. An alternative to corticosteroids is high-dose (2 gm/kg) intravenous gammaglobulin (IVIG), which someexperts consider first-line therapy for pregnancy-asso-ciated ITP due to its lower toxicity profile.11 Though ef-ficacious, the effects of IVIG are transient (durationof 3-4 weeks), and the costs of multiple courses exceed-ingly high. Nevertheless, a regular schedule of IVIGshould be considered when more than 10 mg/day of pred-nisone is required to maintain the platelet count above20,000-30,000/µL. Due to its relatively rapid onset ofaction, IVIG may also be useful in raising the plateletcount in preparation for delivery.

As in the non-pregnant patient, patients refractoryto corticosteroids and IVIG present a difficult manage-ment problem. Occasionally, such individuals will re-spond to combinations of high doses of steroids (methyl-prednisolone, 1 gm) and IVIG.3 As in non-pregnant in-dividuals, remission of ITP is achieved in approximately70% of pregnant women who undergo splenectomy,

though the long-term efficacy of this modality is de-bated.18 If splenectomy is required during pregnancy,most experts recommend that it be performed in the sec-ond trimester, as it has been associated with an increasedincidence of premature labor when performed earlier,and may be technically difficult later in gestation due toobstruction of the surgical field by the gravid uterus.3

Recently, laparoscopic splenectomy has been safely per-formed in several pregnant patients with ITP.19 Little in-formation is available concerning the safety and effi-cacy of third and fourth line agents in refractory ITPduring pregnancy. Azathioprine is relatively contraindi-cated, while danazol and vincristine should be avoided,9

though a report on successful use of the latter in a re-fractory pregnant patient has appeared.20

An area of particular concern in the management ofpregnant patients with ITP is the fetal platelet count andits implications for the mode of delivery. Due to trans-placental passage of maternal IgG, particularly duringthe third trimester, the offspring of patients with ITP mayalso develop thrombocytopenia.3,11 Neonatal plateletcounts below 50,000/µL at delivery occur in 10-25% ofthe offspring of patients with ITP, while counts below20,000/µL occur in 5% of these patients.21 Moreover, nomaternal treatment has been convincingly shown to di-minish the incidence of fetal thrombocytopenia.3,11 Bleed-ing complications may occur in 25-50% of profoundlythrombocytopenic neonates at the time of delivery,3,22

though intracranial hemorrhage is rare.8 Debate hastherefore centered on options to minimize this risk, basedon the untested hypothesis that neonatal head traumaduring passage through the birth canal may precipitateintracranial hemorrhage.

Several studies have attempted to define character-istics of mothers with ITP that predict for delivery of athrombocytopenic neonate. However, there is no con-vincing correlation between the fetal platelet count anda number of maternal characteristics including a historyof prior splenectomy or an increased level of platelet-bound IgG.3,11 A relationship between the severity ofmaternal and fetal thrombocytopenia has been observedin occasional studies,23 but not in the majority of them.3,11

The finding of a relationship between levels of circulat-ing maternal anti-platelet IgG and neonatal thrombocy-topenia22 has not been widely confirmed. This, as wellas a report of widely divergent platelet counts in dizy-gotic twin offspring24 of a patient with ITP, demonstratesthat the pathogenesis of neonatal thrombocytopenia in-volves not only the level of maternal antibody but therate of fetal megakaryopoiesis and ability of the fetalreticuloendothelial system to clear antibody-coated plate-lets.3 Indeed, the most reliable predictor of neonatalthrombocytopenia may be a history of thrombocytope-nia in a prior sibling.25

Hematology 2001 285

Since maternal clinical or laboratory characteristicspredictive of neonatal thrombocytopenia are generallynot available, the fetal platelet count may be determinedeither through fetal scalp sampling during labor or bypercutaneous umbilical blood sampling prior to deliv-ery. Of these, the latter method provides a more reliableestimate, although it is associated with a complicationrate, primarily bleeding and fetal bradycardia, of 0-1%.26

Since this approaches or exceeds the incidence of com-plications from severe thrombocytopenia in the offspringof a patient with ITP, two approaches to management ofthese patients have evolved. One approach, supportedby approximately 60% of perinatologists in the US,27

advocates a trial of labor for patients with ITP withoutprior determination of the fetal platelet count. This ap-proach is based on the belief that severe thrombocytope-nia and bleeding in the offspring of these individuals isuncommon and that there is no evidence that the inci-dence of fetal intracranial hemorrhage is reduced byCesarean section.9,28 A second approach involves inva-sive determination of the fetal platelet count, generallyby percutaneous umbilical blood sampling, followed byCesarean section if the platelet count is < 50,000/µL.16

Regardless of the mode of delivery, cord plateletcounts should be obtained on all offspring of motherswith ITP9 at the time of delivery. Moreover, since theplatelet count may decline for the first 4-5 days afterdelivery, daily monitoring and institution of appropriatetherapy for severe thrombocytopenia, should it developover this interval, is indicated.

Preeclampsia and the HELLP syndromePreeclampsia affects approximately 6% of all pregnan-cies, most often those of primigravidas.3 The criteria forpreeclampsia, though frequently revised, include hyper-tension and proteinuria (> 300 mg protein/24 hours) de-veloping after 20 weeks of gestation.29 Preeclampsia oc-curs most commonly in women less than 20 or greaterthan 30 years old, usually in the third trimester. Preec-lampsia in multiparous women may be associated witha change in partners.3 A genetic role in the developmentof preeclampsia has been suggested but remains poorlydefined.30 Recently, an association of preeclampsia witha genetic predisposition to thrombophilia has also beenproposed.31

Although the clinical manifestations of preeclamp-sia are generally not evident until the third trimester, thepathogenesis appears to involve deficient remodeling ofthe uteroplacental vasculature early in pregnancy, per-haps as a consequence of diminished invasiveness of pla-cental trophoblasts.32 Abnormalities in the expression ofcell adhesion molecules33 and activated metallopro-teases34 by trophoblasts from preeclamptic women havebeen described, but whether these are primary or sec-

ondary to other pathophysiologic events has not beenestablished. The net result, however, is the formation ofan insufficient uteroplacental blood supply, resulting inprogressive hypoxia of the fetoplacental unit as preg-nancy progresses.35 Resultant imbalances in the releaseof prostaglandins by placental and extraplacental tissues,enhanced lipid peroxidation, and other undefined fac-tors may contribute to the hypertension, platelet activa-tion and systemic endothelial dysfunction characteristicof this disorder.30

Up to 50% of patients with preeclampsia developthrombocytopenia, with the severity generally propor-tional to that of the underlying disease. Thrombocytope-nia may be an early manifestation of preeclampsia, evenpreceding other disease manifestations on occasion.3 Thecauses of thrombocytopenia are not well defined, thoughthey likely reflect accelerated platelet clearance and/ordestruction due adhesion of circulating platelets to dam-aged or activated endothelium, activation and consump-tion of platelets as a consequence of activation of thehemostatic system, or clearance of IgG-coated plateletsby the reticuloendothelial system.3

Subclinical activation of the coagulation cascadeoccurs in most patients with preeclampsia. Though rou-tine studies such as the prothrombin time (PT), activatedpartial thromboplastin time (aPTT) and fibrinogen levelare usually normal, more sensitive indices such as fi-brinogen D-dimers and thrombin-antithrombin com-plexes are elevated in many patients who develop throm-bocytopenia.3 Activation of hemostasis alone is not likelyto be the primary cause of thrombocytopenia in all cases,though markers of intravascular coagulation may be as-sociated with more severe intrauterine growth retarda-tion.36

The HELLP (hemolysis, elevated liver function tests,low platelets) syndrome is a variant of preeclampsia thatoccurs primarily in white, multiparous women above theage of 25 years.3 Criteria for this disorder include 1)microangiopathic hemolytic anemia (MAHA), 2) levelsof serum aspartate aminotransferase (AST, SGOT) > 70U/L and 3) thrombocytopenia, with a platelet count be-low 100,000/mL.37 Patients frequently present with epi-gastric and right upper quadrant pain, often leading tomisdiagnosis and referral for evaluation of suspectedintraabdominal processes. Approximately 10% of pa-tients with HELLP develop hypertension and proteinuria,and meet criteria for preeclampsia. Despite their similari-ties, HELLP is associated with a higher rate of maternaland fetal morbidity and mortality than preeclampsia.3

Offspring of mothers with HELLP and preeclamp-sia may also become thrombocytopenic, though this maynot develop until after delivery.3 The pathogenesis of neo-natal thrombocytopenia in these infants is not well un-derstood, but in part may represent the effects of prema-


turity and its complications, such as sepsis and acuterespiratory distress.38 Absent umbilical artery end-dias-tolic velocity, a marker of fetal hypoxemia and acidosis,may be a risk factor for neonatal thrombocytopenia ingrowth-retarded offspring.39 Though elevated levels ofplatelet-associated IgG may be present on the plateletsof these infants, they do not correlate with the develop-ment or severity of thrombocytopenia.3 More recent stud-ies suggest that these infants may manifest impaired re-sponses to thrombopoietin.40

Based on the premise that deficient intravascular pro-duction of prostacyclin and excessive platelet activationunderlies the pathogenesis of these disorders, the valueof prophylactic treatment of unselected and high-riskpregnant women with low dose aspirin in primary pre-vention of preeclampsia was assessed. This intervention,however, yielded no significant reduction in the incidenceof preeclampsia in either group.41,42

Management of preeclampsia and/or HELLP is gen-erally supportive, with the goal of medically stabilizingthe patient prior to delivery.4 Some have argued for con-servative management of preeclampsia, particularly inmilder cases or before 34 weeks gestational age, therebyproviding time for further fetal maturation. However, theonly indication for this approach in severe preeclampsiaor HELLP is to gain additional time (24-48 hours) forfetal lung maturation after administration of betametha-sone.4 Platelet transfusions may be given prior to Cesar-ean section to raise the platelet count above 50,000/µL,with the appreciation that the survival of transfused plate-lets may be short. Coagulopathy resulting from dissemi-nated intravascular coagulation (DIC) may be managedwith fresh frozen plasma, if indicated.3 In almost allcases, manifestations resolve within several days afterdelivery, although in some patients the platelet count de-clines for an additional 24-48 hours.4 A subset of pa-tients display prolonged thrombocytopenia, multiorgan

dysfunction and an increased LDH following delivery,manifestations that may be reversed or ameliorated insome patients by plasma exchange43 or corticosteroids.44

Hence, these interventions should be considered in pa-tients who remain symptomatic for more than 4-5 daysafter delivery and in whom other causes of persistentthrombocytopenia are excluded.

Thrombotic thrombocytopenic purpura (TTP) and thehemolytic uremic syndrome (HUS)TTP and HUS are disorders that share the central fea-tures of MAHA and thrombocytopenia. Though neitherdisease is unique to pregnancy, the incidence of both isincreased in this setting.4,45 In some series, up to 10% ofall cases of TTP have occurred in pregnant patients.

TTP is characterized by a pentad of findings includ-ing MAHA, thrombocytopenia, neurologic abnormali-ties (confusion, headache and seizures, among others),fever and renal dysfunction, though the classic pentad ispresent at the time of diagnosis in less than 40% of pa-tients.4 The clinical manifestations of HUS are similar,though neurologic abnormalities are usually prominentin patients with TTP, while renal dysfunction is moresevere in patients with HUS. Recently, the potential roleof congenital or acquired deficiencies of a specific vonWillbrand factor (vWF)-cleaving protease in the patho-genesis of TTP has been evoked,46,47 although involve-ment of this protease in the pathophysiology of HUS isuncertain. No published information is available con-cerning the regulation of the vWF-cleaving proteaseduring uncomplicated pregnancy, though this issue isaddressed elsewhere in this paper.

TTP and HUS may be difficult to discern from oneanother, as well as from pregnancy-specific causes ofthrombocytopenia such as severe cases of preeclampsiaor the HELLP syndrome.48 Table 2 lists features thatmay be of use in differentiating these disorders. First,

Table 2. Differentiation of pregnancy-associated microangiopathies.*

Thrombo- Coagulo- Hyper- Renal CNS Time ofMAHA cytopenia pathy tension Disease Disease Onset

Preeclampsia + + ± +++ + + 3rd trimester

HELLP ++ +++ + ± + ± 3rd trimester

HUS ++ ++ ± ± +++ ± Postpartum

TTP +++ +++ ± ± +/± +++ 2nd trimester

SLE ± to +++ + ± ± +/++ + Anytime

APS - to ± + ± ± ± + Anytime

AFLP + +/± +++ ± ± + 3rd trimester

Abbreviations: MAHA, microangiopathic hemolytic anemia; CNS, central nervous system; HELLP, hemolysis, elevated liver function tests,low platelets syndrome: HUS, hemolytic uremic syndrome: TTP, thrombotic thrombocytopenic purpura; SLE, systemic lupus erythematosus;APS, antiphospholipid syndrome; AFLP, acute fatty liver of pregnancy; ±, variably present; +, mild; ++, moderate; +++, severe

* Modified from McCrae and Cines, Semin Hematol. 1997;34:148 (reference 4)

Hematology 2001 287

microangiopathic hemolysis is generally more severe inTTP or HUS than preeclampsia or HELLP. Second, TTPdevelops relatively early in pregnancy, with a mean on-set of approximately 23.5 weeks, while the majority ofcases of preeclampsia and HELLP develop in the thirdtrimester.3,4 In contrast, approximately 90% of cases ofHUS occur post-partum, with a mean time to develop-ment after delivery of approximately 26 days. Third, pa-tients with TTP and HUS generally maintain normal lev-els of antithrombin, while most of those with preeclamp-sia and HELLP display modest reductions in antithrom-bin levels, likely due to consumption.4

Unlike preeclampsia or HELLP, the course of preg-nancy-associated thrombotic microangiopathies is notameliorated by delivery; hence, pregnancy terminationis not therapeutic.45 However, TTP in pregnant and non-pregnant patients responds similarly to plasma exchange,with > 75% of patients achieving remission. Chronic re-lapsing TTP may also be precipitated during pregnancy;such cases have been treated with plasma exchange orinfusion, the latter occasionally used, with or withoutaspirin, on a prophylactic basis.49 In contrast, the prog-nosis of pregnancy-associated HUS, like that of sporadicHUS occurring in non-pregnant patients, is poor. Al-though response to plasma exchange occurs infre-quently,50 a therapeutic trial is indicated.

