Acute thrombocytopenia suggesting thrombotic microangiopathy
Authors: Laura M. Venier, Christopher J. Patriquin, Theodore E. Warkentin
Journal: American Journal of Hematology (2022)

A 56-year-old male presented to the emergency department with a four-day history of suprapubic pain and hematuria. The patient started ciprofloxacin 3 days prior to treat a suspected urinary tract infection (UTI) but his symptoms had not improved. History revealed that the patient had had recurrent UTIs for approximately 2 years in the setting of spinal cord injury following a car-pedestrian accident at age of 3 years. A complete blood count (CBC) revealed a low platelet count of 27 × 109/L (reference range, 150–400); the white blood cell (WBC) count was 10.1 × 109/L (reference range, 4.0–11.0), with normal absolute neutrophil count of 6.3 × 109/L (reference range, 2.0–7.5). The hemoglobin was 12.0 mg/dL (reference range, 13.0–18.0). Low platelet count in the context of potential UTI can indicate a life-threatening problem such as septicemia with or without associated disseminated intravascular coagulation (DIC)1 or a serious underlying marrow disorder, such as acute leukemia. DIC can be complicated by hemorrhage, thrombosis, or both, but in this case, it is unclear whether hematuria represented a hemorrhagic manifestation of thrombocytopenia or a consequence of UTI (e.g., hemorrhagic cystitis). The recent administration of ciprofloxacin could indicate possible drug-induced immune thrombocytopenia (D-ITP), although D-ITP usually requires at least 5 days following drug start before thrombocytopenia occurs.2 In contrast, any unexpectedly low platelet count can indicate spurious thrombocytopenia (e.g., ex vivo platelet clumping secondary to traumatic venipuncture) or ethylenediaminetetraacetic acid (EDTA)-induced pseuodothrombocytopenia.3 A key task of the clinician is to ensure thrombocytopenia is properly and timely investigated. This should include confirmatory repeat CBC, reticulocyte count, examination of the peripheral blood film (e.g., assessment for vacuolation and toxic granulation, schistocytes, blasts, etc.), screen for DIC (international normalized ratio [INR], activated partial thromboplastin time [APTT], fibrinogen, d-dimer), screen for thrombotic microangiopathy (TMA) (e.g., lactate dehydrogenase [LDH] in relation to hepatic transaminases, alanine transaminase [ALT], aspartate transaminase [AST] and creatine kinase [CK], haptoglobin, free hemoglobin), screen for infection (e.g., blood/urine cultures), and assessment of critical illness (e.g., serum lactate). One should also obtain prior CBCs, if possible, to determine whether abnormalities are new or present on previous occasions. A family history of thrombocytopenia or other hematological abnormalities should be sought. Additional blood was drawn for repeat CBC, evaluation of coagulation parameters, screening for TMA, measurement of serum lactate, and microbiology (blood/urine cultures). Repeat platelet count was 29 × 109/L, and the peripheral blood film did not reveal platelet clumping or toxic changes. A platelet count obtained 18 months earlier was normal (183 × 109/L). The repeat WBC count was slightly elevated (11.5 × 109/L) with increased neutrophils and monocytes. Mean corpuscular volume (MCV) was 96 fL (reference range, 82–99). Reticulocytes were not increased (absolute value, 39 × 109/L [reference range, 10–86]), and nucleated red blood cells were not observed; however, schistocytes were reported by the laboratory technologist. The repeat hemoglobin was 11.7 mg/dL, confirming mild anemia. The hemoglobin from 18 months earlier was normal (15.1 mg/dL). The repeat CBC confirmed severe thrombocytopenia, and blood film review did not reveal platelet clumping; thus, true thrombocytopenia was confirmed. The finding of schistocytes was of considerable importance, as these can indicate DIC or TMA. The DIC screen showed normal INR (1.0 [reference range, 0.8–1.2]), normal APTT (30 s [reference range, 22–35 s] and elevated fibrinogen (630 mg/dL [reference range, 160–420]). The d-dimer level was 1.66 mg/L