Pseudo-thrombotic microangiopathy due to folate deficiency

Abstract

Classically, deficiencies of vitamin B₁₂ and folate are associated with megaloblastic anaemia. Additionally, vitamin B₁₂ is able to cause a haemolytic anaemia in the form of pseudo-thrombotic microangiopathy (pseudo-TMA). Here, we present a case of a middle-aged woman with a history of Roux-en-Y gastric bypass who presented with dyspnoea and fatigue and was found to have thrombocytopenia and a non-immune haemolytic anaemia. Work-up for haemolytic uraemic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal haemoglobinuria, infection, malignancy and autoimmune conditions was unremarkable. Her haemolytic anaemia and thrombocytopenia resolved with folate replenishment. She was diagnosed as likely having pseudo-TMA secondary to folate deficiency.

Background

Folate is a water-soluble B vitamin (B₉) with many functions in the body. Folate deficiency principally impacts proliferative cells due to its role in nucleic acid synthesis, and clinical manifestations of folate deficiency range from neural tube defects in neonates, to gastrointestinal symptoms like diarrhoea and aphthous ulcers, to bone marrow disorders including megaloblastic anaemia or pancytopenia.¹ Despite efforts to fortify foods with folate, clinical folate deficiency and inadequacy persist, either due to decreased availability (impaired absorption, reduced intake), increased requirement (physiological growth, pathological proliferation, certain drugs) or both.²

Cobalamin, another B vitamin (B₁₂), participates in many of the same metabolic processes as folate, and deficiencies in either can have overlapping clinical presentations. A clinical entity known as pseudo-thrombotic microangiopathy (pseudo-TMA) has been uniquely linked to B₁₂ deficiency and has been elucidated in several case reports.^3–27

Pseudo-TMA is characterised by non-immune haemolytic anaemia, thrombocytopenia and the presence of schistocytes. In primary TMA syndromes such as thrombotic thrombocytopenic purpura (TTP), haemolytic uraemic syndrome (HUS) and atypical HUS, haemolysis occurs as platelet microthrombi complexes precipitate in arterial capillary beds, shearing red blood cells (RBCs).²⁸ This occurs via Willebrand factor-platelet aggregates in the case of TTP, Shiga toxin-mediated microthrombi in the case of HUS, and immunoglobulin or complement complexes in the case of atypical HUS.²⁸ In pseudo-TMA, hypoproliferative bone marrow, structurally weak RBC membranes, and endothelial dysfunction cause haemolytic anaemia and thrombocytopenia due to elevated homocysteine levels.^{29 30}

Although cobalamin deficiency has been linked to pseudo-TMA, we were unable to discover any reported cases of pseudo-TMA as a result of folate deficiency. Only one case report linking severe folate deficiency and non-immune haemolytic anaemia exists.³⁰ Here, we report a case of likely pseudo-TMA secondary to folate deficiency in a woman following gastric bypass.

Case presentation

A woman in her 40s with a medical history of morbid obesity treated with Roux-en-Y gastric bypass (approximately 20 years ago) and alcohol use disorder (consumption of 750–1500 mL of wine daily for approximately 20 years) presented to an outside hospital with shortness of breath and fatigue. On admission at the outside hospital, complete blood count (CBC) was significant for severe macrocytic anaemia, thrombocytopenia and leucocytosis. Haemoglobin was 55 g/L with mean corpuscular volume (MCV) of 111 fL; platelet (PLT) count was 136 x 10⁹/L and white blood cell (WBC) count was 18.8 x 10⁹/L with a differential showing 76% neutrophils, 21% lymphocytes and 3% monocytes. Reticulocyte count was 3.5%. Creatinine was 0.8 mg/dL. International normalised ratio (INR) was 1.3. Vitamin studies showed normal vitamin B₁₂ of 303 pg/mL and undetectable folate. Homocysteine was elevated to 108.1 (reference range 5–15 µmol/L); methylmalonic acid level was not tested. High-sensitivity cardiac troponin was elevated to 14 ng/L (reference range <14 ng/L). Work-up for haemolysis showed an elevated lactate dehydrogenase (LDH) of 1457 U/L, undetectable haptoglobin and negative Coombs’ test. Peripheral smear demonstrated substantial RBC dysmorphic findings with frequent schistocytes, elliptocytes, stomatocytes and anisopoikilocytosis as well as toxic granulations and thrombocytopenia. The patient scored 5 points on the PLASMIC score, placing her at intermediate risk (6%) for severe ADAMTS13 deficiency.³¹ Subsequently, the patient received two units of packed RBCs, was started on oral prednisone, vitamin B₁₂ and folate, and was transferred to our academic centre with concern for TTP for initiation of plasmapheresis.

