Blood Reviews (2008) 22, 115

www.elsevierhealth.com/journals/blre

REVIEW

Cold antibody autoimmune hemolytic anemias

Lawrence D. Petz
*

Emeritus Professor of Pathology and Laboratory Medicine, University of California Los Angeles, StemCyte International Cord Blood Center, Arcadia, California, United States

KEYWORDS
Autoimmune hemolytic anemia; Cold agglutinin syndrome; Paroxysmal cold hemoglobinuria

Summary The cold antibody autoimmune hemolytic anemias (AIHAs) are primarily comprised of cold agglutinin syndrome (CAS) and paroxysmal cold hemoglobinuria (PCH) but, in addition, there are unusual instances in which patients satisfy the serologic criteria of both warm antibody AIHA and CAS (mixed AIHA). CAS characteristically occurs in middle-aged or elderly persons, often with signs and symptoms exacerbated by cold. The responsible antibody is of the IgM immunoglobulin class, is maximally reactive in the cold but with reactivity up to at least 30 C. Therapy is often ineffective, but newer agents such as rituximab have been benecial in some patients. PCH occurs primarily in children, often after an upper respiratory infection. The causative antibody is of the IgG immunoglobulin class and is a biphasic hemolysin that is demonstrated by incubation in the cold followed by incubation at 37 C in the presence of complement. Acute attacks are frequently severe but the illness characteristically resolves spontaneously within a few days to several weeks after onset and rarely recurs. Treatment consists of supportive care, with transfusions frequently being needed. c 2007 Elsevier Ltd. All rights reserved.

Introduction
The immune hemolytic anemias are classied as indicated in Table 1.1 This classication divides these disorders into distinctive categories which have differing clinical manifestations, prognosis and therapy, as indicated in Table 2. The cold antibody autoimmune hemolytic anemias (AIHAs) are primarily comprised of cold agglutinin syndrome (CAS) and paroxysmal cold hemoglobinuria (PCH) but, in addition, there are unusual instances in which patients satisfy the serologic criteria of both warm
* Tel.: +1 626 821 9860; fax: +1 626 821 9660. E-mail address: lpetz@stemcyte.com

antibody AIHA and CAS. Such cases are designated as combined cold and warm AIHA or mixed AIHA. These disorders are diagnosed on the basis of characteristic serologic reactions. The majority of cases of AIHA are mediated by warm-reactive autoantibodies, i.e., antibodies displaying optimal reactivity with human RBC at 37 C and which are usually of the IgG immunoglobulin class. In contrast, CAS is generally caused by IgM autoantibodies which exhibit maximal reactivity at 4 C. The causative in PCH antibody is an IgG immunoglobulin with specicity that differs from that found in CAS. The antibody is best detected in vitro by its ability to cause hemolysis of normal RBC in a twostep procedure, which requires incubation in the

0268-960X/$ - see front matter c 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2007.08.002

2
Table 1 Classication of immune hemolytic anemias.

L.D. Petz

Autoimmune hemolytic anemias (AIHA) Warm antibody AIHA Idiopathic Secondary (e.g., chronic lymphocytic leukemia, lymphomas, systemic lupus erythematosus) Cold agglutinin syndrome Idiopathic Secondary Non-malignant disorders (e.g., mycoplasma pneumoniae infection, infectious mononucleosis, other virus infections) Malignant disorders (e.g., lymphoproliferative disorders) Paroxysmal cold hemoglobinuria Idiopathic Secondary Viral syndromes Syphilis Combined cold and warm AIHA (mixed AIHA) Drug-induced immune hemolytic anemia Drug-related antibody identiable Drug-induced AIHA Alloantibody-induced immune hemolytic anemia Hemolytic transfusion reactions Hemolytic disease of the fetus and newborn

Table 2

Some characteristic features of autoimmune and drug induced immune hemolytic anemias.

