Paroxysmal Cold Hemoglobinuria Case Study

Abstract

Paroxysmal cold hemoglobinuria (PCH) is an acquired hemolytic anemia caused by immunoglobulin G (IgG) antibodies that sensitize red blood cells (RBCs) at cold temperatures by fixing complement to the RBCs causing intravascular hemolysis on rewarming. PCH usually appears in young children as recurrent high fevers, chills, and passage of red-brown urine. The diagnostic test for PCH is the Donath-Landsteiner test, an in vitro assay for biphasic hemolysis. Herein, we present 2 cases of PCH that occurred within 12 months of each other. We quickly diagnosed the second case and treated the patient successfully, in part due to our recognition of its characteristics based on the first case. PCH is a hemolytic anemia for which there is a specific diagnostic test; the timely recognition of this entity by physicians and laboratory staff will allow prompt, supportive therapy and will raise the odds of quick resolution of hemolysis.

paroxysmal cold hemoglobinuria, Donath-Landsteiner antibody, autoimmune hemolytic anemia, cold hemagglutinin, autoantibody, anemia

Case Presentation 1

A 23-month-old girl, previously healthy according to her parents, arrived at a pediatric hospital in the Midwest with an upper respiratory tract infection (URI), decreased activity and appetite, and jaundice; urinalysis results revealed a trace of blood, with 0 to 5 red blood cells (RBCs)/high power field. Three weeks before her arrival at the hospital, she experienced intermittent URIs and recent otitis media treated with antibiotics.

The initial laboratory findings included a hemoglobin of 5.5 g/dL, which rapidly decreased to 3.2 g/dL, and reticulocytopenia with a corrected count of 1.3%. Her peripheral blood smear showed approximately 40% spherocytes, no blasts, no neutrophil erythrophagocytosis, and normal white blood cell (WBC) and platelet morphologic characteristics. She had elevated lactate dehydrogenase (LDH) and elevated bilirubin levels. The results of Mycoplasma and Bartonella serologic tests, along with blood and stool cultures, were all negative.

Serologic investigation undertaken by the transfusion services at 2 independent hospitals yielded a negative direct antiglobulin test (DAT) result and variable reactive antibody screening results. We consulted the Indiana Blood Center Immunohematology Reference Laboratory (IRL) and obtained the results shown in Table 1. We requested that a Donath-Landsteiner test be performed because the antibody was apparent only at lower temperatures, coupled with a low titer and a clinical course that was suspicious for paroxysmal cold hemoglobinuria (PCH). We obtained a fresh serum specimen from the patient, collecting it in 2 serum tubes, and kept it between 24°C and 37°C in a warm water bath to assure that there would be no in vitro binding of the antibody. The specimen was allowed to settle without centrifugation for 2 hours; then, the serum was collected and sent to the IRL for analysis.

Table 1

Regional Reference Laboratory Testing: Case 1

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The hemolysis observed in the group A1 and A2 tubes that contained serum from the patient demonstrated the presence of a biphasic hemolysin, which was not present in the A3 control tube (Image 1). The remaining control groups B and C had negative results and showed no hemolysis, which excluded the possibility of a biphasic cold autoantibody. No hemolysis was observed in the extended incubation testing immediate spin, room temperature, 15°C, and 37°C testing we performed on the specimens; this indicates that thermal amplitude was not demonstrated. These findings indicate a positive Donath-Landsteiner test result; based on these results, the treating physician diagnosed the patient with acute hemolytic anemia secondary to PCH.

Image 1

Donath-Landsteiner test results from patient 1. Tube 1: Patient serum + reagent cells; Tube 2: Patient serum + reagent cells + donor serum (fresh compliment); Tube 3: Donor serum + reagent cells (control); Group A: 4°C for 30 minutes, followed by 37°C for 60 minutes; Group B: 4°C for 90 minutes; Group C: 37°C for 90 minutes (control). Hemolysis was observed in the A1 and A2 tubes and was not observed in the A3 control tube indicating the presence of a biphasic hemolysin. The remaining control groups B and C had negative results and showed no hemolysis.

