To the Editor,

Pediatric 2019 novel coronavirus disease (COVID-19) is characterized by a wide clinical spectrum, including hematological manifestations.¹ Anemia and thrombocytopenia occur mainly in severe forms of the disease and in multisystem inflammatory syndrome² but are rare in asymptomatic and mildly symptomatic patients. Only few reports of autoimmune hemolytic anemia (AIHA), rarely associated with immune thrombocytopenia (ITP), have been described in children with COVID-19.^3-5 Here, we present two pediatric cases of severe cold agglutinin disease and a brief review of the literature.

A 15-year-old male presented with nausea, vomiting, and asthenia. He was febrile, tachycardic, and tachypneic. Clinical examination revealed hepatosplenomegaly. Blood tests showed severe hemolytic anemia (hemoglobin 3.7 g/dl, hematocrit 7.4%, bilirubin 3.51 mg/dl, lactic dehydrogenase [LDH] 425 U/L), positive direct antiglobulin test (DAT) with high title cold agglutinins (IgG+/C3d+), and thrombocytopenia (platelets 77 × 10⁹/L) with anti-platelet antibodies. A nasopharyngeal swab resulted positive for SARS-CoV2. Peripheral blood smear revealed severe anisocytosis and aggregates of red blood cells. Evans syndrome was diagnosed. The patient was promptly transfused with red blood concentrates (RBCs), and intravenous corticosteroids (prednisone 2 mg/kg) were administered, showing only a partial response. On day 7, he thus received a 3-day regimen of intravenous immunoglobulins (IVIG) (0.5 mg/kg/die). Over the next few days, hemoglobin levels rapidly improved. Prednisone was gradually tapered after day 13 and then withdrawn at day 32. The patient was discharged on day 16. Extensive immunophenotyping was performed showing normal results. Figure 1 highlights the clinical and laboratory course of this case together with therapies.

A 2-year-old male child affected by beta thalassemia major received an allogeneic hematopoietic stem cell transplantation (HSCT) at 16 months of age from matched unrelated donor. He developed steroid sensitive chronic graft versus host disease (GvHD). At 3-month follow-up, bone marrow aspirate showed complete donor chimerism. Six months after transplantation, he presented with fever, anorexia, and weakness. Nasopharyngeal swab resulted positive for SARS-CoV2. At admission, he was receiving cyclosporine (CsA) as GvHD prophylaxis. Blood samples showed severe hemolytic anemia (hemoglobin 2.3 g/dl, hematocrit 4.8%, bilirubin 2.94 mg/dl, LDH 510 UI/L) with DAT positive for cold pan-agglutinins (IgA+/IgG+/IgM+/C3c+/C3d+). Severe anisocytosis and aggregates of red blood cells were detected at peripheral blood smear. Corticosteroids (prednisone 2 mg/kg) and RBCs were rapidly initiated. Hemoglobin levels slowly normalized, requiring repeated transfusions, the last on day +23. Concurrently, his dosage of prednisone was gradually reduced from day +34 and withdrawn at day +42. The patient tested persistently positive for SARS-Cov2 on nasopharyngeal swabs until day +35.

AIHA is a rare disorder in children, often secondary to self-limited viral or bacterial infections.⁶ When treatment is required, corticosteroids represent the first-line therapy. Other options include rituximab and IVIG.⁷ This condition is more common in children with innate or acquired immune dysregulation (e.g., autoimmune lymphoproliferative syndrome, post-HSCT), in whom the management is frequently challenging.⁸ Moreover, AIHA can be combined with thrombocytopenia (Evans syndrome).⁷

Since the beginning of the pandemic, AIHA has seldom been described in adults with COVID-19^{9, 10} and appears to be even rarer in children.^3-5 The five pediatric patients reported in literature so far (see Table S1) developed severe anemia during the acute phase of infection (hemoglobin range 2.3–6.3 g/dl). No child presented with symptoms of severe respiratory syndrome. They all received steroids as first-line therapy, while only one child required rituximab, because of refractory course of disease.⁴ According to our clinical practice in ES, IVIG were administered as second-line treatment.¹¹ All patients showed a clinical response within 1 month. One child was previously affected by refractory chronic ITP.³ Our second patient developed AIHA 6 months after allogeneic HSCT, during CsA therapy. The other three patients had no relevant comorbidities. In our first case, immunological investigations excluded underlying immune disorders.