fibrin-equivalent units (reference range, <0.5 mg/L). Although the d-dimer was elevated, the value was not as high as is usually seen in overt decompensated DIC. Following the International Society on Thrombosis and Hemostasis (ISTH) DIC scoring system,1 these results yielded a total DIC score of only 2 points (2 points for platelet count <50 × 109/L; 0 points for normal INR, elevated fibrinogen, and minimally elevated d-dimer). Our local practice is to score 2 points for moderately elevated d-dimer (2.0–9.9 mg/L) and 3 points for greatly elevated d-dimer (>10 mg/L).4 Thus, a DIC disorder did not appear to explain the patient's thrombocytopenia. The serum lactate was normal. However, the LDH measured 967 U/ml (reference range, 120–250), with normal levels of ALT, AST, and CK. The serum creatinine was 1.19 mg/dL (reference range, 0.68–1.24), which was higher than the value 18 months earlier (0.76). Urinalysis revealed presence of blood (quantitated at 3+), as well as 21–30 red cells per high power field (hpf; reference range, 0–3); however, relatively few leukocytes were identified (0-3/hpf, within the normal range). The urinalysis quantitated bacteria as “few.” The normal serum lactate and transaminase levels are consistent with a patient who was normotensive and in no apparent distress. The most striking abnormality was the markedly elevated LDH, with normal levels of CK, AST, and ALT. Moreover, the patient's creatinine was higher than a previous value (1.19 vs. 0.76). The urinalysis was nonspecific, but consistent with symptomatic UTI in a patient who had received 3 days of antibiotics. Formal referral to hematology consultation service was made. The hematologist personally reviewed the blood film, and also noted occasional red cell fragments (schistocytes). No primitive or neoplastic cells were identified. Nucleated red cells (normoblasts) were not identified. Further history was taken, including review of previous CBCs available over a 20-year period, which showed no previous thrombocytopenia, except on one occasion 30 months earlier, when the platelet count transiently fell to 125 × 109/L (nadir) during a 6-day hospitalization for suspected UTI treated with antibiotics (however, despite pyuria, blood and urine cultures were negative). Moreover, there was no known family history of thrombocytopenia and no recent history of acute diarrheal illness. There were no features of rheumatologic or autoimmune disorders. The constellation of clinical and laboratory features can be summarized as new-onset severe thrombocytopenia and mild anemia with schistocytes and elevated isolated LDH, together with increased creatinine over baseline, in the context of acute hematuria and possible UTI. An isolated LDH (i.e., with normal CK and transaminases) points to potential intravascular (or severe extravascular) hemolysis. The clinical picture of anemia and schistocytes corroborated by isolated elevation in LDH is suggestive of microangiopathic hemolysis, a type of anemia that results from physical damage to red cells usually arising from occlusion of microvasculature by fibrin deposition (e.g., DIC) or platelet aggregates (e.g., TMA).5 When there is concomitant thrombocytopenia, the clinical picture is called thrombotic microangiopathy, or TMA.5, 6 This is an important finding as it points to a potentially life-threatening diagnosis such as acquired (immune) thrombotic thrombocytopenia purpura (TTP), a disorder usually caused by antibodies against ADAMTS13.5, 6 One assessment for predicting for ADAMTS13 deficiency in acute TTP is the PLASMIC score7 (Table 1). Haptoglobin Undetectablea The patient's PLASMIC score was 6 points, indicating a high risk for ADAMTS13-deficient TTP (sensitivity, ~85%; specificity, ~90%).8 Moreover, acute TTP was the leading clinical impression of the hematologist. Given also the concerns for UTI—including as a potential trigger of a TMA disorder—the patient was continued on ciprofloxacin (400 mg IV q12h). Further, daptomycin (Cubicin; 4.5 g IV q8h) was also commenced for concern of potential severe sepsis of unknown origin. TTP can be either