Poor fetal outcomes are common in patients withthrombotic microangiopathies, due to placental ischemiaand the morbidity associated with prematurity.45 TTP andHUS may recur in subsequent pregnancies, though thefrequency with which this occurs is uncertain.4 Preg-nancy-associated HUS has also occurred in women witha familial history of this disorder. The long-term mor-bidity of thrombotic microangiopathies, particularlyHUS, is significant, with many patients left with chronicrenal insufficiency and hypertension.51

Acute fatty liver of pregnancyAcute fatty liver of pregnancy (AFLP) affects one ofevery 5,000 to 10,000 pregnancies, typically developingin the third trimester. Though more common in primi-paras, AFLP can initially present after several uncom-plicated pregnancies. This disorder may result from afetal deficiency of long-chain 3-hydroxy-acyl CoA de-hydrogenase (LCHAD) or other enzymes involved in mito-chondrial fatty acid oxidation.52,53 A glutamic acid toglutamine mutation at position 478 of LCHAD appears tobe of particular importance in disease development.53

Clinically, women present with malaise, nausea, epi-gastric and right upper quadrant pain, dyspnea, mentalstatus changes and cholestatic liver abnormalities. Dia-betes insipidus may also occur, and hypoglycemia iscommon and often severe.4 Levels of fibrinogen and an-tithrombin are severely depressed, and a prolonged PT

accompanied by laboratory evidence of “disseminatedintravascular coagulation” is present in 75%, perhapsresulting from impaired synthesis of antithrombin. Theseverity of microangiopathic hemolysis and thrombocy-topenia (if present) is far less than that seen in HELLP,TTP or HUS,54 though up to 50% of patients with AFLPmight also meet clinical criteria for preeclampsia. Diag-nosis is usually made based on clinical criteria, as he-patic imaging studies are generally not specific. Confir-mation may be obtained by staining a liver biopsy speci-men with oil red O. Management consists of correctionof hypoglycemia and electrolyte imbalances, as well asthe underlying coagulopathy. Up to 10 days may be re-quired after delivery for normalization of the coagula-tion status. Fetal mortality remains at 15%, though ma-ternal mortality occurs in less than 5% of cases.55

Miscellaneous Causes ofPregnancy-Associated Thrombocytopenia

As in the non-pregnant setting, HIV infection should beconsidered in any thrombocytopenic patient with appro-priate risk factors.3 Thrombocytopenia may occur in upto 25% of patients with systemic lupus erythematosus(SLE) secondary to platelet destruction due to antiplateletantibodies, circulating immune complexes or othercauses.56 Antiphospholipid antibodies (APLA) may beassociated with an increased risk of preeclampsia in ad-dition to their better-recognized association with throm-bosis and recurrent fetal loss.57 Some patients with APLAhave been described with syndromes resembling HELLP,HUS or TTP, which may not respond in the usual man-ner to standard management.4 Drug-induced thrombocy-topenia occurs in the pregnant as well as the non-preg-nant setting. Unrecognized cocaine use has been associ-ated with the development of a HELLP-like syndromein pregnant women.58 Disseminated intravascular coagu-lation, with attendant thrombocytopenia, may complicateseveral pregnancy-related disorders, including placentalabruption, amniotic fluid embolism, uterine rupture andretention of a dead fetus.3 Diagnosis of these obstetricalemergencies is usually straightforward

Finally, congenital platelet disorders, such as May-Hegglin anomaly, may occasionally remain undiagnoseduntil pregnancy; hence, thorough review of the periph-eral blood smear should be an important component ofthe evaluation of such patients.3 Pseudothrombocyto-penia, an in vitro artifact usually caused by EDTA-de-pendent antiplatelet antibodies, may be transferred frommother to fetus by transplacental passage of the relevantantibody.59 Finally, Type IIb von Willbrand disease maybe associated with thrombocytopenia in pregnant womendue to increased levels of an abnormal vWF moleculethat binds to platelets with high avidity and enhancestheir clearance.3


II. NOVEL APPROACHES TO MANAGEMENT OF

IMMUNE THROMBOCYTOPENIC PURPURA:RESULTS OF RECENT TRIALS

James B. Bussel, MD*

Immune (not idiopathic) thrombocytopenic purpura(ITP) was first described as a disease of immune plate-let destruction many years ago by William Harrington.This was documented by the thrombocytopenic responsein normal volunteers to infusion of plasma from patientswith ITP. In certain individuals, however (6 of 17 in oneof the early studies), infusion of plasma from ITP pa-tients did not cause thrombocytopenia1 in the recipients.Potential explanations for this included the possibilitiesthat 1) the antiplatelet antibody was entirely bound tothe patient’s (donor) platelets and therefore was absentfrom infused plasma; 2) there was a particular charac-teristic of the recipients that made them not susceptibleto the thrombocytopenic effects of the plasma, i.e. theyhad spherocytosis; or 3) the plasma was obtained from apatient who appeared clinically identical to other patientswith ITP but who in reality had another cause of throm-bocytopenia, and therefore did not have antiplatelet an-tibodies at all. This last possibility, the inability to becertain of the diagnosis in individual patients, persiststoday. The lack of specificity of platelet antibody test-ing means that it is still not possible to use platelet anti-body studies to demonstrate that a given individual ei-ther does or does not have ITP. Studies of newer ways oftesting for the presence or absence of platelet antibodiesas well as additional tests to better define the pathophysi-ology of the thrombocytopenia and, indirectly, the diag-nosis, such as thrombopoietin and glycocalicin levels andplatelet reticulocyte counts, are underway.

Even in cases clinically presumed to be ITP the eti-ology is usually unknown. Table 3 contains a list of im-munologic abnormalities or polymorphisms that havebeen associated with ITP. None of them, however, havea very strong association nor are they present in a ma-jority of patients. This emphasizes the difficulty in un-derstanding what causes this disease. Preliminary datausing phage display technology has suggested that anti-platelet antibodies in patients with ITP disproportion-ately select a limited assortment of VH heavy chains.2 Thesame study suggested that this phenomenon occurs as aresult of antigen driven selection, i.e. persistent antigenexposure. This refers to ongoing exposure to platelets.

Persistent infections are also associated with ITP(Table 4). Typically the ITP in these cases would be char-acterized not only as having a “mixed” pathophysiol-ogy, decreased platelet production as well as increasedplatelet destruction, but also as being “secondary” ITP.

* New York Hospital, Division of Pediatrics, 525 E 68th Street,Payson 6, New York NY 10021

Dr. Bussel receives clinical research support from Cangene,Amgen, Genentech, IDEC, Bayer, and Baxter.

Table 3. Potential predispositions and etiologies of immunethrombocytopenic purpura (ITP).

Predispositions

1. hypogammaglobulinemia: combined variable immunodeficiency(CVI), severe combined immunodeficiency disease (SCID), IgAand IgG2 deficiency, but not x-linked agammaglobulinemia(XLA)

2. deficiency of classical pathway of complement components,C4, C2

3. ? certain HLA types, e.g. B8DR3

4. abnormalities of CD95 (Fas pathway)

5. ? other failure of autoimmune lymphocytes

6. ? certain Fc receptor polymorphisms

Etiologies

1. Persistent antigen exposure

2. ? selection of certain heavy chain genes in antibody production

3. unknown tendency to develop autoimmunity: systemic lupuserythematosus (SLE), Evans syndrome

4. abnormal antigen presentation, e.g. in lymphoproliferative statessuch as chronic lymphocytic leukemia (CLL)

5. persistent infections: see Table 4

Table 4. Persistent infections in patients with immunethrombocytopenic purpura (ITP).

Associations of ITP

1. Human Immunodeficiency Virus (HIV): Multiple studies havedocumented:

a. a high incidence of thrombocytopenia in infected patients

b. autoimmune nature of the thrombocytopenia:best evidence: response to treatments (IVIG, IV anti-D) ofimmune thrombocytopenia

c. improvement of the thrombocytopenia with suppression ofthe virus

2. Hepatitis C: Studies have suggested:

a. an incidence of thrombocytopenia in infected patients

b. anecdotal evidence of response to treatments of immunethrombocytopenia

c. improvement of the thrombocytopenia with suppression ofthe virus

3. Helicobacter Pylori: Preliminary studies have postulated

a. a high incidence of Helicobacter pylori in patients with ITP

b. improvement of the thrombocytopenia with eradication of theinfection

Inability to Demonstrate Associations of Infections with ITP

1. Cytomegalovirus (CMV): no increased incidence in ITP and noevidence that eradication influences ITP

2. HTLV1: no increased incidence in ITP

Hematology 2001 289

Nonetheless, there is an immunologic component to thethrombocytopenia in these cases. Two recent studies havesuggested that Helicobacter pylori may promote ITP,3,4

potentially by contributing to either the development and/or the persistence of ITP in infected patients. Therefore,eradication of H. pylori infection may improve the courseof ITP or ameliorate the disease. Evidence that this isthe case was demonstrated in an initial study in whichthrombocytopenia improved in 7 patients with H. pyloriwhose infection was treated.3 This instigated a secondstudy of 30 chronic ITP patients, 13 of whom were foundto be infected with H. pylori. H. pylori was eradicatedafter treatment in 12 of these 13 infected patients; 6 ofthese had improvement in their platelet count.4 In ourongoing study, we have tested 16 chronic ITP patientsand identified 4 that were infected with H. pylori. Therehas been no improvement in the platelet count of the 3who have completed 2 weeks of treatment (prevacid-amoxicillin-biaxin) and an additional 6 weeks of fol-low-up, and who have had their breath test repeated todemonstrate that they have become negative. In addi-tion we have treated 10 patients with the same antimi-crobial combination, the “prevpak,” even though theydid not have H. pylori, and this has not improved theplatelet count in any of them. Updated results will bereviewed. Of note, IVIG almost certainly has antibodiesto H. pylori in it because of the relative ubiquity of thisinfection. This presence of passive antibody in certainpatients is the reason to utilize the breath test to docu-ment infectivity. There will likely be an increased inci-dence of infection in more elderly patients, although itis not known whether this will be related to their ITP.

Table 4 lists other infectious etiologies that have beenexplored in patients with ITP including cytomegalovi-rus (CMV), HTLV1, human immunodeficiency virus-1(HIV1), and hepatitis C. The latter two disorders havebeen well demonstrated to have an “immune” thromb-ocytopenia, and their suppression with antiviral therapyis coincident with an improvement in the platelet countin the majority of cases. This has been most clearlyproven in multiple studies in HIV-infected patients, sug-gested in several small series of hepatitis C patients, andis being tested for in those infected with H. pylori.

There are several specific new developments thataffect treatment of “acute” ITP. Figure 1 (see color page549) is a picture of wet purpura in the mouth of a patientwith a platelet count of 2,000/µL. Virtually all physi-cians would agree that such a patient requires a rapidincrease in the platelet count and various means of us-ing steroids and/or IVIG have been given to patients suchas this one. Recently it has been shown that infusion ofIV antiD at a dose of 75 µg/kg, as opposed to 50 µg/kg,typically will result in a substantial overnight plateletincrease in the majority of Rh+, non-splenectomized

adults and children with ITP.5 This data appears compa-rable to the platelet increases seen with IVIG, given thatthe latter is effective in patients who may not respondwell to IV anti-D, especially Rh- or splenectomized pa-tients. This is of interest because, although both IVIGand IV anti-D cause “Fc receptor blockade” as an im-portant mechanism of acute platelet increase, they mayhave additional immunomodulatory effects that are quitedifferent as a result of differences in the way that theycause “Fc receptor blockade.” Two streams of informa-tion support important differences between IV anti-Dand IVIG.

Recent work in mice has shown that IVIG impactsFc receptor-mediated clearance of antibody-coated par-ticles specifically by upregulating FcRIIb, the inhibitoryFc receptor.6 This was demonstrated both with an anti-FcRIIb antibody and in an FcRIIb knockout model. Inboth situations when FcRIIb is rendered inactive, IVIGcompletely loses its ability to block the thrombocytope-nia induced by infusion of an anti-mouse-platelet anti-body. In contrast, it is presumed that anti-D directly in-teracts with and occupies the activating Fc receptor, i.e.FcRIII. Calculations of the number of molecules of anti-D infused as related to the number of D+RBC allow foran average of approximately 500 molecules of anti-D tobind to each red cell. This type of large immune com-plex would look very different to the FcR system than adoubling or tripling of the serum IgG level in connec-tion with infusion of small quantities of IgG dimers andisohemagglutinins that inconsistently cause a weaklypositive direct antiglobulin test.

