Updates on the Diagnosis and Management of Cold Autoimmune Hemolytic Anemia

INTRODUCTION

Immune-mediated hemolytic anemias can be divided into 2 separate types based on in vitro experiment performed over 80 years ago.¹ The most common form of immune-mediated hemolysis occurs in vitro at 37°so-called warm antibody type hemolytic anemia.² This is covered in other chapters in this volume of Hematology Oncology Clinics of North America. The other is cold hemolytic anemia. Patients with this disorder will have agglutination with or without hemolysis at 3 degree centigrade without adding an antiglobulin (Coombs antiserum) to promote the reaction. Patients were previously classified as having primary cold agglutinin disease usually in the context of an IgM monoclonal protein in the serum that serves to fix complement to the red blood cells. A secondary form of cold agglutinin syndrome, generally self-limited and postinfectious, is historically associated with the Epstein Barr virus or with mycoplasma pneumonia and atypical bacterium.³ In these instances, a polyclonal immunoglobulin M molecule develops as part of the primary immune response to the infection and leads to deposition of complement on the surface of the red blood cell. Some patients will have features of both warm and cold hemolysis with immunoglobulin G and complement on the red cell surface.⁴ This category is completed with paroxysmal cold hemoglobinuria whereby an immunoglobulin G molecule capable of activating complement and leading to intravascular hemolysis develops often as a postinfectious phenomenon historically described with syphilis but seen in pregnancy⁵ and with viral infections including parvovirus.⁶

Cold agglutinins were identified over 100 years ago long before the concepts of immunoglobulins and complement were developed and before the development of the direct antiglobulin test. The 1st monoclonal antibody ever identified was a cold agglutinin from a patient with cold agglutinin disease. Cold agglutinin antibodies are typically immunoglobulin M but rarely may be immunoglobulin G or rarely immunoglobulin A. The target antigen on the red cell surface to which the immunoglobulin binds is Ii, the so-called individuality blood grouping. In neonates and children, the red cells express i but after the age of 18 months all red cells express I.⁷

PATHOPHYSIOLOGY

Primary cold agglutinin disease is responsible for approximately 15% of all autoimmune hemolytic anemias. Cold agglutinins are auto-antibodies that react optimally at temperatures of 3 to 4 degree centigrade. The patient’s serum will agglutinate with all available red cells in a laboratory. The so-called cold agglutinin titer is measured by serial dilutions of the patient’s serum by a factor of 2 and the titer is the most dilute serum capable of causing visual agglutination. A clinically significant titer is usually considered greater than 1:64 (2⁶ dilution of the patient’s serum).⁸ Some cold agglutinins will lead to agglutination at higher temperatures. The temperature at which agglutination still occurs is referred to as the thermal amplitude. The higher the thermal amplitude the more clinically significant the agglutinating antibody is.⁹ If the thermal amplitude exceeds 28 to 30-degree centigrade red cells will agglutinate in acral parts of the circulation even at mild ambient temperatures and often complement fixation and complement-mediated extravascular hemolysis will ensue.¹⁰

Because red cells are positively charged, they naturally repel one another and will naturally disperse when viewed on a glass slide. Because immunoglobulin M is a macromolecule with a molecular weight approaching 1 million Daltons, it can bridge the intercellular distance which is seen on a peripheral blood film is agglutination, as distinct from rouleaux formation. The IgM protein is typically glycosylated.¹¹

The mechanism underlying hemolysis is well understood. Once IgM binds to the surface of the red blood cell via the I antigen-binding site, complement is fixed to the surface of the red blood cell via the classical complement pathway starting with C1. Theoretically, it would be possible for the alternate complement pathway to activate C3 but in practice, this rarely is seen. In sequence, the activation of complement brings complement components 2, 4, and 3 to the surface of the red cell. At physiologic temperatures, the IgM molecule does not remain on the surface of the red cells. The red cell circulates with the 3rd component of complement on the surface. Naturally occurring C3 convertase removes C3a from the red cell and the cells are now circulating with C3b on the surface. The mononuclear phagocyte system is rich in C3b receptors. This system is found in the spleen, the Kupffer cells of the liver, alveolar macrophages, and lymph nodes. As red cells with C3b on the surface interface with the cells binding C3b removal of small parts of the membrane occurs. As these cells repeatedly pass through, more of the red cell membrane is removed resulting in spherocytes. Ultimately sufficient membrane is removed leading to red cell destruction in the extravascular space. Intravascular hemolysis is generally not seen and when intravascular hemolysis occurs it is associated with a major flare in the intensity of hemolysis often triggered by an infection. The entire circulating red cell mass is not susceptible to hemolysis. Enzymes act on C3b and remove C3c leaving the red cells with a coating of C3d. These cells are resistant to hemolysis as no receptors are found in the mononuclear phagocyte system. Fatal hemolysis is rare because C3d coated red blood cells will not undergo extravascular hemolysis.¹²