Image 1

Donath-Landsteiner test results from patient 1. Tube 1: Patient serum + reagent cells; Tube 2: Patient serum + reagent cells + donor serum (fresh compliment); Tube 3: Donor serum + reagent cells (control); Group A: 4°C for 30 minutes, followed by 37°C for 60 minutes; Group B: 4°C for 90 minutes; Group C: 37°C for 90 minutes (control). Hemolysis was observed in the A1 and A2 tubes and was not observed in the A3 control tube indicating the presence of a biphasic hemolysin. The remaining control groups B and C had negative results and showed no hemolysis.

During her hospital stay, the patient received transfusion of 2 units of leukoreduced RBCs and azithromycin in case of Mycoplasma infection. She was discharged 5 days after admission in stable condition. A month after discharge, her baseline health had returned to normal, and her hemoglobin count was 14.0 g/dL. We could attain no further data on this patient, whose case was lost due to lack of additional follow-up.

Case Presentation 2

One year after the diagnosis of patient 1, a 4-year-old girl was admitted to the same hospital; we initially diagnosed this patient as having acute hemolytic anemia. Two weeks before her admission to the hospital, she had had a viral URI followed by acute otitis media. She had been treated with amoxicillin and had developed dark, tea-colored urine within 24 hours.

On her arrival at the hospital, her laboratory findings included a hemoglobin level of 7.1 g/dL; WBC count of 16,200; platelet count of 504,000; and reticulocyte count of 2.2%. Results of a peripheral blood smear showed anemia with spherocytes, no blasts, no neutrophil erythrophagocytosis, and normal WBC and platelet morphologic characteristics. Her serum had an elevated LDH level of 1369 U/L, decreased haptoglobin count of less than 8, and increased total bilirubin level of 2.5 mg/dL.

The hospital transfusion service serologic investigation in which we participated demonstrated a positive DAT result with components of complement (C3b,d only). We submitted a specimen from this patient to the IRL; the results obtained by the laboratory are shown in Table 2. Anti-M was demonstrated in the specimen from the patient on P1 negative, M+ cells at immediate spin (IS), room temperature (RT), and via gel test. Testing also revealed the presence of cold autoantibody material at 15°C and 4°C and that the type of antigen on the RBCs of the patient was M+N+. We ordered a Donath-Landsteiner test to be performed due to suspicion for PCH; the specimen we tested was reactive. The control cells were P1+M-; therefore, we did not detect the anti-M antibody in DL testing. Hence, the treating physician diagnosed this patient with acute hemolytic anemia secondary to PCH.

Table 2

Regional Reference Laboratory Testing: Case 2

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The patient did not require transfusion and was discharged 4 days after admission. Two weeks after discharge, the serum of the patient was retested; it had a hemoglobin level of 11.2 g/dL. As with patient 1, we were able to gather no further laboratory data on this patient due to lack of follow-up.

Paroxysmal Cold Hemoglobinuria

PCH is categorized as a cold autoimmune hemolytic anemia and is caused by an immunoglobulin G (IgG) antibody that sensitizes RBCs at cold temperatures by fixing complement to the RBCs, which causes intravascular hemolysis on rewarming. Historically, episodes of hemoglobinuria could be precipitated by immersing the extremities of the patient into an ice water bath.1 In 1904, Donath and Landsteiner2 demonstrated the mechanism of hemolysis using an in vitro test. They showed that hemoglobinuria resulted from intravascular hemolysis caused by sensitization of the RBCs and attachment of autoantibodies. Testing proved that antibody binding is temperature-dependent and that a labile serum factor (complement) is also necessary for RBC lysis. The procedure described in reference 2, published in 1904, is still used today as the gold standard diagnostic test for PCH and is known as the Donath-Landsteiner (DL) test, after the the coauthors of the published article.

In the past, PCH was primarily a disease that afflicted patients with secondary or tertiary syphilis.3,4 Now, acute PCH occurs most frequently in young children after viral illnesses, such as mumps, measles, chicken pox, Epstein-Barr virus, cytomegalovirus, influenza, parvovirus B19, coxsackievirus A9, and adenovirus.5–9 More commonly, a specific pathogen is not identified.6,9 Rare examples of PCH have been described in patients with other immunologic disorders such as diffuse large B-cell lymphoma and chronic lymphocytic leukemia.10,11

Many theories have been proposed for the mechanism by which viruses stimulate autoantibody formation, including stimulation of abnormal clones of immunologically competent cells by a virus, structural alteration of the RBC membrane by a virus and subsequent production of antibodies to the altered antigens that cross-react with normal erythrocytes, and alteration of T suppressor function that results in autoantibody production.12