Clinical studies support these differences in that theyprovide evidence of important clinical differences be-tween these two seemingly similar treatments that sharean acute mechanism of effect: inhibition of destructionof antibody-coated platelets. By 1991, differences werealready described between the clinical effects of IVIGand IV anti-D, suggesting that different mechanisms ofinhibition of destruction of antibody-coated particleswere operative.7 Further differences were subsequentlydescribed (Table 5) based on different efficacy in HIV-infected patients (anti-D was more effective)8 and therelationship of the acute platelet increase to the durationof response (only for anti-D).8 However despite the ac-cumulation of clinical differences, it was never clear howIVIG was creating its effect since neither tripling theserum IgG concentration nor infusion of a very smallamount of IgG dimers would have been expected to in-hibit FcRIII or FcRI. Of interest, 5 of 11 refractory ITPpatients treated with a monoclonal antibody that inhib-ited FcRIII responded, while 6 did not. Some of the non-responders were transiently IVIG responsive. In addi-tion, several patients transiently responsive to the mono-clonal anti-FcRIII antibody were IVIG non-respon-


sive.9,10 These studies implied that IVIG did not inhibitplatelet destruction via interaction with FcRIII.

IV anti-D has been associated with the rare occur-rence of severe and/or intravascular hemolysis (IVH),estimated to be at a rate < 1%.11 The higher dose of anti-D, 75 µg/kg, was not associated with a greater develop-ment of anemia in the study cited above, though it couldpossibly be associated with a higher incidence of IVH.5

Only 2 mild cases of transient “pink urine” have beenseen at our center, possibly because we do not treat pa-tients with a positive direct antiglobulin test with anti-Dand possibly because steroid premedication is routinelyused at the 75 µg/kg dose. An ongoing study at our cen-ter is exploring the relationship between these issues andother pathophysiologies in connection with hemolysisafter anti-D: preliminary results will be presented asavailable.

Another outgrowth of the studies with IV anti-D andwith IVIG has been consideration of combination treat-ments in especially refractory patients with ITP. Thiswas first considered in 1986 when 2 children with acuteITP who had received, but not responded to, IVIG12 de-veloped intracranial hemorrhages. Combination treat-ments have been used at our center and the results arebeing prepared for publication. The preliminary find-ings support the efficacy of the combination of IVIG,IV methylprednisolone, and the addition of one or bothof either IV anti-D and/or vincristine (Table 6). Thistreatment approach is typically restricted to patientsproven resistant to IVIG and/or steroids alone, or, rarely,in urgent need of an acute platelet increase (see below).The molecular differences in the effects of IVIG and IVanti-D make their combination more rational. In the fu-ture, the addition of thrombopoietin may be importantfor those patients not producing platelets as well andcould be incorporated, if there is demonstration of effi-cacy, into a combination approach. The bottom line ofthis summary is that combination therapy is better thanindividual treatments if the platelet count must be raised

urgently or if there is reason to think that the patient willnot respond well, e.g. if the patient has not responded tosingle treatments administered in the past. Urgency typi-cally is associated with a high risk or presence of seri-ous bleeding. Examples include the presence of a verylow platelet count and one of the following: 1) wet pur-pura (see Figure 1, color pages); 2) ongoing seriousbleeding, e.g. in the GI tract; 3) head trauma, even ifminor; 4) the recent ingestion of an antiplatelet agent;or 5) a co-existing bleeding tendency, e.g. von Willebranddisease or factor XI deficiency. In all of these settings aplatelet transfusion could be considered for transient sta-bilization, but in any event a means of rapidly increas-ing the patient’s own platelet count is mandatory. Emer-gency splenectomy might also be considered in suchsettings, but subjecting a severely thrombocytopenicpatient to a surgical procedure might be hazardous.Moreover, there is no means of predicting whether sple-nectomy will be effective, and splenectomy may wellbe unnecessary if the ITP is an acute and transient event,as for example in childhood ITP, which often resolvesspontaneously. Alternatively, the situation may occur ina refractory patients who have already had their spleensremoved, as in the patient in Figure 1.

Improved means of defining the “true” pathophysi-ology of the thrombocytopenia would be useful, particu-larly in patients with refractory ITP. In addition to a testto confirm the diagnosis of ITP, it might be useful toknow whether the primary pathobiology is a very highlevel of anti-platelet antibodies or a decrease in plateletproduction. While neither pathophysiology is likely tooccur in isolation, one may predominate over the otherin individual cases. It is here that the biologic measuresdiscussed in the introductory paragraph (platelet reticcount, glycocalicin, or thrombopoietin level) could bevery useful if they prove to correlate with clinical events.Preliminary data suggests that the glycocalicin level maycorrelate with the duration of platelet response to IV anti-D suggesting a better effect for a treatment that inhibits

Table 6. Combination IV treatment of refractory immunethrombocytopenic purpura.

1. MEDS/DOSES: IVIG 1 g/kg, Solumedrol 1 g (30 mg/kg), VCR0.03 mg/kg (1-1.5 mg IVP), and/or Anti-D 75 µg/kg

2. > 70% acute response rate in largely refractory patients i.e. noresponse to IVIG alone

3. ORAL MAINTENANCE MEDS/DOSES: Danazol 600-800 mg/kgand Imuran 2 mg/kg (cyclosporin, dapsone, Cellcept, cyclophos-phamide can be substituted if needed)

4. 3-4 month response rate > 70% of responders, long-term plan totaper to low dose danazol and/or imuran—long-term responserate uncertain

Abbreviations: IVIG VCR, intravenous gammaglobulin vincristine

Table 5. Differences between intravenous gammaglobulin(IVIG) and intravenous anti-D (IV anti-D) in immunethrombocytopenic purpura.

IVIG IV anti-D

1. platelet responses in Rh- patients: + -

2. platelet responses in splenectomized pts: + -

3. rate of plt increase: 1 gm/kg & 50 mcg/kg: fast slow(24 hr) (72 hr)

4. relative effect in HIV+ patients - +

5. duration of plt incr related to acute plt incr no yes

6. Fc receptor involved in acute effect ?II ?III

Abbreviations: IVIG, intravenous gammaglobulin; HIV, humanimmundeficiency virus

Hematology 2001 291

platelet destruction if platelet production is increased.An emerging debate involves the best management

of patients who would “normally” undergo splenectomyafter failing an initial course of 1-3 months of prednisone.Data on this topic may include a discussion of the ef-fects of splenectomy, including not only the long-termefficacy but also whether the toxicity has been underes-timated. A randomized, multicenter trial has recentlybeen completed comparing IV anti-D to prednisone andsplenectomy in newly diagnosed adults with ITP. Wehave studied IV anti-D treatment in 28 adults who hadplatelet counts of 30,000/µl or less at study initiation,were not splenectomized, were within 1 year of diagno-sis, and were HIV negative. The median follow up is 2years. Of the 28 initial patients, only 8 have undergonesplenectomy. Twelve (42%) have received no therapyfor over 6 months while maintaining a platelet countgreater than 30,000/µL; 6 maintain platelet counts >100,000/µL on no treatment, even though some receivedtreatment for > 1 year from diagnosis. The best andhighly significant predictor of a good long-term outcome,i.e. no need for any further therapy including splenec-tomy, was a platelet count > 14,000/µL at study initia-tion. These results require confirmation, and it must alsobe determined whether they are in any way specific toanti-D or would be equivalent with any long-term main-tenance treatment. For example, certain physicians woulduse danazol in a similar fashion. Furthermore, the rateof splenectomy success will fall if patients who wouldhave been responders, presumably like the respondersto repeated anti-D in this study, are no longer includedin the pool of patients undergoing splenectomy.

In current practice, many adults and some physi-cians choose to delay splenectomy, and it is not yet clearwhat the outcome of this approach is.13 The most com-mon reason that patients choose to forego splenectomyis the uncertainty of the outcome. At best it appears sple-nectomy is associated with a 60% rate of continued com-plete remission after 5-10 years. However, our data sug-gest that this rate continues to fall as time passes, i.e. noplateau was reached despite 10 years of follow-up. Itmay be that those with late relapses after splenectomyare those who develop de novo ITP again. Similarly, thereare no data to clarify whether those patients who un-dergo but do not respond to splenectomy will derive abenefit in becoming “more responsive” to other treat-ments. At the moment all that is known is that the re-sponse to IV anti-D is generally lost after splenectomy7

(other than as part of combination treatment).

Patients Who Fail to Respond to SplenectomyThere is no accepted plan of treatment in these patients.Management depends in part upon physician and pa-tient preference, which treatments patients respond to,

even if only transiently, and which treatments need to beavoided because their toxicities would become intoler-able for the individual patient. Moreover, if single agenttreatments are considered based upon the pilot seriesfrom which results have been reported, none, with thepossible exception of IV cyclophosphamide,14 has a re-sponse rate of > 30%. In addition, long-term toxicity ofmany of the treatments is not well defined, especially inpatients with ITP who may be less ill than many patientswho have undergone toxic therapies such as autologousmarrow or renal transplantation, in whom the long-termoutcomes of the treatment have been best described.

Avoidance of toxic therapies, though seeminglystraightforward, can be complex. A common set of is-sues is avoidance of steroids because of diabetes, psy-chiatric disturbances, an ulcer or gastritis not secondaryto H. pylori, hypertension, cataracts or osteoporosis; thelatter two issues require specific investigation yearly.Whether there is osteoporosis or cataracts may make thedifference between the at least temporary acceptabilityof a low daily dose of steroids, (5-10 mg/day) versuscomplete avoidance of steroids. Another example is thatof danazol and probably azathioprine, which cannot beused if the transaminases are already abnormal at initia-tion of treatment. These agents rarely cause a problemif there is no ongoing hepatitis, but are likely to worsenunderlying pathology. Another issue regards fertility inwomen of child-rearing potential, in whom danazol maybe considered either useful, because it suppresses mensesthat may be too heavy, or unacceptable. Cyclophospha-mide is more problematic in this regard even though alimited number of infusions may not impair fertility. Ifthere is either pre-existing neurologic impairment or afamily history of a hereditary neuropathy, a single doseof vinca alkaloids may result in substantial neuropathy.It does not appear that continuous infusion is markedlysuperior to bolus administration of vincristine or vin-blastine and is more likely to result in vessicant injury.Other infrequently encountered toxicities are worsen-ing of pre-existing mild leukopenia with agents such asazathioprine; increased hemolysis in patients with orwithout a positive direct antiglobulin test in whom IVanti-D is used; and aseptic meningitis following IVIGinfusion.

There appears to be an important clinical distinc-tion, actually a continuum, between two types of patientswith refractory ITP. One type is the patient who respondsto conventional treatments but cannot discontinue them,while the other type responds poorly, if at all, to anytreatment. The approach to all such patients at our cen-ter is to begin with combination therapy (see Table 6).This is because of the low rate of success of individualagents as well as the slow onset of the platelet increasein patients treated with single agents even if they do re-


spond.15 In general, combination therapy is highly ef-fective at acutely increasing the platelet count, thoughits long-term efficacy is less well defined. Our goal is toacutely increase the platelet count, on an as requiredbasis, until the oral medications result in a stable increasein the platelet count. After several months of a stable (?normal) platelet count on full dosage of the oral medi-cations, e.g. danazol and azathioprine, we attempt totaper down to a single tablet of each or to only one agentto maintain the effect. If this approach does not work, orresults in unacceptable or undesirable toxicity, there areseveral possibilities depending upon many factors. Cy-clophosphamide based therapy, when given as periodicIV infusions, may be effective in refractory ITP. Thistreatment mimics that used in lupus nephritis, and cy-clophosphamide can be administered either as a singleagent or in combination with other agents resemblinglymphoma regimens, e.g. vinca alkaloids and steroidswith or without additional chemotherapeutic agents.16

An advantage appears to be the duration of response inresponders.17 It has been our practice to hold cyclophos-phamide in reserve, despite its apparently lower toxicityin patients with ITP, because of its potential for long-term toxicity including not only sterility and interstitialfibrosis of the bladder but also the possible induction ofsecondary malignancy.

A common approach is for patients to use so-called“alternative” medical therapies. The efficacy of such ap-proaches is uncertain, and data concerning their toxicityis not available. Unsubstantiated claims are abundant.Hopefully more objective information will be accruedwith these treatments over the next several years.

Because of the well-described limitations of con-ventional and alternative therapies, experimental treat-ments may be enthusiastically approached by cliniciansand patients. Table 7 lists a number of treatments thatare unproven but have been used in limited numbers ofpatients or considered for ITP. The primary novel “ex-perimental” therapies to consider at this time arethrombopoietin, anti-CD40 Ligand, rituximab and au-tologous bone marrow transplant. Each of these hasunique advantages and disadvantages. In most cases, theissues are theoretical because there may not be enoughexperience to quantify benefits or risks. The followingis a summary of these newer therapies:

• No one of them is the optimal treatment for everypatient.

• Rituximab has an apparent response rate of up to50%. The advantages appear to be the lack of toxic-ity, beyond first infusion reactions, and the durabil-ity of the responses (> 6 months and counting).

• Anti-CD40 Ligand in current use (IDEC) appearsnot to have the thromboembolic complications thatresulted in another anti-CD40 Ligand (BIOGEN)being withdrawn from trial. Furthermore, some re-sponders have been those unresponsive to all previ-ous treatments who have survived intracranial hem-orrhage.

• Thrombopoietin has not entered clinical trials in ITP.The excitement regarding its use is centered aroundthe possibility that the patients least responsive toother treatments are those in whom platelet produc-tion is particularly poor. However, this remains tobe confirmed.