CLINICAL FEATURES

In most patients, the hemolysis, when it occurs, is completely extravascular therefore clinical features such as fever, flank pain, and dark urine are lacking as these are typical features of intravascular hemolysis. These patients’ clinical features cannot be distinguished from warm immune hemolytic anemia and include indirect hyperbilirubinemia, LDH elevation, reticulocytosis, absent haptoglobin, and spherocytes visible in the peripheral blood film Fig. 1.¹³ Direct antiglobulin testing is positive and specific IgG and complement antibody testing will be negative for immunoglobulin G and positive for the complement. Artifactual changes may be seen as clumps of red cells pass through the aperture of a Coulter counter-type machine. These would include elevations of the mean corpuscular volume to levels that are not physiologic such as an MCV of more than 130 fL can occur. In addition, wild inaccuracy of the red blood cell count may occur. Artifactually depressed red cell counts as low as 0.5 million per femtoliter can be recorded which results in calculating hematocrits of less than 5%. As the hemoglobin is measured after the red cells are lysed inside the machine the value is quite accurate and may be used for all clinical decision making. The direct antiglobulin test (Coombs) is positive for complement. In practice, if the Coombs test is negative cold agglutinin hemolysis would be very unusual. Although the hemoglobin will be accurate in a routinely processed specimen, an unwarmed specimen will give spurious results for rbc number, MCV, and hematocrit.¹⁴

Fig. 1.

Pathophysiology of cold agglutinin in disease.

One should expect patients with cold hemagglutinin disease to have a monoclonal IgM protein however, a peak on the serum protein electrophoresis may not occur and the protein may be detectable only by immunofixation reflecting a size less than 0.2 g/dL. The monoclonal protein typically ranges between 0.5 and 1.5 g/deciliter. A bone marrow biopsy should be performed in all patients, and will demonstrate red cell hyperplasia as well as clonal lymphoplasmacytic cells. The number may be sufficient for a pathologist to designate this as lymphoplasmacytic lymphoma but in many cases, the clone is only detectable using immunohistochemical techniques or flow.¹⁵ As in virtually all forms of lymphoplasmacytic lymphoma, if sufficient tumor cells are detected, a mutation in MYD88 will be detected. This is not required for the diagnosis but is confirmatory and supportive. The diagnosis of lymphoplasmacytic lymphoma in the bone marrow and any level of IgM monoclonal protein fulfills the criteria for a diagnosis of Waldenstrom Macroglobulinemia. However, most patients with cold agglutinin disease do not fulfill the consensus criteria for the treatment of macroglobulinemia unless the hemolysis is sufficient to produce symptomatic anemia.¹⁶

The most common symptomatic manifestation of cold agglutinin hemolytic anemia is symptoms related to the anemia.¹⁷ As in all patients with chronic anemia there is a shift in the oxygen saturation curve due to rising levels of intracellular 2,3 DPG. There-fore, higher levels of oxygen are liberated to the tissues at higher partial pressures of oxygen. In the clinic, one observes well-compensated function and the ability to perform activities of daily living at hemoglobin less than 7 g/deciliter. As red cells circulate through surface veins, they will agglutinate so that flow through the surface vessels is reduced. The deoxygenated agglutinated red cells produce very typical livedo reticularis (Figs. 2–4) on the hands and lower extremities that are completely reversible with rewarming of the skin. The mechanism underlying the development of the Raynaud phenomenon is unclear but likely relates to in vivo agglutination in the digits leading to oxygen deprivation and is commonly seen in cold agglutinin hemolytic anemia. Acrocyanosis is a common accompaniment of cold agglutinin disease but generally can be easily managed with cold avoidance.

