Hemolysis and schistocytosis in the emergency department: consider pseudothrombotic microangiopathy related to vitamin B12 deficiency

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1 Q J Med 2013; 106: doi: /qjmed/hct142 Advance Access Publication 10 July 2013 Hemolysis and schistocytosis in the emergency department: consider pseudothrombotic microangiopathy related to vitamin B12 deficiency N. NOËL 1, G. MAIGNÉ 1, G. TERTIAN 2,4, N. ANGUEL 3, X. MONNET 3,4, J.-M. MICHOT 1,4, C. GOUJARD 1,4 and O. LAMBOTTE 1,4 From the 1 APHP, Service de Médecine Interne, Hôpital Bicêtre, Le Kremlin Bicêtre, France, 2 APHP, Service d Hématologie Biologique, Hôpital Bicêtre, Le Kremlin Bicêtre, France, 3 APHP, Service de Réanimation Médicale, Hôpital Bicêtre, Le Kremlin Bicêtre, France and 4 Faculté de Médecine Paris-Sud 11, Université Paris-Sud, Le Kremlin Bicêtre, France Address correspondence to Professor O. Lambotte, MD, PhD, Service de Médecine Interne, Hôpital Bicêtre, 78, rue du Général Leclerc, F Le Kremlin Bicêtre cedex, France. olivier.lambotte@bct.aphp.fr Received 25 April 2013 and in revised form 14 June 2013 Summary Background: Hemolytic anemia with thrombocytopenia and schistocytosis is suggestive of thrombotic thrombocytopenic purpura (TTP). However, these features can occur in the context of vitamin B12 deficiency. Aim: To identify simple means of distinguishing between TTP and pseudothrombotic microangiopathies related to vitamin B12 deficiency (pseudo- TMA) at the bedside. Design and methods: Retrospective study of patients with pseudo-tma compared with patients with TTP. The patients with pseudo-tma were further compared with other cases of cobalamin deficiency, in order to detect factors associated with microangiopathic hemolysis during vitamin B12 deprivation. Results: Seven patients with pseudo-tma were compared with six patients with TTP. The pseudo- TMA patients had higher median lactate dehydrogenase (LDH) levels (7310 vs IU/l, P = 0.01), a higher platelet count (73 vs /l, P = ), a lower reticulocyte count (13.1 vs /l, P = ) and a lower neutrophil count (1.3 vs /l, P = ). When compared with 21 patients with vitamin B12 deficiency and anemia (but no schistocytosis), the pseudo-tma patients were more likely to present with pernicious anemia [7 out of 21 (33.3%) vs. 5 out of 7 (71.4%), respectively] and had lower vitamin B12 levels (105 vs. 45 mmol/l, respectively). Vitamin supplementation led to hematological improvements in all pseudo-tma patients. Conclusion: In a context of mechanical hemolysis with thrombocytopenia in a patient admitted to the emergency department, very high LDH levels and a low reticulocyte count are strongly suggestive of pseudo-tma and should prompt the physician to screen for cobalamin deficiency. Introduction Thrombotic microangiopathies (TMAs) are rare, severe conditions associated with a mortality rate of 10 20%. 1 The annual incidence of these conditions in France is four per million inhabitants. 2 Given that the lack of rapid plasma exchange is known to be an independent risk factor for shortterm mortality, 2 there is a need for rapid, reliable diagnostic tools that can help avoid treatment delays. 3 In this context, patients presenting in the! The Author Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please journals.permissions@oup.com

