Catherine P. M. Hayward

The Pathogenesis of Venous Limb Gangrene Associated with Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia is one of the most important immunologic drug reactions that physicians must manage. It is caused by a platelet-activating, heparin-dependent IgG antibody and is an important cause of paradoxical arterial and venous thrombotic complications [1-5]. Acute arterial occlusion is an important cause of limb loss in some patients with heparin-induced thrombocytopenia [1, 6-11]. After we observed a patient with heparin-induced thrombocytopenia and deep venous thrombosis develop distal ischemic limb necrosis in the absence of arterial occlusion (venous limb gangrene), we retrospectively reviewed all of our patients with serologically confirmed heparin-induced thrombocytopenia to investigate this problem. We identified eight patients with venous limb gangrene. Each of these patients had a consistent course of events: heparin-induced thrombocytopenia and deep venous thrombosis followed by venous limb gangrene that developed when heparin therapy was discontinued and warfarin therapy was either initiated or continued. Warfarin is a commonly used oral anticoagulant that reduces functional levels of four vitamin K-dependent procoagulant factors: II, VII, IX, and X [12]. Warfarin also reduces levels of two vitamin K-dependent anticoagulant factors: protein C and protein S [12]. Warfarin anticoagulation can cause paradoxical thrombotic events, particularly central skin necrosis in patients with congenital heterozygous protein C deficiency [13-16]. It has been postulated that warfarin-induced skin necrosis is caused by a transient prothrombotic state that results from a faster reduction in the level of the major natural anticoagulant factor (protein C; half-life, 6 hours) than in the level of the major procoagulant factor (prothrombin; half-life, 72 hours). In this report, we describe a novel syndrome in which patients with acute heparin-induced thrombocytopenia and deep venous thrombosis who are treated with warfarin seem to be at risk for developing venous limb gangrene. Laboratory studies suggest that this syndrome is related to a warfarin-induced failure of the protein C anticoagulant pathway to regulate the increased thrombin generation that occurs in patients with heparin-induced thrombocytopenia. Methods Case-Control Studies In the first of two casecontrol studies, we compared patients with heparin-induced thrombocytopenia who developed venous limb gangrene with patients with heparin-induced thrombocytopenia who developed acute limb arterial thrombosis. In the second casecontrol study, we compared patients who developed venous limb gangrene with patients who did not develop venous limb gangrene during warfarin treatment of heparin-induced thrombocytopenia and deep venous thrombosis. Patients Case-patients and controls were identified from review of the clinical and laboratory records of all 158 patients treated for heparin-induced thrombocytopenia (platelet count nadir, <150 109/L) in one of the five Hamilton, Ontario, hospitals during a 15-year period that ended on 31 December 1994. The diagnosis of heparin-induced thrombocytopenia was confirmed serologically in all patients [17, 18]; 133 patients had been included in previous studies [3, 19]. Four patient summaries are included in this report; three of the four patients had venous limb gangrene (patients 1, 2, and 3), and one patient had severe venous limb ischemia that resolved on reversal of warfarin treatment with vitamin K and plasmapheresis (patient 4). Definitions Limb arterial thrombosis was defined as abrupt arterial occlusion of a limb with absent pulses and was verified in all patients by angiography or surgical thrombectomy. Venous limb gangrene was defined as distal ischemic tissue necrosis complicating deep venous thrombosis despite palpable or Doppler-identifiable arterial pulses. Deep venous thrombosis was confirmed by contrast venography or duplex compression ultrasonography in all patients. All pathologic material was reviewed to evaluate the types of vessels involved in the thrombotic process. Central skin necrosis was defined as skin necrosis that occurred in the breast, abdomen, buttocks, or thigh in association with warfarin treatment. Coagulation Studies Serial citrated plasma samples were available from 34 consecutive patients with heparin-induced thrombocytopenia who were treated at one hospital over a 2-year period. These samples were collected and tested according to a local study protocol approved by the hospitals institutional review board. Before testing, all samples were stored in aliquots frozen at 70C. Four of the 34 patients (including patients 2, 3, and 4) developed venous limb gangrene or severe venous limb ischemia; this allowed us to test the hypothesis that warfarin therapy could contribute to venous limb gangrene. Control studies were done by using plasma obtained during a clinical trial of heparin prophylaxis [3] from 59 patients with deep