Fig. 2.

Peripheral blood film showing agglutination.

Fig. 4.

Livedo reticularis of the lower extremities.

EVALUATION

Laboratory

The list of recommended tests when cold agglutinin hemolytic anemia is in the differential diagnosis can be found in Table 1. Specific attention to the mean corpuscular volume for spurious elevation or low hematocrit out of proportion to other parameters is important. The reticulocyte count is typically elevated but, in many instances, whereby the hemolysis is low-grade compensated it may be less than 3%. Due to the loss of membrane from the antibody-coated red cells spherocytes are common. Markers of red cell destruction include elevation of the indirect bilirubin. In all patients that have a positive direct antiglobulin test testing for immunoglobulin G and complement specificity is required. If complement antiglobulin testing is positive cold agglutinin titer should be measured Immunofixation of the serum in urine looking for the monoclonal IgM protein is required. Nonspecific markers of hemolysis including the LDH and haptoglobin should be assessed. Patients will have depression in complement levels and measurement of total hemolytic complement, C3 and C4 are required.

Table 1.

Recommended testing for the suspected diagnosis of cold agglutinin hemolytic anemia

Tests Recommended for AD Assessment
CBC	LDH	Quantitative Immunoglobulins
Reticulocyte count	Bilirubin Total and direct	Haptoglobin
DAT	Peripheral Blood film for agglutination and spherocytes; cold agglutinin titer	CH50, C3, C4
-if + IgG & C3 monospecific DAT	serum protein electrophoresis with immunofixation, if no monoclonal protein can be detected infectious causes should be sought

Bone Marrow

When a monoclonal IgM protein is confirmed, a bone marrow examination is indicated. Assessment for the presence of lymphoplasmacytic lymphoma or clonal B cells that do not meet criteria for overt lymphoma is indicated. Genetic studies of the bone marrow include a search for mutation in MYD88 and if a mutation is found, CXCR4 mutation should be sought. If overt lymphoma is found in the bone marrow staging would include imaging for the presence of mediastinal or retroperitoneal lymphadenopathy.

NATURAL HISTORY OF COLD AGGLUTININ DISEASE

A long-term study in Norway identified 232 patients fulfilling criteria for CAD. The median age at onset was 67 years with a range from 30 to 92. The male to female ratio was 0.54 which was surprising as monoclonal IgM proteins are more common in men. The incidence was 1 case per million per year and the prevalence 16 cases per million population-reflecting the long survival of these patients after diagnosis. The mean initial hemoglobin was 9.2 g/dL ranging from 4.5 to 15.6 g. Median survival from onset was 12.5 years remarkably given the median age of 67 at diagnosis. 91% of the patients had cold-induced circulatory symptoms and 74% reported exacerbation of their anemia during febrile illnesses. 51% had received at least 1 red cell transfusion. In an update published in 2020%, 27% of patients had hemoglobin less than 8 g/dL with a mean LDH of 534 and bilirubin of 2.75 mg/dL. 37% of patients had hemoglobin ranging from 8 to 10 g/dL with an LDH of 450 and a bilirubin of 2.5 mg/dL. 36% of patients had hemoglobin greater than or equal to 10 g/dL. In all subsets, the median IgM level was less than 0.7 g per deciliter.³

Large studies in the United States are based on artificial intelligence analysis of millions of medical records using keywords in the medical record as well as specific laboratory data.¹⁸ The Stanford translational research integrated database retrospectively identified cold agglutinin disease in 29 patients over 16 years. This database contained information on 2.1 million patients seen in the Stanford system. General observations included disease severity fluctuations that resulted in variable symptoms over time. In some patients, the anemia was mild causing little change in the quality of life. But for some, anemia had a substantial impact on the quality of life with symptoms including acrocyanosis, fatigue, dyspnea, weakness, and mild to severe Raynaud. 79% of patients had severe or moderate anemia. The mean and median hemoglobin levels were 8.3 and 8.2 g/dL and ranged from 4.7 to 11.6 g/dL. 72% of the patients had at least 1 severe anemia event, defined as transfusion required, within the 1st year of monitoring. A subset of patients remained severely anemic despite multiple therapeutic interventions. Overall, there were 7.1 severe anemia events per patient-year, 10.8 moderate events per patient-year, 8.0 mild events per patient-year during the follow-up. 65% of patients had at least 1 transfusion. The median number of transfusions was 4.4 per patient-year of follow-up. The average number of red cell units per transfusion was 1.5.