2 1018 N. Noël et al. emergency department with hemolytic anemia, schistocytosis and thrombocytopenia should be immediately screened for thrombotic thrombocytopenic purpura (TTP). However, differential diagnoses for microangiopathic hemolytic anemia have to be considered, such as malignant hypertension and Evans syndrome. 3 Besides these two conditions, rare situations mimicking TMA have been described in the context of vitamin B12 (cobalamin) deficiency. 4,5 These pseudo-tmas 4 can be confused with lifethreatening TTP if a vitamin B12 assay has not been performed. Hence, pseudo-tma patients presenting at the emergency department may be admitted to the intensive care unit (ICU) and undergo unnecessary, invasive treatments (such as plasma exchange). 5 In this study, we sought to describe the characteristics of patients with pseudo-tma related to vitamin B12 deficiency and compare them with patients admitted for TTP to a French tertiary university medical center over the same period. We further sought to identify simple clinical or biochemical features of pseudo-tma that could enable easier recognition of this condition in the emergency department. Methods Study design and setting Bicêtre University Medical Center is a primary and tertiary care institution in the southern suburbs of Paris, France. We retrospectively reviewed the medical records of patients admitted to our emergency department and subsequently transferred to the ICU or to our internal medicine department for hemolytic anemia associated with schistocytosis and thrombocytopenia (whether related to vitamin B12 deficiency or TMA) between 21 January 2005 and 01 January All patients were first identified using the ICD-10 classification for disclosure diagnoses, and the study data were then exhaustively extracted from these patients medical files. Selection of participants Patients were classified as having TTP if they had thrombocytopenia, microangiopathic hemolytic anemia, 3 low ADAMTS13 (A disintegrin and metalloproteinase with thrombospondin motifs) activity (<5%) and anti-adamts13 antibodies. The diagnostic criterion for pseudo-tma was vitamin B12 deficiency-associated anemia in combination with the biochemical and hematological features of hemolysis, schistocytosis and thrombocytopenia. Seven patients were diagnosed with pseudo-tma; their data at admission were compared with those of six patients admitted for TTP over the same period. In order to identify factors associated with microangiopathic hemolysis in a context of vitamin B12 deficiency, the seven patients with pseudo-tma were further compared with schistocytosis-free patients presenting with anemia related to B12 deficiency over the same period. Pernicious anemia was characterized via a gastric biopsy and an assay for anti-intrinsic factor antibodies. Food-cobalamin malabsorption was diagnosed as follows: documented cobalamin deficiency (<150 mmol/l), a sufficient daily intake of vitamin B12 (to exclude nutritional deficiency) and the existence of a predisposing factor (i.e. gastritis, alcoholism, proton-pump inhibitor exposure and metformin exposure). 6,7 This retrospective study was approved by our local institutional review board (Comité de Protection des Personnes Ile-de-France VII, n8pp ). Outcome measures N.N., G.W., G.T. and O.L. collected information on the patients medical history and clinical and biochemical parameters, with a particular focus on neurological symptoms (coma and objective abnormalities on neurological examination, such as hemiparesia and tetraparesia) and acute kidney failure (glomerular filtration rate <30 ml/min). Blood samples were carefully analyzed for hemolysisrelated parameters in particular [i.e. hemoglobin (g/l), lactate dehydrogenase (LDH; IU/l), haptoglobin (g/l) and bilirubin (mmol/l) levels, mean corpuscular volume (MCV, in fl), presence of schistocytes, platelet count, total white cell count (10 9 /l) and neutrophil count (10 9 /l)]. Schistocytes were characterized as previously described. 8 Bone marrow aspiration results were reported, when available. Statistical analysis Results are presented as the median (range) or n (%). A non-parametric Mann Whitney U test and Fischer s exact test or a 2 test (when applicable) were used for intergroup comparisons of continuous and categorical variables, respectively. The threshold for statistical significance was set to P < Data were analyzed using GraphPad Prism software (version 5.01, GraphPad Software Inc., La Jolla, CA, USA).