• Treatment of patients who fail to respond to sple-nectomy is very complex and confusing. The bestsingle review that covers the use of specific treat-ments is that by Dr. McMillian in the Annals of In-ternal Medicine in 1997.15 Subsequent informationhas accumulated slowly and has not provided muchadditional insight. Ideally, these patients should betreated on recognized protocols, whether using com-binations of conventional agents or experimentalones. Since there is no consensus as to the optimalapproach, the outline below of management ismainly directed at what should be either particularlyconsidered or particularly avoided:

• Daily prednisone, even at a very low dose, is prob-lematic and requires careful observance of toxici-ties (bone density, ophthalmologic examinations,blood glucose, blood pressure, etc.).

• The patient population refractory to splenectomywith platelet counts < 20,000/µL is the group of ITPpatients with the highest mortality, even though theadverse outcomes here remain poorly defined.

Table 7. Treatments of refractory immune thrombocytopenicpurpura.

I. Promising treatments:

a. combination chemotherapy: CHOP-like, VCR-IVIG-SM (Table 6)

b. anti-CD40 Ligand

c. rituximab

d. ? thrombopoietin or thrombopoietin like substances

e. autologous bone marrow transplantation

II. Treatments that have an apparent low likelihood of success whenused alone in refractory patients:

a. Interleukin-11 (Neumega)

b. Anti-D

c. Interferon

d. Staph protein A columns

e. Plasmapheresis

Abbreviations: CHOP, cyclophosphamide, adriamycin, vincristine,prednisone; VCR-IVIG-SM, VCR; intravenous gammaglobulin;SM, Solumedrol

Hematology 2001 293

• There is a low rate of response to individual agents,and therefore combination treatments appear supe-rior to the use of single conventional agents. Theoptimal combination to be used remains to be de-fined and almost certainly varies from patient topatient.

• Finally, there is currently no way to define ITP biol-ogy that will allow rational selection of treatmentsaccording to the “biology of the disease.”

III. VON WILLEBRAND FACTOR CLEAVING PROTEASE IN

PATIENTS WITH AND WITHOUT THROMBOTIC

MICROANGIOPATHIES (TTP AND HUS)

Pier M. Mannucci, MD,* and G. Remuzzi, MD

Thrombotic thrombocytopenic purpura (TTP) and thehemolytic uremic syndrome (HUS) are diseases withfeatures in common such as thrombocytopenia,hemolytic anemia and thrombotic occlusions in termi-nal arterioles and capillaries, which form on areas ofdamaged vascular endothelium and are mainly composedof platelets. The triggers of TTP are varied (includingpregnancy, infections, cancer and drugs) but no trigger-ing condition is often apparent. Differentiating clinicalfeatures are the presence of focal neurological symp-toms in TTP and renal impairment in HUS, the latteroften associated with diarrhea and fever in the form thataffects children. In practice these differences are sel-dom clear-cut, and in some instances clinical symptomsmay shift from one syndrome to another. Hence, in indi-vidual cases the differential diagnosis between TTP andHUS based upon the presence or absence of renal orneurological symptoms is often so uncertain that it hasbeen proposed to group the syndromes under the com-prehensive term of thrombotic microangiopathies.

A role for vWF in the pathogenesis of thromboticmicroangiopathies has been recognized for many years.vWF is a large multimeric glycoprotein contained inplasma, platelets and vascular endothelial cells that me-diates the adhesion of platelets to sites of vascular le-sions and, being the carrier protein for coagulation fac-tor VIII, is required for normal factor VIII survival inthe circulation. By using immunohistochemical tech-niques Asada et al showed that in TTP intravascularthrombi and subendothelial hyaline deposits reactedpositively for vWF antigen and negatively for fibrin,1

whereas in HUS fibrin is present and vWF is usuallyabsent; Moake et al2 found abnormally large (ultralarge)multimeric forms of vWF in the plasma of patients with

chronic relapsing TTP. Ultralarge and large multimersare the most biologically active in platelet-vessel wallinteractions and directly induce platelet aggregation inconditions of high shear stress;3 ultralarge multimerswere also found in HUS by Rose et al.4 This abnormal-ity of vWF cannot be taken as a diagnostic marker forthrombotic microangiopathies because, particularly inthe acute phase of these syndromes, ultralarge and largevWF multimers are sometimes lacking,5 perhaps becausethey bind avidly to activated platelets and are clearedfrom plasma.

The mechanism of the relation between ultralargevWF multimers and thrombotic microangiopathies re-mained elusive until Furlan et al6 and Tsai7 independentlydemonstrated that the link is a plasma metalloproteinasethat physiologically cleaves vWF at the peptide bondbetween amino acid residues 842Thr and 843Met in theA2 domain of the subunit.8 The protease degradesultralarge multimers, normally stored in the Weibel-Palade bodies of vascular endothelial cells from whichthey are secreted luminally into plasma and abluminallyinto the subendothelium. Metal ions are needed for fullprotease activity6,7 and transfusion experiments in TTPpatients have shown that the plasma half-life is unusu-ally long (3-4 days).9

In the late 1990s Furlan et al10-12 and Tsai and Lian13

made the intriguing observation that the protease is de-ficient in patients with TTP but measurable in normalamounts in those with HUS. The chronic recurrent formof TTP, characterized by recurrent episodes of thromb-ocytopenia with or without signs of ischemic organ dam-age, is due to the complete deficiency of functionallyactive metalloprotease,10 often inherited as an autoso-mal recessive trait. On the other hand, in the more com-mon acute idiopathic form of TTP, which is usually notfamilial and occurs sporadically, and in ticlopidine-associated TTP protease activity is low because it isinactivated by specific autoantibodies.11-14 According toFurlan and Tsai, HUS, the clinical manifestations ofwhich are not easily distinguishable from TTP unlessthe prototypic renal symptoms are present, can now beclearly distinguished by the presence of normal levelsof metalloprotease activity and by the absence of inhibi-tory antibodies.12,13

These findings are important because they wouldallow these two disorders to be reliably differentiatedfor the first time, with potentially important clinical im-plications. In TTP the therapeutic efficacy of plasma ex-change and its slightly greater efficacy over plasma in-fusion has been demonstrated by a randomized clinicaltrial.15 However, the possibility of distinguishing casesdue to the inherited deficiency of the protease from thosedue to its inactivation by an autoantibody might help tochoose the infusion of plasma only for the inherited cases,* The University of Milan, Via Pace 9, 20122 Milano, Italy


leaving the more costly and cumbersome procedure ofplasma exchange to immunomediated cases. No random-ized clinical trial has yet demonstrated the efficacy ofplasma exchange or infusion in HUS, even though thistreatment is currently recommended and reported to beeffective.16-18 The availability of protease assays mighthelp to establish these recommendations more objec-tively, because cases diagnosed as HUS, particularlythose occurring in adults, might be misdiagnosed casesof TTP, and vice versa.

How specific is the behavior of the protease in throm-botic microangiopathies? Van der Plas et al19 have shownthat in TTP associated with bone marrow transplanta-tion (BMT) plasma levels of the protease are normal.Perhaps BMT-associated TTP and acute sporadic TTPare different disorders with similar clinical manifesta-tions, as also suggested by the fact that there is littleclinical benefit for plasma exchange for BMT-associ-ated TTP. However, the protease was unmeasurable alsoin a typical case of familial HUS reported by te Loo etal.20 In a large series of patients (20 with TTP and 32with HUS) we obtained additional data challenging theviews that low protease means TTP and normal proteasemeans HUS. Although mean protease levels were lowerin TTP than in HUS (p < 0.001), 5 HUS patients hadunmeasurable protease levels, like many patients withTTP (10 of 22).21 Therefore the deficiency of the activ-ity of the protease that cleaves von Willebrand factormay not be specific for TTP.

Whether or not low plasma levels of the proteaseare at least a specific beacon of thrombotic microangio-pathies or also occur in other physiological and patho-logical conditions was not known until recently. Using aprotease assay different from those used by Furlan andTsai, based upon the lower binding affinity to humancollagen type III of protease-cleaved vWF comparedwith uncleaved vWF22 and upon a prolonged incubationof the samples with barium chloride and high concen-trations of urea to avoid the interference of endogenousvWF in the assay, we have evaluated the plasma changes

of the protease in physiological states and in pathologi-cal conditions associated with organ failure or acutephase reactions, conditions often confounding the pic-ture of thrombotic microangiopathies.23 The protease,which ranged between 40% and 170% in healthy indi-viduals, was lower in those aged more than 65 years thanin those of younger ages, was low in newborns but be-came normal within 6 months, and decreased in the lasttwo trimesters of pregnancy compared with the first. Theprotease was also low in cirrhotics and uremics, duringacute inflammation, and fell in the post-operative pe-riod23 (Table 8). Hence, low plasma levels of the pro-tease are not a specific beacon of thromboticmicroangiopathies, because the protease is low in sev-eral other physiological and pathological conditions.

It is not clear whether low plasma levels of proteaseactivity found in so many physiological and pathologi-cal conditions are due to decreased synthesis or increasedturnover or to other mechanisms, although mixing ex-periments have ruled out inactivation by autoantibodiesas a mechanism.23 In severe liver failure decreased syn-thesis is a plausible mechanism, even though there werecases with normal protease levels.23 In uremics the mod-erately reduced protease levels were not related to se-rum creatinine taken as an index of renal function. Inpatients undergoing major abdominal surgery the pro-tease fell consistently in the post-operative period.24 Theprotein seems to be influenced by high levels of estro-gens, because plasma levels decrease progressively fromthe first trimester of pregnancy, albeit moderately.23 Thelow values found in full-term newborns are perhaps an-other phenotypic expression of the immaturity of thenewborn liver, as indicated by the return of the proteaseto normal levels within 6 months of birth.23

In conclusion, we found that low protease valuesmay not be a specific beacon for TTP. There are casesof HUS in which the protease was unmeasurable, al-though this pattern was seen more frequently in TTP. Inboth microangiopathies the protease was sometimes mea-surable, albeit at reduced levels. However, similarly re-

duced values were found in clinical con-ditions other than thrombotic micro-angiopathies, such as liver and renal dis-ease and inflammatory states. Our datachallenge the views that low protease lev-els are highly and consistently specific fora diagnosis of thrombotic micro-angiopathies. The role of the protease inthe pathogenesis of microangiopathies isonly partially elucidated and the useful-ness of protease assays in diagnosis andmanagement is still uncertain.

Table 8. Changes of the von Willebrand factor cleaving protease activity invarious clinical conditions.

TTP

Acute sporadic or chronic relapsing ↓↓↓↓ AGING ↓

Transplantation-associated N NEWBORNS ↓↓↓

Ticlopidine- or Clopidogrel-associated ↓↓↓ PREGNANCY ↓↓

Cancer-associated ↓↓↓ CIRRHOSIS ↓↓↓

HUS UREMIA ↓↓

Acute sporadic N INFLAMMATION ↓↓↓

Chronic relapsing ↓↓↓↓ POST-OPERATIVE PERIOD ↓↓↓

N denotes normal levels

Hematology 2001 295

Addendum: Two articles in the September 15, 2001,issue of Blood report on the purification of the vonWillebrand factor-cleaving protease, and its identifica-tion as a new member of the ADAMTS family of metallo-proteinases. The gene encoding this enzyme is locatedon chromosome 9q34 (Gerritsen et al 2001;98:1654-1661; Fujikawa et al 2001:98:1662-1666). In the sameissue two clinical papers provide additional evidence thatlow levels of protease activity are not specific for TTP(Veyradier et al, 2001;98:1765-1772; Moore et al2001;98:1842-1846).

IV. HEPARIN-INDUCED THROMBOCYTOPENIA

AND THROMBOSIS

Douglas B. Cines, MD*

Heparin continues to be the most common cause of drug-induced, antibody-mediated thrombocytopenia, and man-aging its thrombotic sequelae remains a difficult chal-lenge. Over the past five years, new insights into thepathogenesis of heparin-inducedthrombocytopenia and thrombosis haveemerged, new diagnostic tests havecome into clinical use, and new formsof treatment have been introduced. Al-though these developments provide op-portunities for improved management,they also present new challenges. Thegoal of this session is to critically ex-amine these new developments fromthe perspective of clinical practice Inthis review the abbreviation HIT willbe used to designate patients with as-ymptomatic thrombocytopenia, HITTbe used to refer to those with thrombo-sis and HIT/T will used when referringto patients with either or both presen-tations.

IncidenceAlthough low molecular weight hep-arins (LMWH)1 and other recently de-veloped anticoagulants are less likelythan unfractionated heparin (UH) tocause thrombocytopenia and thrombo-sis, HIT/T has not disappeared. Indeed,the incidence of HIT/T is stable or mayhave increased. This is most likely due

to the greater use of heparin prophylaxis, increased num-bers of aged individuals with chronic vascular diseasereceiving medical care, and the lack of alternatives tohigh dose UH for vascular bypass procedures.

PresentationThe diagnosis must be considered in any patient whodevelops thrombocytopenia, has an otherwise unex-plained fall in the platelet count of at least 30-40%, ordevelops a new thromboembolic complication (TEC) 5-10 days after ongoing heparin exposure2,3 (Table 9). Thediagnosis may be difficult in settings where other causesfor these findings are more common, e.g. in the inten-sive care setting or after coronary artery bypass graft(CABG) surgery.4,5 The onset may be rapid (median time10.5 hrs) in patients exposed to heparin within the pre-ceding three months, whereas the onset is not acceler-ated when the exposure has been more remote.6-8 Theseobservations are congruent with the time course of anti-body disappearance after heparin withdrawal.6

* Dept. of Pathology and Lab Medicine,University of Pennsylvania, 513A Stellar-Chance Laboratories, 422 Curie Blvd.,Philadelphia PA 19104

Table 9. Clinical criteria for estimating likelihood of diagnosis of heparin-inducedthrombocytopenia.