Warm antibody AIHA Clinical manifestations: Variable, usually symptoms of anemia, occasionally Acute hemolytic syndrome Prognosis: Fair, with signicant mortality Most effective therapies: Steroids, splenectorny, immunosuppressive drugs Cold agglutinin syndrome Clinical manifestations: Moderate chronic hemolytic anemia in middle-aged or elderly person, often with signs and symptoms exacerbated by cold Prognosis: Good, usually a chronic and quite stable anemia Most effective therapies: Avoidance of cold exposure, immunosuppressive drugs Paroxysmal cold hemoglobinuria Clinical manifestations: Acute hemolytic anemia, often with hemoglobinuria, particularly in a child with history of recent viral or viral-like illness Prognosis: Excellent after initial stormy course Therapy: Not well dened; steroids empirically and transfusions if required Drug-induced immune hemolytic anemia Clinical manifestations: Variable, most commonly subacute in onset, but occasionally acute hemolytic syndrome Prognosis: Excellent Therapy: Stop drug; occasionally a short course of steroids empirically

cold followed by incubation at 37 C in the presence of complement. AIHA is designated as idiopathic if it is unassociated with any demonstrable underlying disease. In contrast, AIHA is categorized as secondary if it is associated with an additional disorder and there is reason to suspect that the association is not merely fortuitous. The ingestion of some drugs causes hemolytic anemia in which the causative antibody can be shown to have specicity for the drug or its metabo-

lites. Although the antibody reacts with the patients own RBCs, these disorders are not autoimmune disorders because the antibody does not have specicity for autoantigens. In other quite remarkable cases, ingestion of a drug causes the development of red cell autoantibodies, i.e., the antibody in the patients serum and in an eluate from the patients red cells reacts with red cells similarly to autoantibodies in idiopathic AIHAs, and no relationship between the drug and the antibody can be demonstrated in vitro. Such cases are appropriately

Cold antibody autoimmune hemolytic anemias termed drug-induced AIHAs and can be either of warm antibody or cold antibody type.

3 Symptoms The clinical manifestations vary greatly from patient to patient, probably depending on the thermal range of the cold antibody. Symptoms are frequently just those of a chronic anemia which progresses gradually.10 In idiopathic cases, the disorder often remains quite static and generally only progresses slowly in intensity. Clinical ndings are more serious for patients whose cold agglutinin is active at higher temperatures.11 Temperatures of 30 C and lower are normally attained in the supercial skin vessels of those parts of the body exposed to cold air or water.12 The thermal range of the antibody is more important than the agglutination titer for clinical purposes.11,13 Hemoglobinuria Patients may experience hemoglobinuria, particularly in cold weather, and this nding is prominent among manifestations listed in reviews of the disorder. However, its incidence is difcult to determine, and in many patients hemoglobinuria is never observed. This is true even in some patients who develop severe acrocyanosis during cold exposure.7,14 When an episode of intravascular hemolysis does occur after cold exposure, it is not associated with fever, chills, or acute renal insufciency.12,15 This phenomenon contrasts with the marked constitutional symptoms associated with hemoglobinuria in PCH or in those few patients with warm antibody AIHA who have hemoglobinuria. Acrocyanosis Many affected patients complain of acrocyanosis of the ears, the nose tip, ngers, and toes in cold

Cold agglutinin syndrome

Incidence
Cold agglutinin syndrome (CAS) is relatively uncommon compared with warm antibody AIHA (WAIHA), but it occurs much more frequently than PCH. In a series of 347 patients with acquired immune hemolytic anemias (including drug-induced immune hemolytic anemia), 54 patients (15.6%) had CAS.2 Dacie and Worlledge3 reported that 25 percent of their patients with AIHA had CAS, whereas van Loghem et al4 and Dausset and Colombani5 reported an incidence of only 7.7%.

Age and sex

Patients with idiopathic CAS are typically middleaged or elderly. Worlledge6 reported that the average age of 29 patients was 66. In Dacies series, only 3 of 21 patients with idiopathic CAS were younger than 50, whereas 9 of 12 post-pneumonia cases occurred in patients between the ages of 20 and 45.7 Chronic idiopathic cold agglutinin disease occurs only very rarely in childhood.79

Clinical characteristics
Characteristic Clinical Manifestations are indicated in Table 3.
Table 3

Cold Agglutinin Syndrome Characteristic Clinical Manifestations.