On presentation, vital signs were significant for mild sinus tachycardia but were otherwise within normal limits. Review of systems was significant for fatigue, dyspnoea, dark urine, multiple episodes of non-bloody diarrhoea and alopecia. Physical examination was notable for a body mass index of 65.16 kg/m² and aphthous ulcers involving the hard palate. The neurological examination was unremarkable and non-focal, and the patient was alert and oriented to person, place and time.

The CBC demonstrated haemoglobin of 64 g/L with elevated MCV of 101.6 fL and decreased PLT count of 62 x 10⁹/L from 136 x 10⁹/L at outside hospital. Complete metabolic profile was notable for total bilirubin elevated to 2.3 mg/dL with conjugated bilirubin of 1.3 mg/dL but was otherwise unremarkable including creatinine 0.8 mg/dL. Fibrinogen was within normal limits at 376 mg/dL, as was INR at 1.2. Based on vitamin studies at outside hospital, homocysteine laboratory test was repeated which was increased to 46 µmol/L (reference range 5–15 µmol/L), and methylmalonic acid was within normal limits at 0.27 µmol/L (reference range <0.4 µmol/L), indicating folate deficiency. Peripheral blood smear showed leucocytosis with absolute neutrophilia and hypersegmented neutrophils, macrocytic anaemia with marked anisopoikilocytosis, rare schistocytes and thrombocytopenia (figure 1). CT of the abdomen and pelvis with contrast showed postsurgical changes of Roux-en-Y gastric bypass, diffuse hepatic steatosis and no evidence of lymphoma.

Figure 1.

Peripheral blood smear (magnification 50×) with hypersegmented neutrophils, anisopoikilocytosis, occasional reticulocytes, teardrop cells, three to four schistocytes and thrombocytopenia with overall normal morphology.

Given underlying severe haemolytic anaemia, worsening thrombocytopenia and findings of red cell fragments, she was started on plasmapheresis and caplacizumab with concern for TTP. She was simultaneously initiated on prednisone, cobalamin and folate replacement.

The following day, ADAMTS13 level resulted at 62% which is low (reference range >70%) but not consistent with TTP; therefore, plasmapheresis and caplacizumab therapy was terminated. Further work-up for anaemia indicated no evidence of paroxysmal nocturnal haemoglobinuria or pernicious anaemia, negative serologies for HIV and hepatitis C, and hepatitis B serologies consistent with previous vaccination. Beta-HCG was negative. Vitamin and mineral studies showed normal thiamine of 89 nmol/L. Copper and vitamin E levels were within normal limits. 25-Hydroxy vitamin D was undetectable. The patient was continued on prednisone, as well as supplemental calciferol, folate, B₁₂ and thiamine. Given the lack of evidence for other causes of anaemia, it was postulated that this incidence of acute haemolysis and thrombocytopenia was secondary to severe folate deficiency in the setting of inadequate vitamin supplementation status post-Roux-en-Y gastric bypass complicated by alcohol use disorder.

Outcome and follow-up

Over the course of her hospital stay with steroid treatment and vitamin replenishment, the patient’s anaemia improved to a haemoglobin of 78 g/L and MCV improved to 105.7 fL at the time of discharge. Haptoglobin normalised to 62 mg/dL, LDH decreased to 447 U/L and total bilirubin normalised to 0.9 mg/dL. Serum folate levels normalised to 20.2 ng/mL. She was discharged on a prednisone taper and empirical supplementation with thiamine, folate, and vitamins D and B₁₂. After discharge, a gene panel for atypical HUS resulted negative. At follow-up 3 weeks after discharge, haemoglobin had normalised to 127 g/L with MCV of 96.5 fL. PLT and WBC count had both normalised to 211 x 10⁹/L and 8.58 x 10⁹/L, respectively. Likewise, LDH had normalised to 246 U/L. The patient was adherent to the vitamin regimen at follow-up 5 months after hospital discharge with maintenance of blood counts within normal limits; however, she continued to report alcohol use (750 mL wine daily).