A previously unsuspected complication was a high incidence of thrombotic events. Of the 29 patients, 5 suffered venous thromboembolism. In 3 there was a portal vein thrombosis and 2 had acute venous embolism of deep vessels.¹⁹

The largest cohort comes from the Optum integrated claims data set. Using natural language processing and search terms linked to laboratory data, 55,000,000 patients were screened. A total of 608 cold agglutinin disease patients were identified and were matched with 5873 control patients. 70% of patients were more than 65%, 64% were women. 88.8% of patients were Caucasian. Among 255 cold agglutinin disease patients, there were 395 thromboembolic events representing 31.3% of the total cold agglutinin population developing VTE. The control population had an incidence of 20.2%. Among the 31% that developed thrombosis, 18% had 1 event and 13% had more than 1 event. The odds of having a thrombotic event were 1.85 times higher in patients than in controls. The types of venous thromboembolic events were not merely low-risk DVT.¹⁹ Six patients had a portal vein thrombosis, 40 had a pulmonary embolism and 10 had mesenteric thrombosis. There were also 16 arterial emboli in thromboses including 55 myocardial infarctions and 180 for strokes. All of these were statistically more frequent than the controls. I hemoglobin less than 8 g/dL was seen in 23.1%. Hemoglobin from 8.1 to 10 g/dL was seen in 29.5% and 47.4% had hemoglobin greater than 10 g/dL. Abnormal bilirubin or LDH was seen in 91% of patients. Cold agglutinin disease is associated with a significantly increased risk of venous arterial and cerebral thrombotic events. These thrombotic events are not predicted by the severity of the anemia. However, a relationship was noted that patients with thrombotic events had a higher LDH level.

TREATMENT INCLUDING EMERGING THERAPIES FOR COLD AGGLUTININ HEMOLYTIC ANEMIA

The polyclonal postinfectious secondary forms of cold agglutinin hemolysis associated with infectious mononucleosis and mycoplasma pneumonia can be quite severe but are self-limited transient and result in full recovery.²⁰ Most patients require supportive care only and as this tends to occur in younger individuals with a primary symptom of fatigue, most are misattributed to postinfectious malaise and only the most severe instances with profound anemia come to medical attention with a firm diagnosis. As the primary immune response mediated by immunoglobulin M begins to decline at day 14 full recovery begins.¹²

Unfortunately, monoclonal cold agglutinin disease is associated with the production of an IgM monoclonal protein from lymphoplasmacytic cells in the marrow and results in a chronic sustained relapsing-remitting anemia. Historically, treatment has been directed toward the elimination of the lymphoplasmacytic cells responsible for the synthesis of the IgM protein.²¹ Although, in principle, this should result in lesser IgM protein complement fixation on the red cell surface, the reality is attempts to reduce the IgM protein to levels below which hemolysis no longer occurs is quite a challenge. The standard therapies for warm hemolytic anemia including corticosteroids, splenectomy, and intravenous immunoglobulin infusions are typically ineffective: these therapies that tend to affect the interaction between the red cell and the mononuclear phagocyte system, cannot overcome the substantial density of complement on the red blood cells surface There use is, therefore, discouraged in CAD.

Rituximab has been extensively studied in CAD.²² This agent is well known to produce responses in Waldenstrom Macroglobulinemia; however, it is not very effective in cold agglutinin disease. In the report of 35 patients, 19 (52.3%) has had an initial increase in hemoglobin with a median of 1.5 g/dL. However, and 6 of these 19 had a decline of at least 1.5 g/dL after having an initial response. The rituximab was given 375 mg/M2 for 4 doses LDH and bilirubin was measured in 28 of these patients and in 22 there was a persistent elevation of bilirubin or LDH within 12 months of therapy reflecting that Rituximab as a single agent is not very effective for sustained responses.²³ A recent trial of rituximab reported 13 of 16 patients (81%) responded to the therapy. Responders achieved a median increase in hemoglobin levels of 4.5 g/dL.