The autoantibody of PCH is IgG; it has specificity for the P antigen, which is commonly expressed on microorganisms and is the cellular receptor for the parvovirus B19.3 The antibody is termed biphasic hemolysin because it binds optimally at cold temperatures and then causes rapid hemolysis of the RBCs when warmed to 37°C. The antibody binds to the RBCs of the patient in the cooler temperature of the peripheral-body circulation and causes complement-mediated lysis of the RBCs in the warmer temperature of the core-body circulation, resulting in intravascular hemolysis.7

Incidence

In the past, PCH had been a rare diagnosis;13,14 however, more recent reports6,15,16 suggest a higher incidence of this condition. It is even suggested by Heddle7 to be the most common cause of acute autoimmune hemolytic anemia (AIHA) in children.7 The increase in incidence could relate to increased awareness of the disease and of the Donath-Landsteiner test.4

Demographics

Acute PCH usually appears in young children (median age, 5 years).9 Although more common in children, the condition can affect individuals of all ages: the patients in one study9 ranged from age 1 year to 82 years.9 The combined data from 3 reviews indicate a boy-to-girl ratio of 2.1:1 and no discrimination by ethnicity.4

Signs and Symptoms

The typical clinical appearance is that of a child who had had a URI 1 to 2 weeks before seeking treatment.7,9,17 The onset of hemolysis is usually preceded by recurrent high fevers, chills, and passage of red-brown urine.18 Hemoglobinuria, jaundice, and pallor were the most common clinical findings in 42 cases of acute PCH described in the literature.7 Abdominal pain and fever were also common findings.7 Hemoglobinuria resulting from exposure to cold is rare in acute PCH; this was observed in only 1 of 52 patients;9 however, hemoglobinuria can be induced by exposure to cold.9,18

Laboratory Findings

Patients can have severe and rapidly progressing anemia. Hemoglobin levels at hospital admission ranged from 2.5 to 12.5 g/dL, with a mean value of 6.8 g/dL as calculated from the data in 25 published case reports7 that we reviewed. Another investigation team6 reported hemoglobin levels of less than 5 g/dL in 6 of 22 cases (minimum of 4.4 g/dL) and reported hemoglobin levels of less than 5 g/L in 17 of 51 cases.9 A relative reticulocytopenia occurs in some patients with PCH.7,9,16,17 Patients can have abnormal RBC morphologic characteristics, including presence of spherocytes, anisocytosis, poikilocytosis, fragmented RBCs, basophilic stippling, polychromatophilia, autoagglutination, and erythrophagocytosis by neutrophils rather than monocytes.7,9,17

Leukopenia followed by leukocytosis with left shift can occur during acute attacks. However, WBC counts were normal in 25% of 42 cases.7 Platelet counts are usually normal.18

Additional evidence of intra- and extravascular hemolysis is demonstrated by increased LDH, indirect bilirubinemia, decreased haptoglobin, hemoglobinemia, and hemoglobinuria.18 Because PCH can produce acute renal failure, elevated BUN and creatinine levels may be present.18

Differential Diagnosis

The clinical and laboratory findings in cold agglutinin syndrome (CAS) are similar to those that we and others have observed in PCH; however, the results of serologic investigation are usually sufficient to distinguish between these 2 diagnoses. Unlike PCH, CAS rarely affects young children.4 Hemoglobinuria, a common finding in PCH, is not as frequently observed in CAS.4,19 In PCH, the antibody is of the IgG type, it is not enhanced by acidification, and it demonstrates P specificity (which is found on the RBCs of most individuals).7,20 In CAS, the antibody is of the immunoglobulin M (IgM) type, it shows enhanced reactivity in acidic pH, and is specific for the I– or Pr– blood group antigens.7 PCH antibodies are potent biphasic hemolysins of low titer (less than 32) and seldom cause agglutination, whereas CAS antibodies have a high titer (greater than 1000), cause strong agglutination, and seldom cause in vitro lysis.7 The DL test result will be negative in cases of CAS.7

Diagnostic Tests

PCH is caused by a biphasic IgG autoantibody that fixes complement at low temperatures but dissociates at higher temperatures.18 Because of this, the direct antiglobulin test result is positive with anti-C3b,d; however, it is generally negative with anti-IgG unless testing is performed at colder temperatures.3,7,18