congenital, acquired, or even drug-induced.5, 6 Congenital TTP results from germline mutations in the ADAMTS13 gene leading to severe enzyme deficiency. Although presentation in early childhood is common, congenital TTP can also present in adulthood, often complicating pregnancy.9, 10 Acquired TTP is usually caused by autoantibodies that either inhibit or clear ADAMTS13. Although ciprofloxacin has been reported as a potential cause of drug-induced TTP,11 it was continued in our patient, as the temporal onset of thrombocytopenia seemed too soon to implicate it as a biologically plausible trigger. Further, ciprofloxacin was not listed in a systematic review of drug-induced TTP.12 Other diagnoses to explain TMA in our patient, besides TTP, included hemolytic uremic syndrome (HUS), both typical (i.e., Shiga toxin-mediated) and atypical HUS (aHUS), preeclampsia/HELLP (not applicable to this patient), malignant hypertension, systematic vasculitis, or DIC. None of the clinical features, family history, or additional laboratory testing suggested any of these other explanations for TMA. Table 2 summarizes the differential diagnosis of TMA.12-22 Thrombotic thrombocytopenic purpura (TTP) VWF proteolysis caused by ADAMTS13 deficiency/autoantibody-mediated destruction. Drug-induced TMA Quinine, oxaliplatin, vancomycin, gemcitabine, and pentostatin (5 agents with strongest evidence for drug-induced TMA).12 Hemolytic Uremic Syndrome (HUS) Microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. ST-HUS – caused by infection, over 50% of patients experience watery or bloody diarrhea.14 Atypical HUS (aHUS) – caused by genetic and/or autoantibody-mediated impairment of AP complement regulatory proteins16 HELLP (hemolysis/elevated liver enzymes/low platelets) Irregular vascular tone, vasospasm, and coagulation abnormalities possibly related to an autoimmune reaction leading to maternal-fetal imbalance. Features include microangiopathic hemolysis, elevated liver enzymes (intravascular fibrin deposits that incite sinusoid obstruction, congestion, and elevated intrahepatic pressure, potentially causing focal and/or periportal hepatocyte necrosis), and thrombocytopenia (increased platelet consumption and mild-to-moderate deficiency of ADAMTS13).17 Malignant hypertension Classically defined as diastolic blood pressure > 130 mmHg and retinal hemorrhages and/or exudates, and of several potential etiologies (severe primary [essential] hypertension, secondary, e.g., renal parenchymal disease, aortoarteritis/fibromuscular dysplasia, Takayasu's arteritis involving renal artery, etc.), malignant hypertension can be associated with microangiopathic hemolytic anemia due to endothelial injury associated with high shear forces (arteriolar fibrinoid necrosis). Of note, some patients with malignant hypertension-associated TMA have unrecognized congenital abnormalities in the AP system.18 Systemic vasculitis (e.g., scleroderma, systemic lupus erythematosus, Wegener's granulomatosis, etc.) or some glomerulonephritides Pathogenesis obscure, could relate to interactions of vascular endothelial injury and elevated VWF levels.19 Given the potential treatment with therapeutic plasma exchange (TPE), it is important to obtain baseline (pre-treatment) samples for key diagnostic tests, such as ADAMTS13 activity, anti-ADAMTS13 autoantibodies, and other ancillary diagnostics (e.g., anti-nuclear antibodies, non-specific inhibitor, antiphospholipid antibodies, anti-congestive heart failure (CFH) antibodies, free hemoglobin, haptoglobin). It is especially crucial to obtain a baseline plasma sample given the importance of establishing low ADAMTS13 activity (<10%) to support a diagnosis of TTP. Given the working diagnosis of TTP, the patient was started on methylprednisolone (80 mg IV q12h). After obtaining serum for measurement of ADAMTS13 activity, and prior to organizing TPE, he was given 4 units of frozen plasma (FP). A central line was inserted and TPE commenced (1.5 plasma-volume exchange), first with FP as replacement fluid, then switched to cryosupernatant (“cryopoor plasma” [CPP]). However, during