Bortezomib also known to be active in the management of lymphoplasmacytic lym-phoma was administered to 21 CAD patients with hemoglobin of less than 10 g of whom 10 were transfusion dependent. Nineteen were evaluable for response (1 excluded for pulmonary embolism on day 4). There were only 3 complete responses, 3 partial response for an overall response rate of 32% suggesting therapy for this disease remains inadequate.²⁴

Bendamustine plus rituximab is highly active in the management of lymphoplasmacytic lymphoma. A multi-center trial enrolled 45 patients with CAD.²⁵ Complete responses were seen in 40% partial response in 31% no responses in 29% complete responses had a 76% reduction in the IgM level, partial response had a 74% reduction in the IgM level. It is noteworthy that patients that failed to have any response in hemoglobin still had a 55% reduction in IgM but no impact on hemolysis. The median increase in hemoglobin for complete responders and partial responders was 4.4 and 3.9 g respectively. A reduction in the cold agglutinin titer was only observed incomplete responders. For symptomatic patients and those that are transfusion dependent a trial of rituximab plus bendamustine should be considered a first-line consideration in the management of cold agglutinin hemolytic anemia.

Ibrutinib was reported in 13 patients with cold agglutinin hemolytic anemia 11 of whom fulfilled criteria for lymphoplasmacytic lymphoma and the remainder chronic lymphatic leukemia or small lymphocytic lymphoma. Two patients were MYD 88 mutation positive. Eight of the 10 were previously treated. Seven were transfusion dependent. The median rise in hemoglobin was 5.6 g/L (2.5–10.3) for the 13 patients. There were 12 complete responses and 1 partial response for an overall response rate of 100%. The median time to complete response was 6 months. Table 2 summarizes some of the reported response rates to therapy for CAD.²⁶

Table 2.

Response rates reported to therapy for CAD

Agent	Response Rate in %
Rituximab	52–81
Bortezomib	32
Rituximab/Bendamustine	71
Ibrutinib	100
Sutimlimab	87.5

COMPLEMENT INHIBITION IN THE MANAGEMENT OF COLD AGGLUTININ HEMOLYTIC ANEMIA

The theory behind the use of complement inhibition in CAD is sound: In CAD the binding of C3b initiates a cascade of events as discussed earlier leading to the clearance of the red cell extravascularly. Upstream inhibition of complement activation would prevent the deposition of C3 on the surface of the red cell. Downstream complement inhibiting agents such as Eculizumab which prevents the activation of C5 has therefore jas a limited I impact on extravascular hemolysis.²⁷ The antibody TNT 003 in a macrophage cell culture officially blocked C3 deposition on red cells which resulted in the inhibition of phagocytosis in vitro when cold agglutinin plasma was added to suspensions of red cells at concentrations of 100 μg/mL. This in vitro study supported testing of complement inhibition in the prevention of hemolysis.²⁸

Sutimlimab is a humanized monoclonal antibody directed against in a phase 3 trial in 24 patients with CAD, whereby three of the 24 patients did not experience a hematologic response. Overall the mean increase in hemoglobin was 2.6 g and a mean level of 11 g/dL was maintained from week 3 to the end of the 26 week study period, transfusion independence was noted in 71% of patients from week 5 to 26, with normalization of mean bilirubin level by week 3.²⁹ An improvement in quality of life measures was observed, through clinically meaningful reductions in fatigue. Although 92% of patients experienced one adverse event or more, only 38% of these were assessed as being related to Sutimlimab and mostly graded as 1 to 2. It is expected that continuous lifelong therapy would be required in the absence of definitive treatment of the underlying cause.