Platelets

Platelet count > 120 x 103/µL before heparin therapy and < 100 x 103/µL duringtherapy (or decrease of at least 40%)

with

Thrombocytopenia resolving within 10 days after discontinuation of heparin +2

or

Platelet count recovered while receiving another kind of heparin 0

Platelet count recovering while receiving the same kind of heparin -1

Thrombocytopenia not resolving after discontinuation of heparin -2

Thrombosis

Arterial thrombosis without predisposing factor +4

Arterial thrombosis with possible predisposing factor +2

Venous thrombosis during high-dose heparin therapy +2

Other thrombosis +1

Rechallenge

Recurrence of thrombocytopenia after reexposure to heparin +6

No recurrence of thrombocytopenia after reexposure to heparin -1

Other causes of thrombocytopenia

Excluded +2

Another drug possibly responsible 0

Cause other than drug likely (e.g., sepsis) -2

Clinical Group

Definite =6

Probable 3-5

Possible 1-2

Unlikely =0

Reprinted from Am J Clin Pathol. 1999;111:700 with permission.


PathogenesisA number of laboratories have recently reported that al-most all patients with HIT/T have circulating antibodiesto complexes between platelet factor 4 (PF4) and hep-arin at the time of diagnosis (9,10 among others). It is cur-rently thought that the conformation of PF4 released fromactivated platelets is altered by UH>LMWH>other gly-cosaminoglycans, rendering it antigenic.11,12,79 Many HIT/T antibodies are directed towards a limited number ofregions in the modified PF4.13,14 Antigen-antibody com-plexes may form in plasma, but it is likely that the reac-tion occurs predominantly on the surface of activatedplatelets, endothelial cells and macrophages,15,16 each ofwhich can bind PF4 or heparin/PF4 (Figure 2; see colorpage 549). Platelets may be activated further when theFc portion of the antibody occupies the signal transduc-ing receptor FcRγIIA,17,18 releasing procoagulant micro-particles19 and causing platelets to aggregate. Tissue fac-tor expression by endothelial cells and macrophages stimu-lated by HIT/T antibody and products released by acti-vated platelets15,16,20-22 may also contribute to the pro-thrombotic tendency.

Although the diagnostic importance and relevanceof anti-PF4/heparin antibodies has generated consider-able interest, it is more difficult to prove that they actu-ally cause HIT/T. Recently, it has been shown thattransgenic mice expressing both human PF4 and humanFcRγIIA injected with a murine anti-human PF4/hep-arin-specific monoclonal antibody develop severe throm-bocytopenia and disseminated thrombosis after exposureto heparin.23 Thus, the two major salient features of HIT/T are reproduced in this model, but whether this sequenceoccurs in vivo is unproven.

Binding of HIT/T antibodies to platelets is optimalwhen PF4 and heparin are present at approximatelyequimolar ratios.24 Heparin and PF4 are both clearedrapidly from the circulation. Therefore, for HIT/T todevelop PF4 must be released from activated platelets ata time when heparin is also present and these complexesmust persist long enough for antibodies to develop. Theabsence of persistent platelet activation may help to ex-plain why HIT/T does not occur with every exposure toheparin, even in patients with pre-existing anti-PF4/he-parin antibodies5,25 or in those with a previous history ofthe disease,6 and why HITT rarely develops more than10 days after heparin exposure. It has been proposedthat high doses of heparin used during bypass surgerymay dissociate PF4/heparin complexes from platelets.24

However, there remains no simple explanation as to whyonly some patients with HIT develop thrombosis. Al-though the isotype and functional properties of the anti-bodies and the genotype of the platelet Fc receptor maycontribute,26 it is likely that the health of the vasculatureand its response to platelet activation and immune in-

jury are critical factors in the prevalence, timing and lo-calization of thrombosis.

Laboratory testingTests in clinical use either rely upon platelet activationor measure binding of antibodies to PF4/heparin. Whenrelating data published by experienced research groupsto the practice setting it is important to appreciate thatfew laboratories provide clinically validated data. Rather,most rely on standards promulgated by others, which inturn may be driven more by statistical considerationsthan by well-defined clinical criteria validated by expe-rienced physicians. Therefore, it is important that thephysician become familiar with the experience of spe-cific laboratory as well as the published operating char-acteristics of these commonly used tests.

The utility of functional assays is limited by theirsensitivity and availability. Failure to adsorb, inactivate,or exclude residual heparin may invalidate results. The14C-serotonin release assay (SRA), still considered the“gold-standard” in diagnosis, has a positive predictivevalue approaching 100% and a negative predictive valueof approximately 20%.1,27,28 Thus, a negative SRA can-not be used to exclude a diagnosis of HIT/T. The hep-arin-induced platelet activation test and other tests basedon platelet aggregation may be comparable to the SRAin experienced hands, but the test requires careful vali-dation with known positive and negative controls to con-trol for differences in donor platelet responsiveness.29 How-ever, some have found that tests based on platelet aggrega-tion are less sensitive than the SRA (reviewed in 80).

Because of these limitations, increased reliance hasbeen placed on measuring anti-PF4/heparin antibodies,generally by ELISA. FDA-approved diagnostic kits inuse employ PF4 bound to heparin or to polyvinyl sul-fonate and measure IgG, IgA and IgM antibodies, pro-vide similar, but not identical, results.30 In one recentstudy based on a comprehensive clinical scoring systemto assess the likelihood of HIT/T (Table 9), the sensitiv-ity of the ELISA was estimated to be 97% and 93% us-ing low or high cut-off values in patients with a “defi-nite” or “probable” diagnosis27 (Figure 3). Similar find-ings have been reported by most groups (10,31 among oth-ers). The sensitivity of the assay is lower in studies us-ing less rigorously defined pre-analytic clinical inclu-sion/exclusion criteria.32,33 The incidence of delayed se-rologic conversion is unknown.34

In this study, the PF4/heparin ELISA was also posi-tive in 23% of patients who received a comparable courseof heparin but did not develop HIT/T.27 Thus, the speci-ficity of the ELISA was 86% and 100%, the positivepredictive value 93% and 100%, and the negative pre-dictive value 95% and 88%, using the low and high cut-offs, respectively. Even this analysis is an oversimplifi-

Hematology 2001 297

cation. The incidence of “false positive” ELISAs rangesfrom < 5% in patients on hemodialysis, to ~10% of medi-cal and surgical patients receiving heparin,35,36 to approxi-mately half of all patients with recent heparin exposureafter cardiac bypass surgery.5,25,37 Thus, 25 times as manypatients develop anti-PF4/heparin antibodies after CABGas develop HIT/T.38

These findings have several implications. First, it isinappropriate to rely solely on a positive ELISA to makea diagnosis of HIT/T in settings such as CABG, whereasa negative assay may help exclude the diagnosis. Sec-ond, research studies and pharmaceutical trials lackingrigorous clinical diagnostic entry criteria and lackingserologic confirmation by more than one technique arelikely to include patients with other causes of thrombo-sis and thrombocytopenia. Third, there is a need to dis-tinguish, if possible, antibodies with “pathogenic” po-tential from the more prevalent seemingly “non-patho-genic” antibodies in order to develop a more clinicallyuseful assay. Some have suggested that measuring IgGantibodies may represent a first step,81 while others ar-gue that IgM and IgA antibodies may be pathogenic aswell.39 It is also unclear whether the epitopes in PF4 rec-ognized by HIT-antibody contribute to outcome.

“Natural History”Defining the natural history of HIT and HIT/T has be-come an important issue for two reasons. First, the FDAhas relied upon outcomes in historical controls to ap-

prove new therapies. Second, physicians must weigh thebenefits versus the risks of using these newer treatmentsin asymptomatic thrombocytopenic patients.

HITTHistorically, mortality rates approach 20%, with ~10%of patients requiring limb amputations and new TECsdeveloping in ~30%. The risk can be lessened by man-aging co-morbid risk factors and by avoiding surrepti-tious heparin exposure and unwarranted platelet trans-fusions. It is also important to note that many deaths aredue to underlying disease, a distinction that is not ad-dressed adequately in many studies. Nevertheless, it isclear that simply discontinuing heparin is insufficient.Thrombectomy, placement of IVC filters, and the use offibrinolytic agents or alternative anticoagulant agents areappropriate in individual cases. In one study, 3 of 7 pa-tients treated with heparin withdrawal alone died com-pared to 2 of 26 patients who were also treated with avariety of anticoagulants (predominantly dextran, ASAand coumadin).40 Newer forms of therapy are discussedbelow.

HITThe natural history of HIT is less clear. Prospective stud-ies performed prior to 1990 indicated that the incidenceof HITT was approximately 30% that of HIT (reviewedin 41). However, in contemporary practice, HIT is moreoften considered when there is a substantial fall in theplatelet count even in the absence of thrombocytopenia,and it is more generally appreciated that HITT can de-velop after unintended exposure to seemingly trivialamounts of heparin given “by protocol,” e.g. heparin-bonded central lines, flushing solutions, hyperalimen-tation solutions. Nevertheless, in some studies 30-50%of patients with HIT treated solely by withdrawing hep-arin ± coumadin developed TEC within 30 days (80%of which occurred in the first week).40,42,43 On the otherhand, in a contemporaneous study of 108 patients withpositive heparin-induced platelet aggregation tests con-ducted between 1991 and 1994, the incidence of death,amputation and new TECs was reduced from 3/7 (42.9%)patients treated by withdrawing heparin ± ASA to 2/25(8%) patients who received dextran, coumadin andASA.40 In another study, morbidity in 100 consecutivepatients with heparin-dependent antibodies was reducedfrom 61% to 7.4% and the mortality from 23% to 1.1%,which was attributed to frequent platelet counts, totalheparin withdrawal following defined diagnostic crite-ria, and conventional oral antiplatelet and anticoagulanttherapy.8 Similar outcomes have been reported by somegroups,44 while others present a far less sanguine pic-ture.45 It is against this variable background that trialsshowing the efficacy of newer agents have been judged.

Figure 3. Levels of antibodies to heparin-platelet factor 4 (H-PF4), according to clinical score in patients with suspectedheparin-induced thrombocytopenia. Dashed lines representcutoff values of 0.5 or 1 absorbance unite (A492).

Reprinted with permission from Am J Clin Pathol 1999;14:700.


New Developments in Management

Danaparoid sodiumThe introduction of danaparoid sodium (Orgaran) ap-proximately 10 years ago represented the first signifi-cant advance in management. Danaparoid is a low mo-lecular weight glycosaminoglycan composed of heparansulfate (84%), dermatan sulfate (12%) and chondroitinsulfate. Danaparoid has an anti-Xa/antithrombin activ-ity ratio of > 22. The anti-Xa and antithrombin half-livesare ~25 hours and 6-9 hours, respectively. The drug iscleared renally. Suggested dosing schedules for variousindications are published.46,47 Monitoring with anti-Xalevels is recommended for patients weighing < 55 or >90 kg, children, those with renal insufficiency, after 2-3days of use, and during cardiac bypass. Anti-Xa levelsbetween 0.5-0.8 U/mL are appropriate to treat thrombo-sis and do not affect the INR. There is no antidote forbleeding.

There is considerable experience with the use ofdanaparoid in HIT/T. Results have been compiled froman “off-label” compassionate use study involving 708treatment episodes in 666 patients, 65% of whom hadHITT or HIT plus a pre-existing thrombosis.48 In 75%of the patients, the diagnosis was confirmed serologi-cally or by inadvertent re-exposure. Diverse dosageschedules were employed. Danaparoid was given for 1week on average, but the duration of treatment variedwidely. Ninety-three percent of 586 evaluable patientshad a successful outcome defined as an increase in theplatelet count, resolution of DIC, absence of new TECsor stabilization or improvement in ischemia. Danaparoidwas considered have caused thrombocytopenia in 12patients, thrombosis in 9, and death from bleeding orthrombosis in 14.48 All-cause mortality at day 7 in treatedpatients was 5.1% versus 23.5% in those for whom thedrug was requested but not given.47 Five to 15% of HIT/T sera crossreact with danaparoid in vitro, but the drughas been used successfully in some antibody positivepatients47,49 and the importance of excluding cross-reactivity is uncertain.

In a subsequent uncontrolled series, platelet countsreturned to normal in 35 of 37 patients treated withdanaparoid.46 A third study involved 42 consecutive pa-tients with HIT/T diagnosed with a carefully constructedscoring system, and serologically confirmed by plateletaggregation or heparin-induced platelet activation testsin 36.50 24/26 patients with HITT had successful out-comes, with 2 deaths from pre-existing thromboses and4 from the underlying disease, but no new TECs. NoTECs developed in the 20 patients with HIT, 5 of whomhad a history of HIT, i.e. “latent” disease. Major hemor-rhage occurred in 3 patients (6.5%), 2 of whom werealso receiving ASA. Of the 42 patients, 6.5% had cross-

reactive antibodies, which bore no relationship to out-come. In a recently completed controlled study of pa-tients with new-onset HITT, complete clinical resolu-tion of thromboembolic events was observed in 56% ofpatients (n = 25) treated with danaparoid and coumadincompared with 14% in those (n = 17) treated with dext-ran 70 and coumadin.51 Treatment was considered mod-erately or highly effective (partial or complete response)in 88% of danaparoid-treated patients compared with47% of the dextran 70-treated patients. No serious ad-verse events were reported. In a recently reported con-trolled trial, patients treated with danaparoid (2500 anti-FXa units i.v. bolus followed by 400 anti-FXa units perhour i.v. for 4 hours, 300 anti-FXa units per hour i.v. for4 hours, followed by 200 anti-FXa units per hour i.v. asmaintenance dose) fared comparably to patients treatedwith lepirudin (see below).82 The use of danaparoid inthe US has been largely supplanted by the introductionof two FDA-approved drugs for use in HIT/T (see be-low), although it still finds use during cardiopulmonarybypass surgery and is still used widely outside of thiscountry.