Discussion

Our patient presented with acute non-immune haemolytic anaemia and thrombocytopenia in the setting of severe folate deficiency likely due to impaired absorption secondary to Roux-en-Y gastric bypass and reduced intake due to chronic alcohol use disorder and non-adherence to vitamin supplementation. Her presentation of severe folate deficiency was also complicated by episodes of watery diarrhoea, aphthous ulcers, and new-onset, non-scarring alopecia consistent with folate deficiency.¹ In patients undergoing Roux-en-Y gastric bypass, up to 45% develop folate deficiency due to bypass of the proximal small bowel, the main site for absorption.³² Therefore, it is essential for patients to maintain lifelong vitamin and mineral supplementation to maximise absorption opportunity despite altered anatomy. However, our patient was non-adherent to supplementation, and further impaired nutrient absorption with several years of heavy alcohol use.³³ Her laboratory values indicated a deficiency of folate and vitamin D, otherwise, thiamine, vitamin B₁₂, vitamin E and copper were within normal limits. We were able to demonstrate an improvement of her anaemia with folate supplementation, and her folate levels increased from 4.5 ng/mL to 20.2 ng/mL over the course of her hospital stay.

Another important consideration when investigating vitamin deficiencies is the dietary history. The patient’s diet prior to hospitalisation was lacking in sources of folate, and she denied consumption of leafy green vegetables, fresh fruits or juices, legumes, or fortified grains or cereals. The patient reported that although her vitamin supplement non-adherence and alcohol use disorder were long-standing (>15 years), her limited, folate-poor diet was more recent (approximately 1 year up until hospitalisation). She attributed her dietary change to the COVID-19 pandemic and reliance on food delivery options for almost 100% of her meals, mainly fast-food restaurants, consuming one to two total meals per day consisting chiefly of fried chicken or beef. Interestingly, although lack of vitamin supplementation and alcohol use certainly contributed to folate deficiency in this case, it is possible that substantial changes to the patient’s diet further restricted folate stores and were the catalyst for her acute presentation of pseudo-TMA.

Interestingly, prior to this acute event, there was evidence that the patient had chronic macrocytic anaemia, with laboratory values 2 months prior to hospitalisation demonstrating haemoglobin of 95 g/L with MCV of 116 fL. Vitamin B₁₂, thyroid-stimulating hormone, total bilirubin and PLT count were otherwise within normal limits, suggesting macrocytosis secondary to folate deficiency. For patients who have undergone weight loss surgery (WLS), it is recommended routinely screening for folate status and orally supplementing 1000 µg folate daily, as up to 65% of patients post-WLS are folate deficient.³⁴ This case highlights the importance of appropriate follow-up after WLS to screen for nutritional deficiencies, as otherwise there are possible critical consequences.

In reported cases of pseudo-TMA as the result of cobalamin deficiency, it is believed that elevated levels of homocysteine cause endothelial dysfunction, PLT activation and resultant microangiopathy.^{29 30} Homocysteine has also been proposed as a haemolytic toxin.³⁵ Additionally, cobalamin-deficient, macrocytic RBCs are more fragile, resulting in increased intramedullary and intravascular haemolysis.³⁶ Importantly, the metabolism and utility of cobalamin and folate are linked. Cobalamin is needed for regeneration of tetrahydrofolate and conversion of homocysteine to methionine; hence, deficiency in either cobalamin or folate will lead to elevated levels of homocysteine in the serum.³⁷ Therefore, although there have not been reported cases of folate deficiency leading to pseudo-TMA, the underlying mechanism for both folate-induced and cobalamin-induced pseudo-TMA may be very similar, if not the same. At initial presentation, our patient exhibited extremely elevated homocysteine of 108.1 µmol/L which decreased to 46 µmol/L at time of discharge. This may explain in part the resolution of haemolytic anaemia observed in this patient.