SPECIAL CONSIDERATIONS-TRANSFUSIONS AND PERI-OPERATIVE MANAGEMENT

Because there is a pan agglutinin, crossmatch of red cells is challenging and can take many hours.³⁰ It is important to remember that when the anemia is life-threatening it is not necessary to transfuse with crossmatch compatible red cells. If patients have ABO and Rh compatible cells life-threatening anemia leading to cardiovascular decompensation can be prevented. If patients have other Alloantibodies from prior transfusion this will lead to a delayed hemolytic transfusion reaction weeks in the future and the consequences are minimal and the destruction is the transfused red cells only. When patients need transfusions urgently the hematologist in partnership with transfusion medicine may consider transfusing before a crossmatch to preserve the patient’s oxygen-carrying capacity.³¹

Surgical procedures carry special risks for patients with cold agglutinin disease. An in-line blood warmer is often used in the operating room to ensure that any transfused red cells are kept at a temperature that would minimize fixation of IgM to the red cell surface.³² When extracorporeal circulation is required techniques exist for warming of transfused products. In some instances, it is justified to do preoperative plasma exchange to remove as much of the immunoglobulin M from the patient’s plasma as possible when surgery is required and there is insufficient time to wait for the effect of chemoimmunotherapy. Preoperative assessment by the hematologist and discussion with the surgeon to clarify the magnitude and duration of hypothermia during the procedure is required for planning. This will determine the need for plasma exchange preoperatively and whether warming equipment for the extracorporeal circuit is required.

PAROXYSMAL COLD HEMOGLOBINURIA

The diagnostic pathway to this extremely rare complement-mediated intravascular hemolytic disorder follows a similar pathway to cold agglutinin disease. These patients will have a positive complement component of the Coombs test. . When complement is found on the red cell surface by positive complement Coombs test, the patient should have a cold agglutinin titer performed. However, if cold agglutination is not seen the patient should be screened for hemoglobinuria, hemosiderinuria, and free hemoglobinemia. In these patients, a Donath–Landsteiner antibody should also be performed. This disorder shares with cold agglutinin disease a direct antiglobulin test positive for complement. However, the antibody specificity rather than being anti-I, is anti-P. The thermal amplitude is usually less than 20° centigrade. Unlike cold agglutinin disease whereby the hemolysis is extravascular due to C3b, in paroxysmal cold hemoglobinuria activation of the C5 through C9 membrane attack complex leading to cell lysis on rewarming (biphasic antibody) is seen. These patients have intravascular hemolysis leading to dark urine, flank pain, fever, and rigors. In a Canadian study, spanning 124 testing years 3 positive tests were seen in adults and 14 in children. Concordance in the interpretation of the testing was poor.³³ These hemolytic episodes can be seen after infection and are often self-limited.{Tiwari, 2020 #106)^34,35

SUMMARY AND CONCLUSION

Cold agglutinin disease is an extravascular hemolytic process mediated by the presence of a monoclonal IgM protein. The clinical spectrum is quite broad from mild symptomatic compensated hemolysis and manifestations of acrocyanosis to indefinite transfusion dependency. The process is characterized by a complement-specific Coombs positive Hemolytic process. Standard therapies for warm immune hemolytic anemia including corticosteroids, intravenous immunoglobulin infusions, and splenectomy are ineffective. Primary therapy is directed at the suppression of the cells in the bone marrow responsible for the production of the IgM monoclonal protein. Promising studies with the use of complement inhibition as a strategy to interrupt the hemolysis are forthcoming

Fig. 3.

Livedo reticularis of the hands.

KEY POINTS

• Cold agglutinin disease represents a direct antiglobulin-positive hemolytic anemia.

• In cold agglutinin disease, there is a low-grade lymphoplasmacytic clonal process in thebone marrow that produces a monoclonal IgM protein that fixes complement to the red cell and results in extravascular hemolysis.

• Current therapies focus on the reduction of synthesis of the monoclonal IgM protein using rituximab, bortezomib, or bendamustine alone or in combination.

• Emerging therapy designed to block complement activation such as sutimlimab can resultin the resolution of the hemolysis without the use of cytotoxic agents.

CLINICS CARE POINTS

• All patients with a positive Coombs test should be screened for complement mediated hemolysis.

• All patients with a positive complement Coombs should be screen for monoclonal IgM protein in the serum with reduced complement level.

• These patients tend to be corticosteroid resistant.

• Specific precautions are required if surgical intervention with general anesthesia is required.