Recombinant hirudin (lepirudin, Refludin) (r-hirudin)Lepirudin is the first FDA-approved drug for HIT/T.Lepirudin is a recombinant form of hirudin, a ~7 kDapeptide that acts directly on circulating and clot-boundthrombin. The anticoagulant effect lasts ~40 minutes inhealthy individuals.52 Lepirudin is cleared by the kid-ney, necessitating dose adjustments for renal insuffi-ciency. Lepirudin may be given as a slow IV bolus, 0.4mg/kg body wt (up to 110 kg), followed by a continuousinfusion at 0.15 mg/kg body wt (up to 110 kg). Dosesare adjusted to maintain the aPTT between 1.5-2.5 timesbaseline. Anticoagulation can also be monitored usingthe ecarin clotting time (ECT).52 It is recommended thatwhen patients are to be switched to oral anticoagula-tion, the dose of lepirudin be reduced to attain an aPTTratio of 1.5 and discontinued when the INR is > 2.0.

Results from two prospective, historically controlledtrials have been reported. In the first, HIT/T was de-fined by a decrease in platelet count of > 30% or a plate-let count < 100,000/µL,53 but the temporal relationshipwith heparin was unstated. Historical controls consistedof patients with HIT/T identified between 1989 and 1993meeting the same criteria. All patients had the diagnosisconfirmed by heparin-induced platelet activation test-ing and were treated with the “best available care,” whichwas not further defined. One of four treatment regimeswas employed, but the data were pooled for analysis.Patients undergoing CABG were excluded. The groupswere unbalanced with respect to the distribution of sur-gical versus medical patients, with patients in the treat-ment group younger on average but more likely to have

Hematology 2001 299

had multiple TECs. Sixteen patients in the treatment armhad “latent HIT.” The combined endpoints of deaths, newTECs and amputations was reduced from 23% to 9.9%on day 7 and 52.1% to 25.4% on day 35 in the lepirudintreated patients in comparison to the historical controlgroup. The study was not sufficiently powered to dem-onstrate differences in the individual outcomes. Of thestudy patients 32.9% experienced at least 1 bleedingevent and 11 (13.4%) experienced 15 major bleedingevents, but these rates were only slightly higher than incontrols.

The entry criteria in the second study were similarexcept that a > 50% reduction in platelets was required.54

The incidence of death, amputation or new TEC wasreduced from 52.1% in historical controls to 30.9% inlepirudin-treated patients at day 35, a difference that didnot reach statistical significance (p = 0.12), and morebleeding was seen in the lepirudin-treated patients(44.6% vs 27.2%, p < 0.001). It is important to note,however, that the design of both studies created a win-dow of ~1.7 days between enrollment and initiation oftherapy to allow for serologic confirmation. This is theperiod when the risk of thrombosis is greatest. There-fore, it is not surprising that ~45% of TECs developedbefore lepirudin was started and the average combinedadverse rate fell from 5.1% per day in the pre-treatmentperiod to 1.5% per day on therapy.

A subset meta-analysis of patients with HITT fromthese two studies has been reported.55 Of the patients inthe lepirudin arm, 77.3% had HITT compared with 89%of controls, the remainder requiring anticoagulation forpre-existing TECs caused by other conditions. The inci-dence of the primary endpoints of death, amputation andnew TECs was reduced from 52.1% to 22.1% (p = 0.004),due primarily to 17% fewer TECs (10.1% vs 27.1%);there were trends towards a reduction in death (8.9% vs17.6%) and amputations (6.5% vs 10.4%). The combinedevent rate per patient day decreased from 6.1% in thepretreatment period to 1.3% during treatment. There wasmore bleeding requiring transfusion in the lepirudin-treated group, but no fatal bleeding events or intracra-nial hemorrhage. The study was not powered to showbenefit of lepirudin over danaparoid or phenprocoumon.

Antilepirudin antibodies develop commonly.56,57

Some antibodies impair renal clearance of active drugprolonging the aPTT, while others neutralize its activ-ity.58 Re-exposure of antibody positive patients is oftensuccessful, but allergic reactions may occur and frequentdeterminations of the aPTT are indicated. Subcutane-ously administered lepirudin (25 mg BID) has been usedto manage patients with HITT stabilized by intravenoustherapy and to manage patients with HIT.56

ArgatrobanArgatroban is a 509 dalton arginine-based direct throm-bin inhibitor recently approved by the FDA for use inHIT/T. Argatroban inhibits both soluble and clot-boundthrombin. The therapeutic half-life is 46.2±10.2 min.Steady-state activity is attained within 1-2 hours of con-tinuous infusion. Treatment is initiated at a dose of 2 µg/kg/min. The aPTT is checked 2 hours later, and the doseis adjusted (maximum 10 µg/kg/min) to prolong the aPTT1.5-3 times the baseline (maximum 100 sec). The liveris the predominant site of metabolism. The half-life isprolonged 2- to 3-fold in patients with hepatic insuffi-ciency, and the starting dose should be reduced by 75%.There is no antidote for bleeding. Conversion tocoumadin is accomplished by measuring the INR oncombined therapy. When the INR is > 4, argatroban isstopped; the INR measured 4-6 hours later reflects theeffect of coumadin.59

Outcomes in 160 patients with HIT who were treatedwith argatroban have been compared with 147 histori-cal controls, and those in 144 patients with HITT com-pared with 46 historical controls.60 HIT/T was definedby a platelet count < 100/000/µL or a fall of > 50% inthe platelet count on heparin without specific referenceof the temporal relationship to drug onset. Serologic con-firmation was not required. A positive platelet aggrega-tion test or SRA was documented in 50% and 65% ofthe argatroban-treated patients in the HIT and HIT/T arm,respectively, compared with 81% and 35% of the his-torical controls. Eight patients with a history of a posi-tive HIT antibody test (“latent HIT”) were included inthe treatment arm. Historical controls were generallymanaged only with heparin withdrawal and/or oral anti-coagulation. Argatroban therapy was continued untilresolution, institution of alternative anticoagulation, orfor a maximum of 14 days.

Patients with HIT treated with argatroban had a sig-nificant reduction in new TECs at 37 days (8.1% vs22.4%, p < 0.001). The reduction in TECs in patientswith HIT/T was more modest (19.4% vs 34.8%, p =0.044). There was no decrease in death or amputation inHIT or HIT/T, but a significant reduction was seen indeath caused by thrombosis (p = 0.005 and < 0.001, re-spectively). Argatroban was associated with more rapidresolution of thrombocytopenia and no increase in bleed-ing. Diarrhea (11%) and pain (9%) were the major sideeffects. Argatroban has also been used successfully inpatients with HIT/T undergoing percutaneousangioplasty61 and in several patients undergoing extra-corporeal circulation in settings where prolongation ofthe ACT > 400 seconds is not required,62 but it is notapproved for use in coronary bypass surgery.


OthersBivalirudin (Angiomax), a new direct thrombin inhibi-tor with distinct biochemical characteristics, has beenused successfully in patients with recent-onset or latentHIT undergoing percutaneous coronary interventions andin a limited number of patients with HIT/T to treat throm-bosis or for other indications.63 There is anecdotal expe-rience using glycoprotein IIb/IIIa antagonists as adjunc-tive therapy.64,65

Cardiac bypass surgeryCABG is a hazardous procedure in the setting of HIT/T.Ideally, surgery should be delayed for several months topermit anti-PF4/heparin antibodies to dissipate,6 at whichtime bypass can often be performed safely using UH,with or without concurrent aspirin.8,66-69 Indeed, theremust be a clear and compelling reason to proceed withsurgery more expeditiously, because other approachesto anticoagulation require more complicated monitor-ing and are associated with significant rates of seriousbleeding. However, it is not always possible to delaysurgery, and it has been our experience that after an ini-tial fall in titer, the ELISA may remain positive at a lowlevel for many months in some patients, precluding asimple “watch-and-measure-and-wait” approach.

It has been posited that the concentrations of hep-arin used in bypass may dissociate PF4/heparin com-plexes, rendering them less antigenic. Residual heparinis rapidly cleared with protamine. Thus, if heparin ex-posure is strictly limited to the bypass itself by scrupu-lously avoiding the use of heparin-bonded central lines,heparin flushes, and other surreptitious exposures, anti-genic PF4/heparin complexes may remain in the circu-lation for only a short time. A retrospective analysis ofpatients undergoing CABG managed in this way showedthat none developed HIT/T despite pre-existing anti-PF4/heparin antibodies and clear evidence of antibody pro-duction post-procedure.5 Nevertheless, there are no cri-teria to gauge the safety of exposing patients with knownheparin-dependent antibodies in this way, and the riskof complications developing in the perioperative periodwhen platelets have been activated must not be underes-timated. Therefore, alternative forms of anticoagulationare recommended in patients with a recent history ofHIT/T.

The largest published experience is with dana-paroid.48,70-72 Various dosing regimes have been proposedfor the patient and to prime the pump. Current recom-mendations are that anti-Xa levels should be maintainedin the range of 1 U/mL throughout the procedure by in-tra-operative monitoring73-75 with dose adjustments forbleeding or thrombosis.76 After surgery, danaparoid isdiscontinued until hemostasis is attained and then re-sumed to achieve anti-Xa levels of 0.15-0.8 U/mL. Not-

withstanding adherence to these guidelines, the risk ofbleeding during CABG remains considerable.

Experience with lepirudin has been limited by thefact that the drug cannot be reliably monitored with theACT or aPTT at concentrations required for bypass (> 4µg/mL), although some patients have been managed suc-cessfully in this way.77 A number of protocols have beenemployed. One approach is to give a bolus of additionallepirudin to prime the pump (0.2 mg/kg) and begin acontinuous infusion (0.5 mg/min) 5 minutes prior to by-pass. Dosing is modified at 5 minute intervals so as tomaintain the ECT at ~400 seconds monitored online withthe Thrombolytic Assessment System or monitoredmanually (albeit at less frequent intervals) based on anin-house standard curve. The initial dose is modified ifthe patient is already on lepirudin.78 Therapy must bebased on clinical observation of hemostasis as much ason laboratory measurement. Adjustments for obesity andimpaired renal function may be required.

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33. Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preec-lampsia is associated with abnormal expression of adhesionmolecules by invasive cytotrophoblasts. J Clin Invest.1993;91:950.

34. Graham CH, McCrae KR. Expression of gelatinase andplasminogen activator activity by trophoblast cells isolatedfrom the placentae of normal and preeclamptic women. Am. J.Obstet. Gynecol. 1996;175:555.

35. Redman CWG. Current topic: pre-eclampsia and the placenta.Placenta. 1991;12:301.

36. Schjetlein R, Haugen G, Wisloff F. Markers of intravascular

coagulation and fibrinolysis in preeclampsia: association withintrauterine growth retardation. Acta Obstet Gynecol Scand.1997;76:541.

37. Sibai BM. The HELLP syndrome (hemolysis, elevated liverenzymes, and low platelets): Much ado about nothing? Am JObstet Gynecol. 1990;162:311.

38. Burrows RF, Andrew M. Neonatal thrombocytopenia in thehypertensive disorders of pregnancy. Obstet Gynecol.1990;76:234.

39. Baschat AA, Gembruch U, Reiss I, Gortner L, Weiner CP,Harman CR. Absent umbilical artery end-diastolic velocity ingrowth-restricted fetuses: A risk factor for neonatal thrombocy-topenia. Obstet Gynecol. 2000;96:162.

40. Roberts IA, Murray NA. Neonatal thrombocytopenia: newinsights into pathogenesis and implications for fetal manage-ment. Curr Topics Micro Immunol. 2001;13:16.

41. Caritis S, Sibai B, Hauth J, et al, NICHHD Network ofMaternal-Fetal Medicine Units. Low dose aspirin to preventpreeclampsia in women at high risk. N Engl J Med.1998;338:701.

42. Collaborative Low-dose Aspirin Study in Pregnancy Collabora-tive Group: CLASP: A randomised trial of low-dose aspirin forthe prevention and treatment of pre-eclampsia among 9364pregnant women. Lancet. 1994;343:619.

43. Martin JN Jr, Files JC, Blake PG, Perry KG Jr, Morrison JC,Norman PH. Postpartum plasma exchange for atypicalpreeclampsia-eclampsia as HELLP (hemolysis, elevated liverenzymes and low platelets) syndrome. Am J Obstet Gynecol.1995;172:1107.

44. Martin JN, Perry KG, Blake PG, May WA, Moore A, RobinetteL. Better maternal outcomes are achieved with dexamethasonetherapy for postpartum HELLP (hemolysis, elevated liverenzymes, and thrombocytopenia) syndrome. Am J ObstetGynecol. 1997;177:1011.