Symptoms and Signs Symptoms of Chronic Anemia Fatigue Dyspnea on Exertion Weakness Dark Urine (especially in cold weather) Acrocyanosis (occasionally leading to gangrene of digits) Pallor and Jaundice Hepatosplenomegaly and Lymphadenopathy may be present. Laboratory Findings Mild to Moderate Anemia Prominent autoagglutination, especially at cold temperatures. Abnormal RBC Morphology Modest degrees of Spherocytosis, Anisocytosis, Poikilocytosis, Polychromatophilia Reticulocytosis Jaundice Hemoglobinuria Erythroid Hyperplasia in the bone marrow

4 temperatures, a condition that vanishes quickly on warming.11,16 These symptoms are caused by autoagglutination of the patients erythrocytes in the skin capillaries causing local blood stasis. Gangrene Occasionally actual gangrene, particularly of a digit or digits, has been observed.17,18 Although the occurrence of gangrene after prolonged exposure to cold would appear to be a logical consequence of vascular insufciency caused by autohemagglutination, there is some question as to whether the presence of cryoglobulins may not be an additive factor. Raynauds phenomenon The acrocyanosis associated with cold agglutinins may be distinguished from the much more common Raynauds phenomenon that may be idiopathic or associated with diseases such as collagen vascular disorders.10 In the former, the exposed distal ngers or toes turn a dusky blue that either quickly reverts to normal on warming or may progress to blanching after more prolonged or more severe cold exposure. In Raynauds phenomenon, the digits characteristically turn white from vasospasm and then, as ischemia is resolving, turn blue; this change may be followed by a hyperemic phase in which the exposed parts become reddened. Further, exposure to moderate degrees of cold in patients with cold agglutinins will cause all digits to be equally affected, whereas Raynauds phenomenon may cause marked blanching of one or two ngers with the others remaining normal. Other physical ndings Pallor and jaundice may be present and the intensity of these ndings depends on the rate of hemolysis and the ability of the liver to excrete bilirubin. Hepatosplenomegaly and lymphadenopathy are not prominent ndings.11 Dacie7 indicates that the spleen is palpably enlarged only in a minority of patients, whereas Schubothe12 states that usually the spleen and sometimes the liver are slightly enlarged. Cutaneous necrosis is an unusual manifestation of cold agglutinin syndrome.1922

L.D. Petz erty distinguishes cold autoagglutination from rouleaux formation and also from autoagglutination which occurs in some patients with warm antibody AIHA. In the latter case, the autoagglutination does not resolve at 37 C. The agglutination creates problems in making satisfactory blood lms and performing blood counts.11,2326 However, it should be stressed that clinically insignicant cold autoantibodies will also cause autoagglutination. Such antibodies have a low thermal amplitude and do not cause hemolysis.27 Therefore, the mere observance of cold autoagglutination is not diagnostic of cold agglutinin disease, and the cold antibodies must be further characterized as described below. A rather common diagnostic error is over-diagnosis of CAS in patients who have benign cold antibodies and pathogenic warm autoantibodies. Peripheral blood RBC morphology is less abnormal than in patients with warm antibody AIHA, with lesser degrees of anisocytosis and poikilocytosis.7 Similarly, spherocytosis is less intense but has been noted by Leddy and Swisher,28 Schreiber et al29 and Rosse.30 Leukocyte and platelet counts are usually normal. Bilirubinemia of mild degree is common. Reticulocytosis usually is in proportion to the severity of hemolysis. Bone marrow Bone marrow examination shows a variable degree of erythroid hyperplasia and a slight but denite increase of lymphoid cells.11 Several authors have reported lymphoid proliferation in some cases which may become progressively more extensive, especially in the terminal stages of the disease.12,31,32 Diagnostic Serologic Tests for CAS. The diagnosis must be considered in all patients with acquired hemolytic anemia who have a positive direct antiglobulin test (DAT) using anti-C3 and a negative DAT using anti-IgG. A practical initial serum screening procedure is to test the ability of the patients serum to agglutinate saline-suspended normal red cells at 20 C (or room temperature) after incubation for 3060 minutes. This recommendation is based on the fact that the sera of essentially all patients series who have CAS cause agglutination of saline-suspended red cells at 20 C. If this screening test is negative, cold agglutinin syndrome is extremely unlikely; if positive, further studies are necessary to determine the thermal amplitude of the antibody. When CAS appears to be a possible diagnosis on the basis of the preceding evaluation, studies of the thermal range of reactivity of the antibody in