the second TPE procedure using CPP, the patient developed symptoms/signs of a possible transfusion reaction, so the replacement fluid was switched to solvent-detergent plasma (SDP); three SDP exchanges proceeded uneventfully. The patient's platelet counts progressively increased, and elevated LDH and creatinine levels returned to baseline [Figure 1]. Admission blood/urine cultures yielded no growth. The patient's dramatic platelet count recovery, and progressive decrease in LDH levels, indicated response to TPE. Further, negative blood cultures argued against bacteremia/septicemia as the initial explanation for thrombocytopenia. The switch from FP to CPP replacement fluid reflected theoretical considerations, that is, lower von Willebrand factor (VWF) levels in CPP could be helpful in a disorder like TTP that features platelet-FWF microaggregates. The subsequent switch to SDP was performed in case the patient's transfusion reaction was related to plasma proteins within CPP. Corticosteroid therapy was switched to prednisone (100 mg daily); he also received dalteparin thromboprophylaxis and aspirin 81 mg daily (both beginning after the platelet count rose above 50 × 109/L), cefazolin (2 g IV q12h), and ciprofloxacin (400 mg IV q12h). The patient was discharged after 6 TPE procedures, with a presumptive diagnosis of TTP. However, results of ADAMTS13 activity and antibodies were pending at discharge. Although caplacizumab had recently been approved by Health Canada for treatment of TTP, its use was not considered by the treating physicians. At that time (as well as currently [February 2022]), caplacizumab was not commercially available in Canada, and could only be accessed via compassionate-use program to patients with confirmed TTP (i.e., ADAMTS13 activity < 10%). The overall clinical course appears consistent with acute TTP, albeit with some atypical features. For example, our patient's platelet count nadir (27 × 109/L) was higher than many patients with confirmed TTP. Further, our patient did not have expected reticulocytosis. Nonetheless, the current paradigm of TTP is that severe reduction in ADAMTS13 activity is required for diagnosis, and hence the hematologist awaited the results with interest. Following discharge, the ADAMTS13 activity, measured using a pre-TPE serum sample, was reported as 74% (<10% activity is characteristic of TTP). Further, anti-ADAMTS13 antibodies were not detected. These findings prompted the hematologist to reconsider the (presumptive) diagnosis of acute TTP. Accordingly, the hematologist reassessed the differential diagnosis of TMA, and investigated for aHUS by sending blood to the national reference laboratory at Hospital for Sick Children for genetic testing for pathogenic variants in complement proteins. This revealed the patient to be heterozygous for the pathogenic variant c.1429+1G>C, in the complement factor I (CFI) gene. This finding pointed to a correct diagnosis of aHUS, with the predisposing genetic abnormality of a loss-of-function mutation in CFI, a regulator of complement activity. Interestingly, CFI is a plasmatic factor,22 which may help explain the dramatic response to TPE. This also illustrated that though predictive scoring systems like the PLASMIC score may help guide initial treatment decisions, confirmatory testing is crucial to ensure proper diagnosis. The alternative pathway (AP) of the complement system, along with the classical and lectin pathways comprise part of the innate immune system.14, 16, 22 Figure 2 summarizes the complement AP. C3, a central player in all three pathways, is an abundant plasma protein that can undergo spontaneous activation (“tickover”) or become activated following recognition of foreign or altered host molecular patterns, initiating the AP. Hydrolyzed C3 binds to complement factor B (CFB) to form the precursor to the AP C3 convertase, C3(H2O)B (not shown in figure). Complement factor D (CFD) cleaves CFB within the C3(H2O)B complex, to create the active C3 convertase (C3[H2O]Bb), which cleaves other C3 molecules into C3a and C3b. C3a is an anaphylatoxin which