3 Pseudothrombotic microangiopathy due to vitamin B12 deficiency 1019 Results Comparison of pseudo-tma and TTP patients Seven patients were admitted for pseudo-tma during the study period. They had a median (range) hemoglobin level of 42 (35 79) g/l, a MCV of ( ) fl, a reticulocyte count of 13.1 ( ) 10 9 /l and a platelet count of 73 (38 145) 10 9 /l (Table 1). Schistocytosis was found in all seven patients. Three of the seven were admitted to the ICU for profound anemia and suspected TTP. One patient received 10 (ineffective) plasma exchanges. These data were compared with those from the six TTP patients (Table 1). All but one were transferred to the ICU prior to hospitalization in our internal medicine department. The TTP patients were younger than the pseudo-tma patients [median (range) age: 35.3 [ ] vs. 72 [43-78], respectively; P = 0.05). Five of the TTP patients (80%) had major organ involvement (neurological impairment in four of the six patients (66.6%) and severe kidney failure in two cases (33.3%)]. Importantly, none of the pseudo-tma patients had acute kidney failure, but two displayed confusion. Table 1 Comparison of pseudo-tma and TTP groups Compared with the TTP patients, pseudo-tma patients had higher platelet counts [median (range): 12.5 (8 41) vs. 73 (38 145) 10 9 /l, respectively; P = ), lower white blood cell counts [7.0 ( ) vs. 3.4 ( ) 10 9 /l, respectively; P = ] and lower neutrophil counts [5.1 (3.4 10)] vs. 1.3 ( ) 10 9 /l, P = ]. The most marked differences were related to hemolytic features: reticulocyte counts were significantly lower in pseudo-tma patients than in TTP patients [13.1 ( ) vs ( ) 10 9 /l, respectively; P = ] and LDH levels were significantly higher [7310 ( ) vs ( ) IU/l, respectively; P = 0.01]. Characteristics of patients with anemia and vitamin B12 deficiency In order to better characterize the pseudo-tma condition, we performed an overall, descriptive analysis of the patients admitted for anemia and vitamin B12 deficiency to the internal medicine department. Patients who were not subsequently hospitalized were not included in the analysis. A total of 28 patients had cobalamin deficiency-related anemia Pseudo-TMA (n = 7) TTP (n = 6) P-value Clinical characteristics Male, n (%) 5 (71.4) 2 (33.3) 0.17 Age at hospitalization (years) 72 (43 78) 35.2 ( ) 0.05 Caucasian origin, n (%) 3 (42.9) 4 (66.7) 0.59 Neurological symptoms, n (%) 2 a (28.6) 4 (66.6) 0.17 Acute kidney failure, n (%) 0 (0) 2 (33.3) 0.10 Hematological characteristics Hemoglobin (g/l) 42 (35 79) 69 (49 110) 0.07 Schistocytes, n (%) 7 (100) 6 (100) 1 Reticulocyte count (10 9 /l) 13.1 ( ) ( ) Mean corpuscular volume (fl) ( ) 92 (82 118) 0.05 Platelet count (10 9 /L) 73 (38 145) 12.5 (8 41) White blood cell count (10 9 /l) 3.4 ( ) 7.0 ( ) Neutrophil count (10 9 /l) 1.3 ( ) 5.1 (3.4 10) Neutropenia (< /l), n (%) 2 (28.6) 0 (0) 0.15 Pancytopenia, n (%) 2 (28.6) 0 (0) 0.15 Hemolysis and biochemical patterns LDH (IU/l, Normal <480) 7310 ( ) 1460 ( ) 0.01 Haptoglobin (g/l, Normal >0.2) 0.1 ( ) 0.08 ( ) 0.75 Bilirubin (mmol/l, Normal <10) 21 (16 49) 25 (5 85) 0.72 Outcome Length of hospital stay (days) 14 (5 26) 34 (9 45) 0.03 a The neurological symptom for these two patients was mild confusion (probably caused by anemia). Results are shown as the median (min max) or number of patients, n (%).