45. Esplin MS, Branch DW. Diagnosis and management ofthrombotic microangiopathies during pregnancy. Clin ObstetGynecol. 1999;42:360.

46. Furlan M, Robles R, Galbusera M, et al. von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura andthe hemolytic-uremic syndrome. N Engl J Med. 1998;339:1578.

47. Tsai H-M, Lian ECY. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenicpurpura. N Engl J Med. 1998;339:1585.

48. Brostrom S, Bergmann OJ. Thrombotic thrombocytopenicpurpura: a difficult differential diagnosis in pregnancy. ActaObstet Gynecol Scand. 2000;79:84.

49. Ezra Y, Rose M, Eldor A. Therapy and prevention of thromboticthrombocytopenic purpura during pregnancy: A clinical studyof 16 pregnancies. Am J Hematol. 1996;51:1.

50. Mokrzycki MH, Rickles FR, Kaplan AA, Kohn OF. Thromboticthrombocytopenic purpura in pregnancy: Successful treatmentwith plasma exchange. Blood Purif. 1995;13:271.

51. Dashe JS, Ramin SM, Cunningham FG. The long termconsequences of thrombotic microangiopathy (thromboticthrombocytopenic purpura and hemolytic uremic syndrome) inpregnancy. Obstet Gynecol. 1998;91:662.

52. Ibdah JA, Yang Z, Bennett MJ: Liver disease in pregnancy andfetal fatty acid oxidation defects. Mol Gen Metab. 2000;71:182.

53. Ibdah JA, Bennett MJ, Rinaldo P, et al. A fetal fatty-acidoxidation disorder as a cause of liver disease in pregnantwomen. N Engl J Med. 1999;340:1723.

54. Vigil-De Gracia P. Acute fatty liver and HELLP syndrome: twodistinct pregnancy disorders. Int J Gynaecol Obstet.2001;73:215.

55. Bacq Y: Acute fatty liver of pregnancy. Semin Perinatol.1998;22:134.

56. Budman DR, Steinberg AD. Hematologic aspects of systemic


lupus erythematosus. Ann Int Med. 1977;86:220.57. Wilson WA, Gharavi AE, Koike T, et al. International consensus

statement on preliminary classification criteria for definiteantiphospholipid syndrome. Arth Rheum. 1999;42:1309.

58. Campbell D, Parr MJA, Shutt LE. Unrecognized “crack”cocaine abuse in pregnancy. Br J Anesth. 1996;77:553.

59. Chiurazzi F, Villa MR, Rotoli B. Transplacental transmission ofEDTA-dependent pseudothrombocytopenia. Haematologica.1999;84:664.

II. Novel Approaches to Management ofImmune Thrombocytopenic Purpura:Results of Recent Trials

1. Shulman NR, Weinrach RS, Libre EP, Andrews HL. The role ofthe reticuloendothelial system in the pathogenesis of idiopathicthrombocytopenic purpura. Trans Assoc Am Phys.1965;78:374-90.

2. Roark JH, Bussel JB, Cines DB, Siegel DL. Genetic analysis ofautoantibodies in ITP reveals evidence of clinical expansionand somatic mutation. Submitted manuscript.

3. Gasbarrini A, Francechi F, Tartaglione R, Landolfi R, Pola P,Gasbarrini G. Regression of autoimmune thrombocytopeniaafter eradication of Helicobacter pylori. Lancet. 1998;352: 878.

4. Emilia G, Longo G, Luppi M, et al. Helicobacter Pylorieradication can induce platelet recovery in idiopathic thromb-ocytopenic purpura. Blood. 2001;97:812-814.

5. Newman GC, Novoa MV, Fodero EM, Lesser ML, WoloskiBMR, Bussel JB. A dose of 75 ug/kg of i.v. anti-D increases theplatelet count more rapidly and for a longer period of time than50 ug/kg in adults with immune thrombocytopenic purpura. BrJ Hem. 2001;112: 1076-1078.

6. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatoryactivity of IVIG mediated through the inhibitory Fc receptor.Science. 2001;291:484-6.

7. McFarland JG, Aster RH, Bussel JB, Gianopoulos JG, DebresRS, Newman PJ. Prenatal diagnosis of neonatal alloimmunethrombocytopenia using allele-specific oligonucleotide probes.Blood. 1991;77:2276-2282.

8. Scaradavou A, Woo B, Woloski BMR, et al. Intravenous anti-Dtreatment of immune thrombocytopenic purpura: experience in272 patients. Blood. 1997;89:2689-2700.

9. Clarkson SB, Bussel JB, Kimberly RP, Valinsky J, NachmanRL, Unkeless JC. Treatment of refractory immune thrombocy-topenia purpura with an anti-Fc receptor antibody. New Eng JMedicine. 1986;314:1236-1239.

10. Bussel JB, Kimberly RP, Clarkson SB, Nachman RL, ValinskyJ, Unkeless JC. Infusion of a monoclonal anti-FcR III inpatients with refractory ITP. Neo-Adjuvant Chemotherapy.1990; 169:883-890.

11. Gaines AR. Acute onset hemoglobinemia and/or hemoglobin-uria and sequelae following Rho(D) immune globulin intrave-nous administration in immune thrombocytopenic purpurapatients. Blood. 95:2523-2529.

12. Bussel JB, Graziano JN, Kimberly RP, Pahwa S, Aledort L. IVanti-D treatment of immune thrombocytopenic purpura:Analysis of efficacy, toxicity, and mechanism of effect. Blood.1991;77:1884-1893.

13. Portielje JEA, Westendorp RGJ, Kluin-Nelemans HC, Brand A.Morbidity and mortality in adults with idiopathic thrombocy-topenic purpura. Blood. 2001;97:2549-2554.

14. Reiner A, Gernsheimer T, Slichter SJ. Pulse cyclophosphamidetherapy for refractory autoimmune thrombocytopenic purpura.Blood. 1995;85:351-358.

15. McMillan R. Therapy for adults with refractory chronicimmune thrombocytopenic purpura. Ann Intern Med.1997;126:307-314.

16. Figueroa M, Gehlsen J, Hammond D, et al. Combinationchemotherapy in refractory immune thrombocytopenic purpura.N Engl J Med. 1993;328:1226-1229.

17. McMillan R. Long-term outcomes after treatment for refractoryimmune thrombocytopenic purpura. N Engl J Med.2001;344:1402-1403

III. Von Willebrand Factor Cleaving Proteasein Patients with and without ThromboticMicroangiopathies (TTP and HUS)

1. Asada Y, Sumiyoshi A, Hayashi T, Suzumiya T, Kaketani K.Immunohistochemistry of vascular lesions in thromboticthrombocytopenic purpura, with special reference to factor VIIIrelated antigen. Thromb Res. 1985;38:469-479.

2. Moake JL, Rudy CK, Troll JH, et al. Unusually large plasmafactor VIII: von Willebrand factor multimers in chronicrelapsing thrombotic thrombocytopenic purpura. N Engl J Med.1982;307:1432-1435.

3. Moake JL, Turner NA, Stathopoulos NA, Nolasco LH, HellumsJD. Involvement of large plasma von Willebrand factor (VWF)multimers and unusually large VWF forms derived fromendothelial cells in shear stress-induced platelet aggregation. JClin Invest. 1986;78:1456-1461.

4. Rose PE, Enayat SM, Sunderland R, Short PE, Williams CE,Hill FG. Abnormalities of factor VIII related protein multimersin the haemolytic uraemic syndrome. Arch Dis Child.1984;59:1135-1140.

5. Mannucci PM, Lombardi M, Lattuada A, et al. Enhancedproteolysis of plasma von Willebrand factor in thromboticthrombocytopenic purpura and the hemolytic uremic syndrome.Blood. 1989;74:978-983.

6. Furlan M, Robles R, Lämmle BL. Partial purification andcharacterization of a protease from human plasma cleaving vonWillebrand factor to fragments produced by in vivo proteolysis.Blood. 1996,87:4223-4234.

7. Tsai HM. Physiologic cleavage of von Willebrand factor by aplasma protease is dependent on its conformation and requirescalcium ion. Blood. 1996;87:4235-4244.

8. Dent JA, Berkowitz SD, Ware J, Kasper CK, Ruggeri ZM.Identification of cleavage site directing the immunochemicaldetection of molecular abnormalities in type IIA vonWillebrand disease. Proc Natl Acad Sci USA. 1990;87:6306-6310.

9. Furlan M, Robles R, Morselli B, Sandoz P, Lämmle B.Recovery and half-life of von Willebrand factor-cleavingprotease after plasma therapy in patients with thromboticthrombocytopenic purpura. Thromb Haemost. 1999;81:8-13.

10. Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P,Lämmle B. Deficient activity of von Willebrand factor-cleavingprotease in chronic relapsing thrombotic thrombocytopenicpurpura. Blood. 1997;89:3097-3103.

11. Furlan M, Robles R, Solenthaler M, Lämmle B. Acquireddeficiency of von Willebrand factor-cleaving protease in apatient with thrombotic thrombocytopenic purpura. Blood.1998;91:2839-2846.

12. Furlan M, Robles R, Galbusera M, et al. Von Willebrand factor–cleaving protease in thrombotic thrombocytopenic purpura andthe hemolytic-uremic syndrome. N Engl J Med.1998;339:1578-1584

13. Tsai H-M, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenicpurpura. N Engl J Med. 1998;339:1585-1594.

14. Rock GA, Shumak KH, Buskard NA, et al. Comparison ofplasma exchange with plasma infusion in the treatment ofthrombotic thrombocytopenic purpura. Canadian ApheresisStudy Group. N Engl J Med. 1991;325:393-397.

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15. Tsai H-M, Rice L, Sarode R, Chow T, Moake JL. Antibodyinhibitors to von Willebrand factor metalloproteinase andincreased binding of von Willebrand factor to platelets inticlopidine-associated thrombotic thrombocytopenic purpura.Ann Int Med 2000; 132: 794-799.

16. Dundas S, Murphy J, Soutar RL, Jones GA, Hutchinson SJ,Todd WT. Effectiveness of therapeutic plasma exchange in the1996 Lanarkshire Escherichia coli 0157:H7 outbreak. Lancet.1999;354:1327-1330.

17. Rock G, Shumak K, Kelton J, et al. Thrombotic thrombocy-topenic purpura: outcome in 24 patients with renal impairmenttreated with plasma exchange. Transfusion. 1992;32:710-714.

18. George JN. How I treat patients with thrombotic thrombocy-topenic purpura-hemolytic uremic syndrome. Blood.2000;96:1223-1229.

19. van der Plas RM, Schiphorst ME, Huizinga EG, et al. vonWillebrand factor proteolysis is deficient in classic, but not inbone marrow transplantation associated thrombotic thrombocy-topenic purpura. Blood. 1999;93:3798-3802.

20. te Loo DMWM, Leutchenko E, Furlan M, Rosendaal GPM, vanden Henvel LPWJ. Autosomal recessive inheritance of vonWillebrand factor-cleaving proteasse. Pediatr Nephrol.2000;14:762-765.

21. Mannucci PM, Galbusera M, Noris M, Canciani MT, RemuzziG. Does deficiency of VWF-cleaving protease activitydiscriminate thrombotic thrombocytopenic purpura (TTP) fromhemolytic uremic syndrome (HUS)? Abstract presented at theAnnual Meeting of the American Society of Nephrology andpublished in abstract form in J Am Soc Nephrol, 2001.

22. Gerritsen H, Turecek P, Schwarz HP, Lämmle B, Furlan M.Assay of von Willebrand factor (VWF)-cleaving protease basedon decreased collagen binding affinity of degraded VWF: a toolfor the diagnosis of thrombotic thrombocytopenic purpura(TTP). Thromb Haemost. 1999;82:1386-1389.

23. Mannucci PM, Canciani MT, Forza I, Lussana F, Lattuada A,Rossi E. Changes in health and disease of the metalloproteasethat cleaves von Willebrand factor. Blood 2001; 98: 2728-2733.

IV. Heparin-Induced Thrombocytopeniaand Thrombosis

1. Warkentin TE, Levine MN, Hirsh J, et al. Heparin-inducedthrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med.1995;332:1330-1335.

2. Calaitges JG, Liem TK, Spadone D, Nichols K, Silver D: Therole of heparin-associated antiplatelet antibodies in theoutcome of arterial reconstruction. J Vasc Surg. 1999;29:779-786.

3. George JN, Alving B, Ballem P. Platelets. In McArthur JR,Benz EJ, eds. Hematology 1994: The Educational Program ofthe American Society of Hematology. 1994;66-68.

4. Baughman RP, Lower EE, Flessa HC, Tollerudd J. Thrombocy-topenia in the intensive care unit. Chest. 1993;104:1243-1247.

5. Bauer TL, Arepally G, Konkle BA, et al. Prevalence of heparin-associated antibodies without thrombosis in patients undergo-ing cardiopulmonary bypass. Circulation. 1997;95:1242-1246.

6. Warkentin TE, Kelton JG. Temporal aspects of heparin-inducedthrombocytopenia. N Engl J Med. 2001;344:1286-1292.

7. Kapsch D, Adelstein E, Rhodes G, Silver D. Heparin-inducedthrombocytopenia, thrombosis and hemorrhage. Surgery.1979;86:148-155.

8. Almeida JI, Coats R, Liem TK, Silver D. Reduced morbidityand mortality rates of heparin-induced thrombocytopeniasyndrome. J Vasc Surg. 1998;27:309-316.