venous thrombosis but not heparin-induced thrombocytopenia. We used one-stage assay techniques to measure levels of the following vitamin K-dependent procoagulant factors: prothrombin (factor II), factor VII, and factor X [20]. Protein C activity was determined by using a functional assay from Diagnostica Stago (Wellmark Diagnostics Ltd., Guelph, Ontario, Canada). Free protein S levels were measured by precipitation of the protein S bound to C4b-binding protein with polyethylene glycol [21]; the supernatant that contained the free protein S was measured by enzyme-linked immunosorbent assay [22] using antibodies to protein S (Affinity Biologicals, Hamilton, Ontario, Canada). Thrombin-antithrombin complex levels were determined by using an enzyme-linked immunosorbent assay [23] (Behring Diagnostics, Montreal, Quebec, Canada) to evaluate in vivo thrombin generation [24]. Antithrombin was measured by use of a chromogenic factor Xa inhibition method (Chromogenix, Helena Laboratories, Mississauga, Ontario, Canada) [25]. Evaluation for factor V Leiden was performed by using a functional assay for resistance to activated protein C [26] and by direct demonstration of the mutation at the DNA level [27]. Statistical Analysis We used both quantitative and qualitative measures to compare groups. Because quantitative measures tended to have skewed distributions, medians rather than means were used as summary statistics and in comparisons between groups. The Mann-Whitney test was used to compare medians, and associated nonparametric methods [28] were applied to estimate 95% CIs on differences between medians (MINITAB Release 10.5, Xtra software, Minitab, Inc., State College, Pennsylvania). Binary variables were summarized as proportions and were compared between groups by using the Fisher exact test [29]. Differences between groups for the binary variables were represented as risk ratios with CIs according to the method of Thomas [30]. For cases in which the risk ratio was zero or infinite, StatXact (Cytel Software Corp., Cambridge, Massachusetts) was used to calculate the exact 95% CIs for the odds ratios. The nonzero upper or lower bound that StatXact computed was then used to produce a quadratic Equation that, when solved, provided the expected event count for the first comparison group. The corresponding upper or lower bound for the risk ratio was then calculated by recomputing the 2 2 frequency table. Selected Case Reports Patient 1 A 56-year-old woman developed heparin-induced thrombocytopenia (platelet count nadir, 37 109/L) that was recognized on day 8 of intravenous heparin therapy given for bilateral, idiopathic proximal deep venous thrombosis of the lower limbs and pulmonary embolism. Heparin therapy was discontinued that day, and 20 mg of warfarin was given daily for 2 consecutive days. The following day, the patient had an international normalized ratio (INR) of 9.4 and developed necrosis involving eight toes, despite palpable pedal pulses (Figure 1). The patient was managed conservatively; sloughing of the gangrenous tissues was followed by healing without the need for amputation. Figure 1. Gangrene of lower-limb digits in patient 1. Patient 2 A 49-year-old woman received treatment with heparin and warfarin for 19 days because of idiopathic proximal deep venous thrombosis of the left lower limb. At that time, progressive symptoms of pain and swelling in the left lower limb prompted measurement of the platelet count, which was found to be 69 109/L (nadir); heparin therapy was discontinued. The INR was therapeutic at 2.2, and warfarin, 7.5 mg/d, was given for the next 2 days. The platelet count returned to normal within 4 days of discontinuation of heparin therapy. However, venous limb gangrene involving the distal left foot was first seen on the third day after heparin therapy ended (Figure 2 A). The INR was 7.2, protein C activity was less than 0.01 U/mL (normal, 0.65 to 1.29 U/mL), and free protein S level was markedly reduced (0.06 U/mL; normal, 0.24 to 0.62 U/mL). The prothrombin level was only moderately reduced (0.26 U/mL; normal, 0.5 to 1.6 U/mL), and thrombin-antithrombin complex levels were elevated (16.6 ng/mL; normal, <4.2 ng/mL). The patient required a below-the-knee amputation. Occlusive venous thrombi were noted in small venules and medium-sized veins, and arteries were normal (Figure 2 B, C, and D). Figure 2. Findings in patient 2. Patient 3 A 35-year-old woman received heparin prophylaxis for injuries sustained in a motor vehicle accident. She had no fractures or soft tissue injury to her left lower limb; she developed proximal deep venous thrombosis of the left lower limb on day 8 of subcutaneous heparin prophylaxis in association with a decrease in platelet count from 216 10 (9)/L (day 6) to 144 109/L (day 7). however, heparin-induced thrombocytopenia was not suspected, and intravenous therapeutic-dose heparin was started; the platelet count measured 3 days later was 30 10 (9)/L (platelet count nadir, 20 109/L). Heparin therapy was discontinued

Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function.

Summary.  Background: Closure time (CT), measured by platelet function analyzer (PFA‐100®) device, is now available to the clinical laboratory as a possible alternative or supplement to the bleeding time test. Aim: On behalf of the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH‐SSC), a working Group was formed to review and make recommendations on the use of the PFA‐100 CT in the evaluation of platelet function within the clinical laboratory. Methods: The Medline database was searched to review the published information on the PFA‐100 CT in the evaluation of platelet disorders and platelet function. This information, and expert opinion, was used to prepare a report and generate consensus recommendations. Results: Although the PFA‐100 CT is abnormal in some forms of platelet disorders, the test does not have sufficient sensitivity or specificity to be used as a screening tool for platelet disorders. A role of the PFA‐100 CT in therapeutic monitoring of platelet function remains to be established. Conclusions: The PFA‐100 closure time should be considered optional in the evaluation of platelet disorders and function, and its use in therapeutic monitoring of platelet function is currently best restricted to research studies and prospective clinical trials.

Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH

Background: Light transmission aggregometry (LTA) is the most common method used in clinical and research laboratories to assess platelet function. However, the method has never been standardized. Objectives: As the first step towards development of methodological guidelines, the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) undertook a large, detailed, global survey of LTA practices. Methods: Members of ISTH and of External Quality Assurance in Thrombosis and Haemostasis organizations were invited to complete a 129 item, online questionnaire. Results were analyzed anonymously to participant identities. Results: The online supplement for this article (http://www.isth.org/Publications/OfficialCommunications/PlateletPhysiology/LightTransmissionAggregometry/tabid/201/Default.aspx) contains the full details of the study findings. 359 (244 clinical, 115 research) laboratories from 48 countries participated in the survey. LTA was widely used to assess inherited or acquired bleeding disorders. Common practices were identified in sample collection, processing and analysis and although some are generally considered acceptable, others are not ideal. The agonist concentrations used for LTA varied, and many laboratories used ADP, collagen, epinephrine and Ristocetin, at more than one concentration, in addition to arachidonic acid. The parameters commonly used to assess LTA responses were maximal amplitude or % aggregation, which was considered particularly important, in addition to the presence of a ‘secondary wave’, deaggregation, shape change and a measure of the lag phase. However, many laboratories did not have appropriate reference intervals. Conclusions: This is the largest and most detailed survey of LTA practices ever undertaken. It shows a very high variability in LTA practices worldwide, and, as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of ISTH methodological guidelines for LTA.

Diagnostic utility of light transmission platelet aggregometry: results from a prospective study of individuals referred for bleeding disorder assessments.

Summary.  Background: Light transmission aggregometry (LTA) is commonly performed to assess individuals for bleeding disorders. Objectives: The goal was to evaluate the incidence and spectrum of platelet function abnormalities in a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. Patients/methods: Subjects were healthy controls and patients from a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. LTA was performed by standardized methods using platelet‐rich plasma adjusted to 250 × 109 platelets L−1. Maximal aggregation data were analyzed to determine the likelihood of detecting a platelet function disorder by LTA, and the sensitivity and specificity of LTA for platelet disorders. Results: The incidence of false positive LTA among subjects excluded of having bleeding disorders was similar to healthy controls. Abnormal LTA was more common in subjects with bleeding disorders and the likelihood of a bleeding disorder was significantly increased (odds ratio 32) when maximal aggregation was reduced with two or more agonists. Receiver operator curve analyses indicated that LTA had high specificity and moderate sensitivity for detecting inherited defects in platelet function and that the LTA agonists 1.25 μg mL−1 collagen, 6 μM epinephrine, 1.6 mM arachidonic acid and 1.0 μM thromboxane analogue U44619 detected most inherited disorders with abnormal LTA. Conclusions: LTA is valuable for detecting platelet function abnormalities among individuals referred for bleeding problems, particularly when the test indicates abnormal responses to multiple agonists.

Variability in clinical laboratory practice in testing for disorders of platelet function Results of two surveys of the North American Specialized Coagulation Laboratory Association

Disorders of platelet function are important causes of abnormal bleeding that require laboratory tests for diagnosis. Currently there are limited guidelines on how to perform clinical testing for these disorders. The goal of our study was to obtain information on how disorders of platelet function are currently evaluated in clinical laboratories. Two patterns-of-practice surveys were distributed to laboratories of the North American Specialized Coagulation Laboratory Association (NASCOLA). The information collected was analyzed to determine practices and common problems. Forty-seven NASCOLA laboratories participated and 54% completed both surveys. The majority of the laboratories that responded performed more than 50 aggregation tests per year, mainly using platelet rich plasma based methodologies. A minority performed testing for platelet secretion and dense granule abnormalities. While platelet aggregation results were reviewed in various ways, laboratories most commonly issued a combined report containing quantitative values (% aggregation and/or slope) and a qualitative interpretation. Although laboratories used similar agonists for aggregation testing, the final agonist concentrations varied widely. Several approaches were also used to obtain reference intervals. Comments offered by the participants indicated that performing, and interpreting platelet function tests were challenging for many clinical laboratories. Although common practices have evolved, there is considerable variability in the diagnostic test procedures used by clinical laboratories to evaluate disorders of platelet function. These patterns-of-practice surveys illustrate a need for guidelines and recommendations for clinical laboratories performing tests of platelet function.

