Management of Mixed Warm/Cold Autoimmune Hemolytic Anemia: A Case Report and Review of Current Literature

Background Mixed warm/cold autoimmune hemolytic anemia (AIHA) is a rare diagnostic entity with limited therapeutic options. Previous literature has described the diagnostic difficulty in this pathology and the limited response rates to corticosteroids. Furthermore, there is limited evidence regarding the use of rituximab in this condition. Methods Alongside our case report, we conducted a scoping review of case reports/case series describing mixed AIHA, their treatment, and clinical outcomes since 2000. Inclusion criteria included a confirmed diagnosis of mixed AIHA (confirmed warm antibodies and cold agglutinins based on DAT). Case Summary/Results. We present a case of mixed AIHA in an 83-year-old female presenting with extensive, bilateral pulmonary embolisms and left renal vein thrombosis. The patient underwent extensive workup with no identifiable provoking etiology. Initial treatment involved prednisone therapy was transitioned to rituximab upon diagnosis of mixed AIHA. The patient demonstrated a mixed response with stable hemoglobin and transfusion independence; however, with persistently elevated hemolytic indices following completion of rituximab treatment. Our literature review identified 16 articles; two were excluded for unavailable clinical details. The most commonly associated conditions included autoimmune conditions (n = 5, 26%) and lymphoproliferative disorders (n = 3, 12%). The most common treatment involved corticosteroids; seven studies involved the use of rituximab. Conclusion Mixed AIHA represents a complex diagnosis and optimal management is not well established. Consistent with our case, recent literature suggests a promising response to rituximab and a limited response to steroid treatment. Given the limited literature, additional studies are required to elucidate optimal management of this unique pathology.


Introduction
Mixed warm/cold autoimmune hemolytic anemia (AIHA) is a rare diagnostic entity with limited therapeutic options. Previous literature has described the diagnostic difculty in this pathology and a limited response to steroid therapy. Furthermore, there is limited evidence regarding the use of rituximab in this condition. In this case report, we describe a case of severe mixed warm and cold autoimmune hemolytic anemia with a complicated diagnosis which was treated with both steroids and rituximab with a clinical response evidenced by stability in hemoglobin and improvement in hemolytic indices. Further, we conducted a review of recent literature involving case reports and series of mixed warm/cold AIHA and their clinical courses.

Research Aim
(i) To highlight the diagnostic difculties within mixed warm/cold autoimmune hemolytic anemia and use of rituximab as a management strategy within this rare pathology