4 1020 N. Noël et al. Table 2 Clinical and biochemical characteristics of our patients with anemia and vitamin B12 deficiency Pseudo-TMA (n = 7) No schistocytosis (n = 21) P-value Clinical characteristics Male, n (%) 5 (71.4) 10 (47.6) 0.27 Age at hospitalization (years) 72 (43 78) 54 (27 92) 0.22 Caucasian origin, n (%) 3 (42.9) 16 (76.2) 0.17 Mild confusion, n (%) 2 (28.6) 4 (19) 0.59 Acute kidney failure, n (%) 0 (0) 0 (0) 1 Hematological characteristics Hemoglobin (g/l) 42 (35 79) 61 (31 116) 0.19 Hemoglobin <60 g/l, n (%) 5 (71.4) 10 (47.6) 0.27 Schistocytes, n (%) 7 (100) 0 (0) < Reticulocyte count (10 9 /l) 13.1 ( ) 26.0 ( ) 0.22 Mean corpuscular volume (fl) ( ) ( ) 0.46 Platelet count (10 9 /l) 73 (38 145) 111 (24 307) 0.27 White blood cell count (10 9 /l) 3.4 ( ) 4.7 ( ) 0.17 Neutrophil count (10 9 /l) 1.3 ( ) 2.9 ( ) 0.01 Neutropenia (< /l), n (%) 2 (28.6) 0 (0) 0.01 Pancytopenia, n (%) 2 (28.6) 0 (0) 0.01 Hemolysis and biochemical patterns LDH (IU/l, N < 480) 7310 ( ) 2950 ( ) 0.08 Haptoglobin (g/l, N > 0.2) 0.1 ( ) 0.09 ( ) 0.39 Bilirubin (mmol/l, N < 10) 21 (16 49) 25.5 (4 51) 0.93 Vitamin levels Vitamin B12 (mmol/l, N > 150) 45 (15 111) 105 (15 145) 0.07 Folic acid (nmol/l, N > 15) 17.5 (1 34) 9 (1 54) 0.90 Etiology of vitamin B12 deficiency Pernicious anemia, n (%) 5 (71.4) 7 (33.3) 0.08 Food cobalamin malabsorption, n (%) 1 (14.3) 10 (47.6) 0.12 Nutritional deficiencies, n (%) 1 (14.3) 2 (9.5) 0.72 Post-surgical malabsorption, n (%) 0 (0) 2 (9.5) 0.40 Outcome of vitamin B12 therapy Reticulocyte increase (day) 5 (1 9) 5 (2 10) 1 Length of hospital stay (days) 14 (5 26) 10 (2 37) 0.23 Results are shown as the median (min max) or number of patients, n (%) (i.e. the seven pseudo-tma patients and 21 patients without schistocytosis). The median (range) age of the 28 patients was 56 (27 92) years. Overall causes of deficiency were pernicious anemia (n = 12), food-vitamin B12 malabsorption (n = 11), nutritional deficiency (n = 3) and gastric surgery (n = 2). Although six patients presented with mild confusion, none had symptoms of sensorimotor neuropathy or kidney failure. When we compared the seven pseudo-tma patients with the schistocytosis-free 21 patients, we did not observe any significant difference in age at admission or the gender ratio. Although the two groups had similar hemoglobin levels and platelet counts, the pseudo-tma group had a lower neutrophil count and a greater frequency of pancytopenia (Table 2). Bone marrow aspiration was performed in four pseudo-tma patients and 12 schistocytosisfree patients and yielded similar results in the two groups (i.e. megaloblastosis, dyserythropoiesis and/ or hypersegmented neutrophils). Consistently, the vitamin B12 levels in the pseudo-tma group were half those in the non-pseudo-tma group [median (range): 45 (15 111) vs. 105 (15 145) mmol/l, respectively; P = 0.07]. The main cause of vitamin B12 deficiency in the pseudo-tma group was pernicious anemia. Overall, the treatment consisted of vitamin B12 (1000 mg/day) and folic acid supplementation. For the 28 patients as a whole, the reticulocyte count (reflecting bone marrow regeneration) rose to 293 (92 684) g/l on Day 5 (range: 1 9). All patients improved with vitamin supplementation. Discussion We described seven patients with vitamin B12 deficiency associated with pseudo-tma and compared

5 Pseudothrombotic microangiopathy due to vitamin B12 deficiency 1021 their characteristics with those of six TTP patients. A presentation of pseudo-tma appears to be associated with very low vitamin B12 levels and pernicious anemia. Very elevated LDH levels and a low reticulocyte count were always found. These features are more suggestive of pseudo-tma than TTP and should prompt the physician to screen for vitamin B12 deficiency. Vitamin B12 deficiency is frequent in the general population and can affect up to 15% of the elderly. 9,10 Moreover, pseudo-tma accounts for 2.5% of the hematological disorders related to vitamin B12 deficiency, 6 although few cases have been reported. 