9. Amiral J, Bridey F, Wolf M, et al. Antibodies to macromolecu-lar platelet factor 4-heparin complexes in heparin-induced

thrombocytopenia: a study of 44 cases. Thromb Haemost.1995;73:21-28.

10. Greinacher A, Amiral J, Dummel V, Vissac A, Kiefel V,Mueller-Eckhardt C. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of plateletaggregation test, heparin-induced platelet activation tests, andplatelet factor 4/heparin enzyme-linked immunosorbent assay.Transfusion. 1994;34:381-385.

11. Arepally G, Cines DB. The pathogenesis and clinical relevanceof heparin-induced autoantibodies. In Shoenfeld Y, ed. TheDecade of Autoimmunity. Amsterdam; Elsevier Science:1999;157-168.

12. Newman P, Chong B. Further characterization of antibody andantigen in heparin-induced thrombocytopenia. Br J Haematol.1999;107:303-309.

13. Suh J-S, Aster RH, Visentin GP. Antibodies from patients withheparin-induced thrombocytopenia/thrombosis recognizedifferent epitopes on heparin:platelet factor 4. Blood.1998;91:916-922.

14. Ziporen L, Li ZQ, Park KS, et al. Defining an antigenic epitopeon platelet factor 4 (PF4) associated with heparin-inducedthrombocytopenia (HIT). Blood. 1998;92:3250-3259.

15. Pouplard C, Iochmann S, Renard O, Colombat P, Amiral J,Gruel Y. Induction of monocyte tissue factor expression byantibodies to heparin-platelet factor4-complexes developed inheparin-induced thrombocytopenia. Blood. 2001;97:3300-3302.

16. Arepally GM, Mayer I. Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to expresstissue factor and secrete interleukin-8. Blood. 2001;98:1252-1254.

17. Kelton JG, Sheridan D, Santos A, et al. Heparin-inducedthrombocytopenia: laboratory studies. Blood. 1988;72:925-30.

18. Taylor SM, Reilly MP, Schreiber AD, Chien P, Turkosh JR,McKenzie SE. Thrombosis and shock induced by activatingantiplatelet antibodies in human FcRγIIA transgenic mice: theinterplay among antibody, spleen, and Fc receptor. Blood.2000;96:4524-4260.

19. Hughes M, Hayward PM, Warkentin TE, Horsewood P,Chorneyko KA, Kelton JG. Morphological analysis ofmicroparticle generation in heparin-induced thrombocytopenia.Blood. 2000;96:188-194.

20. Visentin GP, Ford SE, Scott JP, Aster RH. Antibodies frompatients with heparin induced thrombocytopenia/thrombosis arespecific for platelet factor 4 complexed with heparin or boundto endothelial cells. J Clin Invest. 1994;93:81-88.

21. Cines DB, Tomaski A, Tannenbaum S. Immune endothelial cellinjury in heparin-associated thrombocytopenia. N Engl J Med.1987;316:581-589.

22. Hebert J-M, Savi P, Jeske WP, Walenga JM. Effect ofSR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of humanendothelial cells. Thromb Haemost. 1998;80:326-331.

23. Reilly MP, Taylor SM, Hartman NK, et al. Heparin-inducedthrombocytopenia/thrombosis in a transgenic mouse modelrequires human platelet factor 4 and platelet activation throughFcγRIIa. (abstract) Blood. In press.

24. Horne III MK, Hutchinson KJ. Simultaneous binding of heparinand platelet factor-4 to platelets: Further insights into themechanism of heparin-induced thrombocytopenia. Am JHematol. 1998;58:24-30.

25. Visentin GP, Malik M, Cyganiak KA, Aster RH. Patients treatedwith unfractionated heparin during open heart surgery are athigh risk to form antibodies reactive with heparin:platelet 4complexes. J Lab Clin Med. 1996;128:376-383.

26. Carlsson LE, Santoso S, Baurichter G, et al. Heparin-inducedthrombocytopenia: New insights into the impact of the FcγRIIa-R-H131 polymorphism. Blood. 1998;92:1526-1531.


27. Pouplard C, Amiral J, Borg J-Y, Laporte-Simitsidis S,Delhousee B, Gruel Y. Decision analysis for use of platelet-aggregation tests, carbon 14C-serotonin release assay, andheparin-platelet factor 4 enzyme-linked immunosorbent assayfor diagnosis of heparin-induced thrombocytopenia. Am J ClinPathol. 1999;111:700-706.

28. Chong BH, Burgess J, Ismail F. The clinical usefulness of theplatelet aggregation test for the diagnosis of heparin-inducedthrombocytopenia. Thromb Haemost. 1993;69:344-350.

29. Eichler P, Budde U, Haas S, et al. First workshop for detectionof heparin-induced antibodies: Validation of the heparin-induced platelet activation test (HIPA) in comparison with aPF4/heparin ELISA. Thromb Haemost. 1999;81:625-629.

30. Izban KF, Lietz HW, Hoppensteadt DA, et al. Comparison oftwo PF4/hepain ELISA assays for the laboratory diagnosis ofheparin-induced thrombocytopenia. Semin Hemostas Thromb.1999;25(Suppl. 1):51-56.

31. Suh J-S, Malik MI, Aster RH, Visentin GP. Characterization ofthe humoral immune response in heparin-induced thrombocy-topenia. Am J Hematol. 1997;54:196-201.

32. Walenga JM, Jeske WP, Wood JJ, Ahmad S, Lewis BE, BakhosM. Laboratory tests for heparin-induced thrombocytopenia: amulticenter study. Semin Hematol 1999;36 (Suppl. 1):22-28.

33. Look KA, Sahud M, Flaherty S, Zehnder JL. Heparin-inducedplatelet aggregation vs. platelet factor 4 enzyme-linkedimmunosorbent assay in the diagnosis of heparin-inducedthrombocytopenia-thrombosis. Am J Clin Pathol. 1997;108:78-82.

34. Harenburg J, Wang L, Hoffman U, Huhle G, Feuring M.Improved laboratory confirmation of heparin-inducedthrombocytopenia type II. Am J Clin Pathol. 2001;111:432-438.

35. Arepally G, Reynolds C, Tomaski A, et al. Comparison of PF4/ELISA with the 14C-serotonin release assay in the diagnosis ofheparin associated thrombocytopenia/thrombosis. Am J ClinPathol. 1995;104:648-654.

36. Amiral J, Peynaud-Debayle E, Wolf M, Bridey F, Vissac A-M,Meyer D. Generation of antibodies to heparin-PF4 complexeswithout thrombocytopenia in patients treated withunfractionated or low-molecular-weight heparin. Am JHematol. 1996;51:90-95.

37. Warketin TE, Sheppard JI, Horsewood P, Simpson PJ, MooreJC, Kelton JG. Impact of the patient population on the risk forheparin-induced thrombocytopenia. Blood. 2000;96:1703-1708.

38. Singer RL, Mannion JD, Bauer TL, Armenti FR, Edie RN.Complications from heparin-induced thrombocytopenia inpatients undergoing cardiopulmonary bypass. Chest.1993;1436:1440.

39. Amiral J, Wolf M, Fischer A-M, Boyer-Neumann C, Vissac A-M, Meyer D. Pathogenicity of IgA and/or IgM antibodies toheparin-PF4 complexes in patients with heparin-inducedthrombocytopenia. Br J Haematol. 1996;92:954-959.

40. Nand S, Wong W, Yuen B, Yetter A, Schmulbach E, Fisher SG.Heparin-induced thrombocytopenia with thrombosis: Incidence,analysis of risk factors, and clinical outcomes in 108 consecu-tive patients treated at a single institution. Am J Hematol.1997;56:12-16.

41. Warkentin TE. Clinical presentation of heparin-inducedthrombocytopenia. Semin Hematol. 1998;35 (Suppl. 5):9-16.

42. Warkentin TE, Kelton JG. A 14-year study of heparin-inducedthrombocytopenia. Am J Med. 1996;101:502-507.

43. Wallis DE, Quintos R, Wehrmacher W, Messmore H. Safety ofwarfarin anticoagulation in patients with heparin-inducedthrombocytopenia. Chest. 1999;116:1333-1338.

44. Pouplard C, May M-A, Iochmann S, et al. Antibodies to plateletfactor 4-heparin after cardiopulmonary bypass in patientsanticoagulated with unfractionated heparin or a low-molecular-weight heparin. Clinical implications for heparin-induced

thrombocytopenia. Circulation. 1999;99:2530-2536.45. Wallis DE, Workman DL, Lewis BE, Steen L, Pifarre R, Moran

JF. Failure of early heparin cessation as treatment for heparin-induced thrombocytopenia. Am J Med. 1999;106:629-635.

46. Greinacher A, Alban S. Heparinoids as alternative forparenteral anticoagulation therapy in patients with heparin-induced thrombocytopenia. Hamostaseologie. 1996;16:41-49.

47. Magnani HN. Orgaran (danaparoid sodium) use in thesyndrome of heparin-induced thrombocytopenia. Platelets.1997;8:74-81.

48. Magnani HN. Heparin-induced thrombocytopenia (HIT): Anoverview of 230 patients treated with Orgaran (Org 10172).Thromb Haemost. 1993;70:554-561.

49. Warkentin TE. Danaparoid (Orgaran) for the treatment ofheparin-induced thrombocytopenia (HIT) and thrombosis:effects on in vivo thrombin and cross-linked fibrin generation,and evaluation of the clinical significance of in vitro cross-reactivity (XR) or danaparoid for HIT-IgG. (abstract) Blood.1996;88 (Suppl. 1):626a.

50. Tardy-Poncet B, Tardy B, Reynaud J, et al. Efficacy and safetyof danaparoid sodium (ORG10172) in critically ill patients withheparin-associated thrombocytopenia. Chest. 1999;115:1616-1620.

51. Chong BH, Gallus AS, Cade JF, et al. Prospective randomizedopen-label comparison of danaparoid with dextran 70 in thetreatment of heparin-induced thrombocytopenia with thrombo-sis. A clinical outcomes study. Thromb Haemost. In Press.

52. Potzsch B, Hund S, Madlener K, Unkrig C, Mueller-BerghausG. Monitoring of recombinant hirudin: Assessment of a plasma-based ecarin clotting time assay. Thromb Res. 1997;86:373-383, 1997

53. Greinacher A, Volpel H, Janssens U, et al. Recombinant hirudin(Lepirudin) provides safe and effective anticoagulation inpatients with heparin-induced thrombocytopenia. A prospectivestudy. Circulation. 1999;99:73-80.

54. Greinacher A, Jansenns U, Berg G, et al. Lepirudin (recombi-nant hirudin) for parenteral anticoagulation in patients withheparin-induced thrombocytopenia. Circulation. 1999;100:587-593.

55. Greinacher A, Eichler P, Lubenow N, Kwasny H, Luz M.Heparin-induced thrombocytopenia with thromboemboliccomplications: meta-analysis of 2 prospective trials to asses thevalue of parenteral treatment with lepirudin and its therapeuticaPTT range. Blood. 2000;96:846-851.

56. Huhle G, Hoffman U, Hoffman I, Liebe V, Harenberg JF, HeeneDL. A new therapeutic option by subcutaneous recombinanthirudin in patients with heparin-induced thrombocytopenia typeII: A pilot study. Thromb Res. 2000;99:325-334.

57. Eichler P, Friesen H-J, Lubenow N, Jaeger B, Greinacher A.Antihirudin antibodies in patients with heparin-inducedthrombocytopenia treated with lepirudin: incidence, effects onaPTT, and clinical relevance. Blood. 2000;96:2373-2378.

58. Huhle G, Liebe V, Hudek R, Heene DL. Anti-R-hirudinantibodies reveal clinical relevance through direction functionalinactivation of R-hirudin or prolongation of R-hirudin’s plasmahalflife. Thromb Haemost. 2001;85:936-938.

59. Sheth SB, DiCicco RA, Hursting MJ, Jorkasky DK. Interpretingthe international normalized ratio (INR) in individualsreceiving argatroban and warfarin. Thromb Haemost. 85:435-440, 2001

60. Lewis BE, Wallis DE, Berkowitz MD, et al. Argatrobananticoagulant therapy in patients with heparin-inducedthrombocyotpenia. Circulation. 2001;103:1838-1843.

61. Matthai WH Jr. Use of argatroban during percutaneouscoronary interventions in patients with heparin-inducedthrombocytopenia. Semin Hemost Thromb. 1999;25 (Suppl1):57-60.

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62. Kawada T, Kitagawa H, Hoson M, Okada Y, Shiomura J.Clinical applications of argatroban as an alternative anticoagu-lant for extracorporeal circulation. Hematol/Oncol Clin N Am.2000;14:445-457.

63. Campbell KR, Mahaffey KW, Lewis BE, et al. Bivalirudin inpatients with heparin-induced thrombocytopenia undergoingpercutaneous coronary intervention. J Invasive Cardiol.2000;12 (Suppl. F):14F-19F.

64. Koster A, Kukucka M, Bach F, et al. Anticoagulation duringcardiopulmonary bypass in patients with heparin-inducedthrombocytopenia type II and renal impairment using heparinand the platelet glycoprotein IIb-IIIa antagonist tirofiban.Anesthesiol. 2001;94:245-251.

65. Walenga JM, Jeske WP, Wallis DE, et al. Clinical experiencewith combined treatment of thrombin inhibitors and GPIIb/IIIainhibitors in patients with HIT. Semin Thromb Hemostas.1999;25(Suppl 1):77-81.

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