Case Report
Our case involved an 83-year-old female with a past medical history signifcant for provoked deep vein thrombosis (DVT) following an ankle fracture, hypertension, and osteoporosis endorsing a period of worsening fatigue and exertional dyspnea over the preceding several weeks. Upon admission, the patient was found to have elevated liver enzymes as well as D-Dimer greater than 4000 μg/L (<500 μg/L) which prompted further workup for thromboembolic disease. Computed tomography angiography identifed extensive bilateral pulmonary emboli and a large left renal vein thrombus. Doppler venous US of the legs identifed a left-sided proximal DVT. In addition, the complete blood count (CBC) showed a polychromic and normocytic anemia with a hemoglobin of 59 g/L requiring red cell transfusion, leukocytes of 13.7 × 10 9 /L, platelets of 277 × 10 9 /L, and reticulocytes of 57 × 10 9 /L (23-90 × 10 9 /L). Further investigations demonstrated evidence of hemolysis including elevated lactate dehydrogenase (LDH) of 1347 U/L (<140 U/L), total and direct bilirubin of 37 μmol/L and 15 μmol/L, respectively (<20 μmol/L and <5 μmol/L), and decreased haptoglobin of less than 0.08 g/L (0.30-2.00 g/L). On the peripheral smear, there were marked spherocytes and mild Howell-Jolly bodies with signifcant agglutination. Initial hemolysis workup included a Direct Antibody Test (DAT) which was invalid twice due to positive controls. Investigations revealed positive cold autoantibody at 4 degrees Celsius (°C) and negative ones at 37°C. Due to clinical suspicion of cold autoimmune hemolysis, the patient was subsequently started on folic acid supplementation, and cold avoidance was recommended. Secondary etiologies including infection were investigated including HIV, EBV, and hepatitis B serologies, all of which were negative as well as SARS-CoV-2 PCR. Malignancy workup including CT chest, abdomen, and pelvis did not reveal evidence of malignancy. A bone marrow biopsy was also performed, demonstrating erythroid hyperplasia without evidence of a lymphoproliferative disorder, and paroxysmal nocturnal hemoglobinuria markers were also sent and found to be negative.
Due to ongoing clinically signifcant hemolysis, the patient was started on a trial of prednisone at a dose of 1 mg/ kg for four days. Unfortunately, the patient continued to have signifcant hemolysis demonstrated by ongoing transfusion requirements as well as elevated hemolytic markers, and additional investigations were undertaken. Due to ABO type discrepancies, samples were sent to our reference lab at Canadian Blood Services for further analysis. Te patient's red cells were found to be reactive by immediate spin with diferent monoclonal ABO antisera (anti-A, anti-B, and anti-A, B) due to the presence of a cold autoantibody. Te determination of the cold antibody titre was unable to be performed and therefore remained unknown in this case. Te interference was not eliminated by the washing patient's red blood cell suspension three times with prewarmed saline and retesting. Te valid ABO typing was obtained by using the prewarmed technique and incubation at 37°C for an hour. A valid DAT result was obtained from testing the patient's dithiothreitol (DTT)treated cells. Te patient's plasma was reactive with all papain-treated cells tested by the indirect antiglobulin testing including autocontrol. An acid eluate prepared from the patient's red cells was reactive with all cells tested by the gel method indicating the presence of a warm autoantibody. Cold autoabsorption with rabbit erythrocytes stroma was also performed to remove the cold autoantibody, and the adsorbed plasma was still reactive, but the strength of the reaction was reduced by gel. A diagnosis of both cold and warm autoantibodies was confrmed, while all major clinically signifcant alloantibodies were excluded.
Based on these fndings, prednisone was discontinued, and the patient was treated with rituximab 375 mg/m 2 for a total of four doses. Te prednisone course was limited to four days due to the discovery of cold antibodies as well as ongoing signifcant anemia. Te patient began demonstrating signs of response after 2 doses of rituximab evidenced by decreasing transfusion requirements, improved hemoglobin stability, and improvement in hemolytic indices. Follow-up at 5 months after rituximab completion demonstrated ongoing hemoglobin stability with evidence of compensated hemolysis (Table 1).

Discussion
Mixed warm/cold autoimmune hemolytic anemia is a relatively rare entity and current evidence regarding optimal management of these cases is limited. Furthermore, there is considerable variability between sources in diagnostic criteria which further complicates the diagnosis and management of this pathology [1].
Previous studies have found that mixed warm/cold AIHA account for 6.5-8.3% of AIHA cases and approximately 50% of which are idiopathic without an identifable secondary etiology [2,3]. Interestingly, an association between mixed AIHA and SLE has been described; however, this was not observed in our case [4,5]. Mayer et al. investigated 2194 patients with detected warm autoantibodies and found only 2 patients, less than 0.1%, had features consistent with mixed warm/cold AIHA which makes the true prevalence of this pathology difcult to ascertain [1].
Te use of steroids in the management of this condition has been previously described with a mixed response, and in several cases, patients required splenectomy due to ongoing hemolysis (Table 2) [1,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. We conducted a literature review of case reports published and available on PubMed since the year 2000 with confrmed mixed AIHA (confrmed warm antibodies and cold agglutinins based on DAT) and their clinical course ( Table 2). Additional case reports were excluded for unavailable clinical details [19,20]. Rituximab, a monoclonal CD20 antibody, is another treatment that has recently been employed as an efcacious therapeutic strategy amongst warm and cold AIHA [21][22][23][24]. Rituximab has been described for the management of mixed AIHA, and in a case report, a patient responded with resolution of hemolysis after two courses of rituximab following initial management with prednisone [8]. Our study similarly demonstrated an initial response following one course of rituximab with stabilization in hemoglobin, and will continue to be observed for long-term remission.
In summary, mixed warm/cold AIHA represents a complex diagnosis, and the optimal management of these cases has not been well elucidated. Case series report largely limited early response following steroid therapy, and recent case reports suggest a promising response to rituximab. However, given the limited literature surrounding this topic, additional investigations would be required to further elucidate the optimal management of this pathology.  Response with stabilization in hemoglobin

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Disclosure
Te manuscript was already published as an Abstract at the Canadian Transfusion Medicine Society conference [25].

Conflicts of Interest
Te authors declare that there are no conficts of interest.