Laboratory ndings
Autoagglutination Autoagglutination of anticoagulated blood samples that occurs quickly as blood cools to room temperature is characteristic and is frequently the rst observation made to suggest the diagnosis. Autoagglutination is intensied by cooling the blood to 4 C and is reversed by warming to 37 C. This prop-

Cold antibody autoimmune hemolytic anemias saline and albumin are indicated. It is also convenient to determine possible li blood group specicity of the antibody simultaneously. The titer of the cold agglutinin in CAS is invariably highest at 4 C and progressively decreases at higher temperatures. Of particular note are the reactions at 30 C. If the reaction is positive in saline or albumin, the antibody may well be of pathogenetic signicance, i.e., it may be causing short red blood cell survival in vivo. Utilizing clinical information, the results of the DAT, and the preceding screening tests, a reasonably condent assessment of the presence or absence of CAS may be made. CAS may be diagnosed if the following are present: (1) Clinical evidence of acquired hemolytic anemia; (2) a positive DAT caused by sensitization with C3; (3) a negative DAT using anti-IgG; (4) the presence of a cold autoagglutinin with reactivity up to at least 30 C in saline or albumin; and (5) a cold agglutinin titer (at 4 C) P 256, except in exceptional cases. An alternative diagnosis must be sought for patients who do not satisfy all these criteria.

Management
Avoidance of cold Therapy for cold agglutinin syndrome is often unsatisfactory. It is fortunate that the disorder is frequently not severe and, instead, results in a chronic mild hemolytic anemia with a hemoglobin level of 912 gm per dL. Such patients require no therapy other than avoidance of cold.33 If patients go out in cold weather, they must clothe themselves well, including earmuffs, warm socks, and warm mittens. These measures will prevent the development of acute hemolytic crises but even with strict avoidance of cold, some hemolysis usually persists. Patients with moderate degrees of anemia should usually be urged to learn to tolerate the symptoms rather than embark on therapeutic trials that have greater potential risk than probable benet. Corticosteroid therapy Corticosteroids are less effective in cold agglutinin syndrome than in warm antibody syndromes.33 Lack of success of corticosteroid therapy in cold agglutinin syndrome has been reported by Pisciotta,34 Firkin et al,35 Dausset and Colombani,5 and Dacie.33 However, some case reports do indicate that corticosteroids can be effective, often in patients with atypical clinical and serological ndings.29,36,37 Immunosuppressive drugs Cyclosphosphamide and chlorambucil have resulted in favorable responses in a minority of patients with CAS.12,38,39 In patients who do not respond satisfactorily to therapy with immunosuppressive drugs, bone marrow depression and temporary worsening of anemia result. Therefore, it is wise to begin with low doses orally, e.g., daily doses of chlorambucil of 2 or 4 mg per day. Biweekly blood counts including a reticulocyte count should be obtained, and the daily dose may be increased by 2 mg every 2 months until favorable effects or limiting adverse reactions result. The latter effects usually consist of marrow suppression, although anorexia and nausea may limit dosage as well. If a favorable response does result, maintenance therapy may be advisable, although an alternative approach is to use therapy intermittently as needed.38,40 Fludarabine Jacobs41 described a patient with idiopathic cold agglutinin disease who initially responded to cyclophosphamide-containing chemotherapy but subsequently became refractory to this agent. Treatment was changed to udarabine, 25 mg/mm2 of body surface area daily for 5 days intravenously.

Course and prognosis

The prognosis in patients with idiopathic CAS is signicantly better than it is in patients with warm antibody AIHA. Most commonly, the patient has a chronic, fairly stable anemia that is mild or moderate in severity. For example, in only 2 of the 12 patients reviewed by Ferriman et al14 did the hemoglobin fall below 7.4 gm/dl. Similarly, Schubothe12 describes the course of the disease as monotonous, and patients may survive many years, suffering minimal disability. He reported 16 patients followed for several years, and every patient had a hemoglobin of 9 mg/dl or greater at some time during the period, and in only 2 patients did the hemoglobin fall below 6.5 gm/dI at any time. This nding contrasts strikingly with hemoglobin values in warm AIHA or PCH. Most patients tolerate their mild or moderate anemia quite well. In more severe cases, however, death may ensue from complications of slowly progressive anemia, or from complications of blood transfusion. Sometimes, after a considerable time, a generalized malignant lymphoproliferation may occur which is manifested in the bone marrow morphology, as described above. Occasionally, an intensication of hemolysis with an increase of the thermal amplitude up to the range of the internal body temperature seems to lead to a fatal complication.