Severe AIHA has to be considered within the spectrum of pediatric COVID-19 complications. ITP and bilinear cytopenia (Evans syndrome) can also be associated with SARS-Cov2, even if rarely reported in the pediatric population so far.^{3, 4, 12-15} The underlying pathogenic mechanism could be related to the predominant immunological and inflammatory activation secondary to SARS-CoV-2 infection.^{1, 2, 16} Furthermore, in adults immune-mediated cytopenia seems to be more common in the moderate-to-severe respiratory form of COVID-19.¹⁶ On the contrary, affected children did not present any respiratory involvement. Molecular mimicry has also been proposed as potential mechanism for ITP.¹⁶ Indeed, Angileri et al. reported the structural affinity and potential cross-reactivity between erythrocyte membrane protein Ankyrin-1 and viral protein spike.¹⁷

The severity of anemia appears not to be dependent on the patient's immunological status. Regardless, children with an underlying immune dysregulation may require particular attention considering a specific susceptibility to autoimmune manifestations during infections. Moreover, a protracted course of AIHA, possibly related to a delayed resolution of the infection, can occur in immunosuppressed children, as emblematically observed in our transplanted patient.

So far, the relation between COVID-19 and immune-mediated cytopenia remains unclear. Similar to other viral infections, SARS-CoV-2 can act as a trigger and has to be considered during pandemic among the viral causes of new-onset AIHA. In a pediatric setting, clinical management can be challenging, particularly in patients with an innate or acquired disorder of immune regulation.

致编辑，

儿科 2019 年新型冠状病毒病 (COVID-19) 的特点是临床表现广泛，包括血液学表现。¹贫血和血小板减少症主要发生在疾病的严重形式和多系统炎症综合征²中，但在无症状和轻度症状患者中很少见。在 COVID-19 的儿童中，仅有很少的自身免疫性溶血性贫血 (AIHA) 报告很少与免疫性血小板减少症 (ITP) 相关。^3-5在这里，我们介绍了两例严重的冷凝集素病儿科病例并简要回顾了文献。

一名 15 岁男性出现恶心、呕吐和虚弱。他发热、心动过速和呼吸急促。临床检查发现肝脾肿大。血液检查显示严重溶血性贫血（血红蛋白 3.7 g/dl，红细胞比容 7.4%，胆红素 3.51 mg/dl，乳酸脱氢酶 [LDH] 425 U/L），直接抗球蛋白试验（DAT）阳性，具有高标题冷凝集素（IgG+/C3d+ ) 和血小板减少症 (血小板 77 × 10 ⁹/L) 与抗血小板抗体。鼻咽拭子检测结果呈 SARS-CoV2 阳性。外周血涂片显示严重的红细胞不均匀和红细胞聚集。埃文斯综合征被诊断出来。患者立即输注红血球 (RBC)，并给予静脉注射皮质类固醇（泼尼松 2 mg/kg），仅显示部分缓解。因此，在第 7 天，他接受了为期 3 天的静脉内免疫球蛋白 (IVIG) 方案（0.5 mg/kg/die）。在接下来的几天里，血红蛋白水平迅速改善。强的松在第 13 天后逐渐减量，然后在第 32 天停用。患者于第 16 天出院。进行了广泛的免疫表型分析，结果正常。图 1 突出显示了该病例的临床和实验室过程以及治疗方法。