is chemoattractant for neutrophils and monocytes. C3b has three roles: it binds to pathogens or altered host cells, promoting their phagocytosis (opsonization); it can form more C3 convertase (“amplification”); and it binds to C3 convertases to form C5 convertase (e.g., C3bBbC3b). Bb within the C5 convertase cleaves C5 into C5a (another anaphylatoxin), and C5b, which together with other complement proteins (C6, C7, C8, and several C9 moieties), forms the membrane attack complex (MAC). Several regulatory proteins prevent excessive activation of the AP.14, 16, 22 C3b is inhibited primarily by CFI but also by complement factor H (CFH), membrane cofactor protein (MCP/CD46), complement receptor 1 (CR1), and C4b-binding protein (C4BP). aHUS arises when there are hereditary (~90%) or acquired (~10%) abnormalities affecting the AP.14 Genetic mutations associated with aHUS typically impair normal functioning of AP regulators due to loss-of-function (LOF) mutations (e.g., CFH, CFI, MCP); however, gain-of-function (GOF) mutations in C3 or CFB can also cause disease (Table 2).14, 22 Autoantibodies against CFH represent an acquired abnormality of the AP that can lead to aHUS.14, 22 In approximately 30%–40% of aHUS patients no causative genetic abnormality is identified.22 Our patient was found to have a pathogenic CFI mutation, variant c.1429+1G>C. CFI is a serine protease that acts as the primary inhibitor of C3b. It requires a cofactor, such as MCP or CFH, for effective enzyme activity. Normally, CFI-mediated C3b inactivation arrests C3 convertase assembly and excessive fluid-phase C3b activity. CFI converts C3b to create iC3b and C3dg, which are enzymatically inactive, yet can still serve as opsonins. The specific CFI genetic variant identified in our patient affects the light chain (splicing site) of the CFI protein.23, 24 We are aware of two previous reports23, 24 implicating c.1429+1G>C as predisposing to aHUS. Interestingly, one of these patients also responded promptly to TPE,24 consistent with our experience. Eculizumab (monoclonal antibody that inhibits C5) is a treatment option if our patient developed recurrent aHUS in the future.24, 25 Given his response to plasma and an estimated relapse rate of 10%–30%,26 the patient and his treating physician were comfortable with ongoing monitoring of TMA parameters (with prompt reevaluation were he to develop recurrent infection). Although data are limited, the patient's variant does not appear to be high-risk for recurrence following eculizumab discontinuation27; given his full resolution of TMA parameters, Ministry of Health (Ontario, Canada) approval for eculizumab would not have been granted, despite a known pathogenic variant. Our patient illustrates how clinical and laboratory evaluation of acute thrombocytopenia led to recognizing a TMA state, with suspicion for acute TTP. Despite response to TPE, this presumptive diagnosis was no longer tenable after normal ADAMTS13 activity was demonstrated. This prompted evaluation for aHUS risk factors, leading to identification of a rare LOF mutation (c.1429+1G>C) in the AC regulatory protein, CFI. We thank Ms. Jo-Ann I. Sheppard for preparing Figure 1. Theodore E. Warkentin has received lecture honoraria from Alexion and Instrumentation Laboratory (Werfen); has provided consulting services to Aspen Canada, Aspen Global, CSL Behring, Ergomed, Instrumentation Laboratory (Werfen), and Octapharma; has received research funding from Instrumentation Laboratory, and has provided expert witness testimony relating to HIT and non-HIT thrombocytopenia. Christopher J. Patriquin has received advisory board and speaking honoraria from Alexion, Apellis, BioCryst, Octapharma, Sanofi, and Sobi. He has also served as site investigator for interventional trials sponsored by Alexion and Apellis. Ms. Laura M. Venier declares no relevant conflicts of interest. The patient provided written permission for this report (institutional approval is not required for case reports in our medical community). All data generated or analyzed during this study are included in this published article.