6 The treatment was repeated every 28 days for a total of three courses. This resulted in a clinical remission with a normal hemoglobin, reticulocyte count and bilirubin that had lasted 4 years by the time of publication. Although udarabine therapy has been known to cause AIHA,42 Jacobs41 nevertheless suggested that consideration should be given to udarabines use in patients with idiopathic CAS who are refractory to conventional alkylating agents. Rituximab A number of reports indicate that Rituximab43 is an effective drug in some cases of CAS.4447 Berentsen et al48 reported on 37 courses of rituximab administered prospectively to 27 patients with primary chronic CAS. Fourteen of 27 patients responded to their rst course of rituximab, and 6 of 10 responded to re-treatment. In both groups combined, responses were achieved after 20 of 37 courses, giving an overall response rate of 54%. The investigators observed 1 complete and 19 partial responses. Two nonresponders and 3 patients who experienced relapse received second-line therapy with interferon-alpha combined with a new course of rituximab, and 1 nonresponder and 2 patients who experienced relapse achieved partial responses. Responders achieved a median increase in hemoglobin levels of 40 g/L (4 g/dL). Median time to response was 1.5 months, and median observed response duration was 11 months. The authors concluded that rituximab is an effective and well-tolerated therapy for CAS. Schollkopf et al49 performed a phase II multi center trial to investigate the effect of rituximab in CAS, including 20 patients studied from October 2002 until April 2003. Thirteen patients had idiopathic CAS and seven patients had CAS associated with a malignant B-cell lymphoproliferative disease. Rituximab was given in doses of 375 mg/m2 at days 1, 8, 15 and 22. Sixteen patients were followed up for at least 48 weeks. Four patients were excluded after 8, 16, 23 and 28 weeks for reasons unrelated to CAS. Nine patients (45%) responded to the treatment, one with complete response, and eight with partial response. Eight patients relapsed, one patient was still in remission at the end of follow-up. There were no serious rituximab-related side-effects. The authors concluded that their data study conrms previous ndings of a favourable effect of rituximab in patients with CAS. However, they suggested that few patients will obtain complete remission and, in most patients, the effect will be transient. Other reports also indicated that rituximab is effective in achieving remissions in patients with idiopathic and secondary CAS.5054

L.D. Petz Plasma exchange There is some logic to the use of plasma exchange in those hemolytic anemias caused by IgM antibodies, since IgM has a predominantly intravascular distribution, and experience has been favorable with treatment of IgM-induced hyperviscosity syndrome and hemostatic defects in Waldenstroms macroglobulinemia. However, the results in patients with cold agglutinin syndrome have been far from impressive and improvement, when it occurred, has generally been only temporary. Further, dealing with cold agglutinins with a high thermal range necessitates special procedures to prevent autoagglutination during the procedure. McLeod et al,55 reviewed 14 reported cases of cold agglutinin syndrome that had been treated with plasma exchange. They stated that most patients responded with rapid and substantial reductions in antibody titer and that elevations of hemoglobin level and/or decreases in transfusion requirement were seen in many patients. However, the improvement in many cases was transient and apheresis therapy did not confer signicant longterm benet. Thus, the procedure appears to offer a modest degree of temporary benet in some patients with cold agglutinin syndrome. Splenectomy Splenectomy is generally considered ineffective in patients with cold agglutinin syndrome. McCurdy and Rath reported failure in one patient.56 Bell et al.57 described the outcome in three patients; two died of sepsis and one died of lymphoma. Pirofsky15 described one patient who derived no clinical benet. However, other investigators have described marked clinical improvement after splenectomy, particularly in patients whose antibody caused lysis of enzyme-treated red cells at 37 C.33,58,59 Intravenous immunoglobulin (IVIG) Rather little data are available concerning the use of IVIG in cold agglutinin syndrome. A review of cases of AIHA treated with IVIG was developed by the University HealthSystem Consortium. Of the 77 cases of AIHA evaluated, all but one were warm antibody AIHA.60 The exception was a patient with cold agglutinin syndrome who was refractory to this therapy.61 Therapeutic failures Although chronic idiopathic CAS is often a mild disorder, there are a number of reports of more serious disease and the failure of a variety of therapeutic maneuvers. For example, Seldon et al62 reported a fatal case of acute low-titer wide-thermal-range cold agglutinin syndrome.