Congenital platelet disorders: overview of their mechanisms, diagnostic evaluation and treatment

Summary.  The bleeding problems associated with common and rare inherited platelet disorders illustrate the importance of platelets to normal haemostasis. At sites of injury, platelets normally adhere, undergo activation, secretion and aggregate formation, and they provide the membrane surface for the assembly of coagulation to generate thrombin. The causes of inherited disorders that alter platelet haemostatic functions are quite diverse, ranging from defects in receptors critical to platelet adhesion and aggregation, to defects in signalling molecules or in transcription factors important for production of functional platelets. The mechanisms of impaired platelet function are largely unknown for the more common disorders that alter platelet activation, secretion and the secondary wave of platelet aggregation. The diagnostic evaluation of congenital platelet disorders has been challenging as some ‘platelet‐type’ bleeding symptoms, such as bruising, are quite common in the general population. Moreover, the diagnostic tests used by clinical laboratories to evaluate disorders of platelet function have not been standardized. In individuals recognized to have an inherited defect in platelet function, therapy is important for controlling and preventing bleeding episodes. Presently, there are a number of choices to consider for the management of bleeding symptoms, including menorrhagia. This paper reviews the causes, diagnostic evaluation and therapies for common and rare congenital platelet disorders.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey

Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach.

Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder linked to a region on chromosome 10 that includes PLAU, the urokinase plasminogen activator gene. QPD increases urokinase plasminogen activator mRNA levels, particularly during megakaryocyte differentiation, without altering expression of flanking genes. Because PLAU sequence changes were excluded as the cause of this bleeding disorder, we investigated whether the QPD mutation involved PLAU copy number variation. All 38 subjects with QPD had a direct tandem duplication of a 78-kb genomic segment that includes PLAU. This mutation was specific to QPD as it was not present in any unaffected family members (n = 114), unrelated French Canadians (n = 221), or other persons tested (n = 90). This new information on the genetic mutation will facilitate diagnostic testing for QPD and studies of its pathogenesis and prevalence. QPD is the first bleeding disorder to be associated with a gene duplication event and a PLAU mutation.

Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: A survey of North American specialized coagulation laboratories

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. As HIT is considered a clinico-pathologic entity, laboratory practices have an important role in diagnosing or excluding HIT. It was the objective of this study to assess the current status of laboratory testing for HIT in North America. An online survey consisting of 67 questions related to laboratory testing for HIT was developed by the North American Specialized Coagulation Laboratory Association (NASCOLA), and distributed to its 59 members. The survey included queries about HIT test ordering practices, HIT immunoassay and activation assays performed, and reporting practices. Data was collected from the 44 NASCOLA laboratories who responded. Of these sites, 88% performed immunoassays for HIT, commonly using commercial assays. However, sites varied in practices related to use of controls, immunoglobulin class of antibody detected, and in result interpretation and reporting. Platelet activation assays for HIT were performed by 36% of sites, commonly using assays of serotonin release (50%) or heparin-induced platelet aggregation (43%). Sites varied in the use of washed platelets versus platelet-rich plasma, controls, and heparin concentrations. This survey is the first comprehensive assessment of patterns of practice in HIT testing among diagnostic coagulation laboratories in North America. We observed site-specific variability of testing methods encompassing all stages of testing, including pre-analytical handling, testing methodologies, and result interpretation and reporting. The variability in HIT platelet activation assay methods among institutions indicates a need for proficiency testing to assess assay performance, and for consensus guidelines on HIT laboratory testing.

An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts

Light transmission platelet aggregation tests are important for diagnosing platelet function defects. However, uncertainties exist about the best procedures to determine aggregation reference intervals. We investigated methods for determining reference intervals for light transmission aggregation tests, using the % maximal aggregation values for prospectively collected data on healthy control samples. Reference intervals for samples tested at 250 x 10(9) platelets/l were determined by mean +/- 2 standard deviations and non-parametric analyses. To establish reference intervals for tests on thrombocytopenic subjects, regression analyses were used to estimate 95% confidence limits for % maximal aggregation, according to sample platelet counts, using data for control samples diluted to match the platelet count of undiluted thrombocytopenic patient platelet-rich plasma samples. For samples tested at 250 x 10(9) platelets/l, non-parametric analyses described 95% of data for healthy control samples better than mean +/- 2 standard deviations. For samples tested at lower counts, to match thrombocytopenic samples, the % maximal aggregation was influenced by platelet count and derived limits were wider at very low platelet counts for almost all agonists. With ristocetin, it proved feasible to test samples with very low platelet counts to exclude Bernard-Soulier syndrome and type 2B von Willebrand disease. Non-parametric analyses should be the preferred method to establish light transmission aggregation reference intervals for samples tested at normal platelet counts. The derived limits for thrombocytopenic samples provide guidance for evaluating thrombocytopenic platelet function disorders, including which agonists to test, based on the sample platelet count.