4,5 Interestingly, when compared with vitamin B12-deficient patients not displaying schistocytosis, patients in the pseudo-tma group had a lower neutrophil count and a higher incidence of pancytopenia. This more severe presentation may be related to the 2-fold lower vitamin B12 levels seen in pseudo-tma patients. According to the literature, pernicious anemia accounts for the majority of cases of pseudo-tma, 4,5 whereas malabsorption tends to be associated with a milder presentation. To the best of our knowledge, this is the first study to have compared pseudo-tma and TTP patients. Interestingly, over a 4-year period, the same number of patients with each condition was admitted to the emergency department emphasizing that although these conditions are infrequent, they can be encountered in routine practice. Our results are consistent with the fact that pseudo-tma related to cobalamin deficiency must be considered as a differential diagnosis of TTP in a setting of microangiopathic hemolytic anemia (along with autoimmune hemolysis and malignant hypertension). 3 Although pseudo-tma may mimic life-threatening TTP, 5 there are some important clinical differences. Acute kidney failure is suggestive of TTP and was not observed in our seven pseudo- TMA patients. None of the latter had severe, acute neurological symptoms (such as drowsiness or coma, which are symptoms of TTP) but two did show confusion which can also be a symptom of TTP. The combination of a low reticulocyte count and very high LDH levels is suggestive of intramedullary hemolysis, 6 whereas blood smear features such as macro-ovalocytosis and hypersegmented polymorphonuclear cells suggest megaloblastic hematopoiesis. However, a reticulocyte count and a blood smear analysis are not always among the first battery of laboratory investigations performed at admission especially at night. Here, we observed that LDH levels were much higher in pseudo-tma related to vitamin B12 deficiency, whereas bilirubin levels were not as high as expected. 11 This may be related to the fact that LDH enzymes are released during the destruction of nucleated cells (i.e. young erythroblasts in the bone marrow) but not the destruction of mature red blood cells. Furthermore, erythroid progenitors produce little hemoglobin and only low levels of free bilirubin can be released. Finally, patients with pseudo-tma displayed a high MCV. In the setting of hemolytic disorders, a high MCV can be observed due to high reticulocyte count, which is not the case in intramedullary hemolysis. Indeed, physicians should be aware that in case of hemolysis, a high MCV despite a low-tonormal reticulocyte count is strongly suggestive of B12 of folic acid deficiency rather than TTP. This study emphasizes the existence of schistocytosis in both pseudo-tma and TTP. In TTP, schistocytes result from mechanical fragmentation on fibrin strands. However, cytoskeletal fragility is responsible for erythroblast fragmentation in dyserythropoiesis (such as in vitamin B12 deficiency) and major differential diagnoses (such as other acquired dyserythropoiesis or hematological malignancies) should also be considered. 8 We suggest that the bone marrow cytology should be analyzed in cases of aregenerative anemia with schistocytosis. Our study had a number of limitations. First, this was a retrospective analysis conducted in a single center (explaining the low number of enrolled patients). However, this process guaranteed the uniformity of the collected data. The small sample size means that some observed trends toward differences between pseudo-tma patients and the other patients with cobalamin deficiency might become statistically significant in a larger series (e.g. vitamin B12 levels or the proportion of patients with pernicious anemia). Despite these caveats, we believe that our results provide a useful means of rapidly differentiating between TTP on one hand and vitamin B12 deficiency-associated pseudo-tma on the other, in order to adjust patient management in an emergency setting. In conclusion, pseudo-tma related to vitamin B12 deficiency may mimic TTP. However, the marked elevation of LDH and the absence of an elevated reticulocyte count should guide the physician toward a diagnosis of pseudo-tma related to vitamin B12 deficiency. Prompt recognition of pseudo-tma should help to avoid the administration of unnecessary and invasive therapies. Acknowledgements N.N., G.M., G.T., N.A., X.M., J.M.M. collected the data; N.N. and O.L. performed the analysis and wrote the manuscript; N.N., G.M., G.T., N.A.,

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