一名患有重型 β 地中海贫血症的 2 岁男童在 16 个月大时接受了来自匹配的无关供体的异基因造血干细胞移植 (HSCT)。他开发了类固醇敏感的慢性移植物抗宿主病 (GvHD)。在 3 个月的随访中，骨髓抽吸物显示出完全的供体嵌合体。移植后六个月，他出现发烧、厌食和虚弱。鼻咽拭子对 SARS-CoV2 呈阳性反应。入院时，他正在接受环孢素 (CsA) 作为 GvHD 预防。血样显示严重溶血性贫血（血红蛋白 2.3 g/dl，血细胞比容 4.8%，胆红素 2.94 mg/dl，LDH 510 UI/L），DAT 冷泛凝集素（IgA+/IgG+/IgM+/C3c+/C3d+）阳性。在外周血涂片中检测到严重的红细胞不均匀和红细胞聚集。皮质类固醇（泼尼松 2 mg/kg）和红细胞迅速启动。血红蛋白水平慢慢恢复正常，需要反复输血，最后一次输血是在第 +23 天。同时，他的泼尼松剂量从+34天开始逐渐减少，并在+42天停药。该患者的鼻咽拭子检测 SARS-Cov2 持续阳性，直到第 +35 天。

AIHA 是一种罕见的儿童疾病，通常继发于自限性病毒或细菌感染。⁶当需要治疗时，皮质类固醇代表一线治疗。其他选择包括利妥昔单抗和 IVIG。⁷这种情况在先天性或获得性免疫失调的儿童中更为常见（例如，自身免疫性淋巴增生综合征，HSCT 后），在这些儿童中，管理常常具有挑战性。⁸此外，AIHA 可合并血小板减少症（埃文斯综合征）。⁷

自大流行开始以来，AIHA 很少在 COVID-19 ^9、10的成人中出现，在儿童中似乎更为罕见。^3-5迄​​今为止，文献中报道的 5 名儿科患者（见表 S1）在感染的急性期（血红蛋白范围 2.3-6.3 g/dl）出现严重贫血。没有儿童出现严重呼吸系统综合症的症状。他们都接受了类固醇作为一线治疗，而只有一个孩子需要利妥昔单抗，因为病程难治。⁴根据我们在 ES 中的临床实践，IVIG 被用作二线治疗。¹¹所有患者均在 1 个月内出现临床反应。一名儿童以前曾受到难治性慢性 ITP 的影响。³我们的第二名患者在 CsA 治疗期间异基因 HSCT 后 6 个月出现 AIHA。其他三名患者没有相关的合并症。在我们的第一个案例中，免疫学研究排除了潜在的免疫疾病。

必须在儿科 COVID-19 并发症范围内考虑严重 AIHA。ITP 和双线性血细胞减少症（埃文斯综合征）也可能与 SARS-Cov2 相关，尽管迄今为止儿科人群中很少报道。^{3, 4, 12-15}潜在的致病机制可能与继发于 SARS-CoV-2 感染的主要免疫和炎症激活有关。^{1, 2, 16}此外，在成人中，免疫介导的血细胞减少似乎在 COVID-19 的中度至重度呼吸形式中更为常见。¹⁶相反，受影响的儿童没有出现任何呼吸道受累。分子模拟也被提议作为 ITP 的潜在机制。¹⁶事实上，Angileri 等人。报道了红细胞膜蛋白 Ankyrin-1 和病毒蛋白尖峰之间的结构亲和力和潜在的交叉反应性。¹⁷

贫血的严重程度似乎不取决于患者的免疫状态。无论如何，考虑到感染期间对自身免疫表现的特定易感性，潜在免疫失调的儿童可能需要特别注意。此外，免疫抑制的儿童可能会出现延长的 AIHA 病程，这可能与感染的延迟消退有关，正如我们在移植患者中所观察到的那样。

到目前为止，COVID-19 与免疫介导的血细胞减少之间的关系仍不清楚。与其他病毒感染类似，SARS-CoV-2 可以作为触发因素，在大流行期间必须考虑到新发 AIHA 的病毒原因。在儿科环境中，临床管理可能具有挑战性，特别是在患有先天性或获得性免疫调节障碍的患者中。