In the single documented case report of folate-induced haemolytic anaemia, there were similarities to this case report.³⁰ Both patients had a history of alcohol use disorder and initially presented with severe macrocytic anaemia (haemoglobin <60 g/L) and relatively mild thrombocytopenia (>100 x 10⁹/L) with severely decreased folate levels (<2.5 ng/mL), but vitamin B₁₂ was otherwise within normal limits. Zhang et al³⁰ did not report investigation for TTP or HUS as causes for the presentation, but both cases had negative work-up for autoimmune haemolytic anaemia, paroxysmal nocturnal haemoglobinuria and haematological malignancy.

A shared theme in reports of pseudo-TMA is the initial use of plasmapheresis to treat suspected TTP. The prompt recognition of TTP and initiation of plasmapheresis are critical because without treatment, mortality reaches 80%–90%.¹⁰ However, treatment of pseudo-TMA with plasmapheresis is not only ineffective but also puts patients at unnecessary risk of complications of plasmapheresis, resulting in longer hospital stays and increased expenditure.⁹ Plasmapheresis may cost upwards of $2500/day for a health system to administer, whereas folate supplementation costs only several dollars per day. Moreover, in a systemic review of pseudo-TMA due to cobalamin deficiency by Tran and Tran, 13 of 14 patients underwent line placement and plasma exchange that was ultimately not indicated, with 2 of 14 patients experiencing severe complications of this therapy.¹⁰ Additionally, Tran and Tran noted that all 14 patients studied demonstrated universal depression of the reticulocyte proliferation index (RPI), as was seen in our patient (decreased to 0.66). Depressed levels of RPI should raise suspicion for hypoproliferation secondary to pseudo-TMA, and raise suspicion for folate or vitamin B₁₂ deficiency. Overall, consideration of pseudo-TMA in the differential diagnosis of TMA, with implementation of RPI query as a diagnostic clue, holds promise to save hundreds of thousands of dollars in healthcare cost with reduced morbidity and mortality.

It is important to consider what other aetiologies may account for this presentation. Although the Coombs’ test was negative in our patient, about 10% of patients with warm-type autoimmune haemolytic anaemia will have a negative result, either due to non-IgG antibodies like IgA and IgM that do not interact with the anti-IgG or C3d Coombs reagent, or low-affinity antibodies that are capable of inducing haemolysis but are not detected by routine screening.³⁸ An underlying autoimmune condition or atypical HUS not identified on the gene panel obtained is possible but unlikely given intact renal function. Myeloproliferative or myelodysplastic syndromes are possible but less likely given relatively acute thrombocytopenia and acute-onset haemolytic anaemia. Moreover, there was no evidence on CT imaging or blood smear to support a haematological malignancy. Because the patient received empirical supplementation with both folate and cobalamin, it is uncertain if correction of the haemolytic anaemia was due to replenishment of folate, or if cobalamin treatment also contributed to resolution of symptoms. Likewise, it is impossible to retrospectively determine if steroid treatment and plasmapheresis also contributed to reversal of TMA.

Severe folate deficiency may represent a rare cause of pseudo-TMA. Exploration of vitamin levels in patients presented for presumed TTP, especially those at risk of impaired absorption or reduced intake, may reduce unnecessary plasmapheresis, exposure to donor plasma and healthcare cost. As vitamin deficiencies are largely reversible with low-risk treatment, and evidence supports the amelioration of pseudo-TMA with supplementation, it may be beneficial for clinicians to consider more extensive metabolic causes when working up the cause of TMA, and use the RPI as a distinguishing feature of pseudo-TMA versus true TMA. Hopefully, future studies will better elucidate the mechanism for folate-induced haemolytic anaemia as seen in this patient.

Learning points.

• Severe folate deficiency may present as pseudo-thrombotic microangiopathy.

• Consideration of vitamin deficiencies may reduce unnecessary plasmapheresis and associated morbidity and cost.

• Appropriate follow-up after weight loss surgery is important to screen for nutritional deficiencies, as otherwise there are possible critical consequences.

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