The diagnostic test for PCH is the DL test. This test involves in vitro testing for hemolysis with the serum of the patient, washed group-O cells that express the P antigen, and fresh normal serum (a source for complement).21 Blood should be collected in prewarmed tubes that do not contain anticoagulant and should be maintained at 37°C during the collection procedure to prevent autoadsorption of the antibody and hemolysis.7 According to the AABB Technical Manual,21 the DL test should be performed as follows: 1 group of samples is incubated at 0°C to 4°C, another at 37°C, and the third incubated first at 0°C to 4°C for 30 minutes and then at 37°C for 60 minutes.21 The DL test result is considered positive when serum specimens from the patient show hemolysis only in the groups of specimens that were incubated first at 0°C to 4°C and then at 37°C.21 Specificities for the DL antibodies were not determined in either of the case reports we present herein because it is not general practice to type the serum of the patient for the P antigen. This antibody is rare, and the antiserum is not readily available for antigen confirmation.

Treatment and Prognosis

The prognosis for PCH is highly favorable. Acute attacks are often severe but typically resolve spontaneously within a few days to several weeks and rarely recur.17 Usually, only supportive care is needed, which may include transfusion with RBCs.4,7 The patient should be kept warm; a blood warmer has even been used during transfusion.7 P-negative blood has been used for transfusions in patients with PCH in whom hemolysis was severe and prolonged; however, its effectiveness is difficult to evaluate because of the unpredictable natural course of the disease. An RBC survival study has shown22 that P-positive blood is useful for transiently raising the hemoglobin level of the patient; however, the long-term survival of RBCs is most effective when P-negative blood is transfused. Corticosteroids are often administered to patients; however, the effectiveness of these drugs is difficult to evaluate due to the transient nature of hemolysis.7

Summary

The typical presentation is that of a child who during the preceding 1-2 weeks had suffered from what appeared to be an undefined or “flu-like” illness. Usually the onset of hemolysis is signaled by a recurrence of fever and then the passage of red-brown urine. This urine contains the iron bearing, oxygen transporting, protein pigment of blood called hemoglobin, which is released when red blood cells are prematurely destroyed. The presence of hemoglobin in the urine (hemoglobinuria) causes the dark brown color of the urine. Hemoglobinuria, hemoglobinemia (hemoglobin in the plasma), jaundice and pallor are common clinical findings in acute PCH and, of particular significance is that hemoglobinuria is found in almost all acute cases in childhood.

Abdominal pain and fever are also common findings. Approximately 25 percent of cases have palpable liver and spleen. Although hemoglobinuria may be induced by exposure to cold, such an occurrence is rare in acute PCH. In acute PCH, hemolysis typically lasts for a few days only and recovery is usually uninterrupted.

The older medical literature describes chronic syphilitic PCH as a rather benign disease that rarely caused severe chronic anemia. However, acute attacks of hemolysis and hemoglobinuria were well known and were characterized by the sudden onset of shaking chills, fever, malaise, abdominal distress, aching pains in the back or legs, arid nausea. Usually, hemoglobin was present in the first specimen of urine passed after the onset of symptoms, and the interval between chilling and the development of symptoms ranged from a few minutes to eight hours. The extent of cold exposure could be surprisingly slight and, in some cases, a history of undue exposure to cold was not elicited.

Individuals with PCH often develop the classic symptoms of anemia- the medical term for low levels of circulating red blood cells. These symptoms may include fatigue, difficulty breathing upon exertion (dyspnea) and abnormal or extreme paleness of the skin (pallor). In addition, affected individuals may develop chills, fever, abdominal pain, and pain or aching in the legs or lower back. Less common findings associated with PCH include headaches, vomiting and diarrhea.

Following an episode, affected individuals usually develop the signs of hemolysis including yellowing of the skin, whites of the eyes and mucous membranes (jaundice). In some cases additional symptoms may occur including tingling in the hands and feet, a condition marked by a feeling of coldness or numbness of the hands, nose and ears in response to cold temperatures (Raynaud’s phenomenon), or a skin condition marked by reddening and itching of the skin in response to cold temperatures (cold urticaria). In extremely rare cases, the kidney may become involved and kidney (renal) failure has been reported in a few cases.

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