Cold antibody autoimmune hemolytic anemias High-dose corticosteroids, cyclophosphamide, and plasmapheresis failed to control hemolysis. Similarly, Rousey and Smith63 reported a patient who had idiopathic cold agglutinin syndrome with a low titer IgM cold agglultinin with anti-PR specicity, and who died because of refractory hemolysis. The patient required frequent transfusions (25 units of RBC per week). All therapies that were attempted failed including prednisone, cyclophosphamide, azathioprine, IVIG, plasmapheresis and danazol. Serologic studies documented rising titers of the IgM cold agglutinin and immediately prior to death, the thermal amplitude reached 37 C.

7 young children. The reason why acute transient PCH appears to be a more common type of childhood AIHA than it was several decades ago is uncertain, but probably relates to greater awareness of the disorder and more frequent use of the Donath-Landsteiner test, especially in children with acute AIHA with hemoglobinuria.

Classication
Dacie75 has suggested that PCH should be classied into three categories, i.e., chronic syphilitic, chronic non-syphilitic and acute transient. In modern times, chronic PCH is very rare and almost all cases are of the acute and transient type. PCH may also be classied as idiopathic or secondary.

Paroxysmal cold hemoglobinuria (PCH)

Incidence
Many reports emphasize that paroxysmal cold hemoglobinuria (PCH) is an unusual disease. Howard et al64 noted only two occurrences in 38 years and 298,878 admissions at the Montreal Hospital. Becker65 noted that the diagnosis was made in only I patient in 20 years at the University of Chicago clinic, during which time 130,000 patients were admitted to the hospital and 382,799 patients were seen as out-patients. Pirofsky15 encountered only 2 occurrences over a I5-year period at Bellevue Hospital in New York and at the University of Oregon hospitals and clinics. PCH was diagnosed in only 4 of 34 children with acute AIHA by Habibi8 and only one of 17 children reviewed by Buchanan et al.66 Subsequent reports suggested a somewhat higher incidence. Dacie and Worlledge3 reported 15 cases out of a total of 195 patients with AIHA (5.1 percent) and van Loghem4 and his associates reported an incidence of 10 percent in their 168 patients. Johnsen et al67 encountered 3 cases within a period of 7 months, and especially remarkable is the report of Bird et al68 who diagnosed this disorder in 3 children within a period of just 16 days. In more recent years, PCH has become recognized as being a relatively frequent cause of acute transient AIHA in childhood. Sokol et al69 stated that 17 of 42 (40%) of patients less than 16 years of age with AIHA had formed Donath-Landsteiner antibodies, and Gottsche et al70 reported an inci dence in children with AIHA of 32% (22 of 68). Wynn et al71 reported six cases of children presenting with PCH occurring in a 3-year period on the north-west of England, and Lambert et al72 reported six episodes of biphasic hemolysis in four children over a 1-year period. Indeed, both Nordhagen et al73 and Heddle74 have suggested that PCH is one of the most common causes of acute AIHA in

Race, sex, and age

There does not appear to be any particular racial distribution for the development of PCH. Puzzling information is the fact that combined data in three reviews70,74,76 indicate that 52 of 77 children were boys, a boy-to-girl ratio of 2.1:1. Acute PCH typically presents in young children. In the series of Sokol et al77 the median age at presentation was 5 years. In one study, all of 22 patients with PCH were less than 9 years old, no cases being seen amongst 531 adults with AIHA.70 Although particularly common in children, the disease affects patients of all ages, and the patients in the series of Sokol et al77 ranged from 1 to 82 years.

Symptoms and signs

Characteristic clinical manifestations are indicated in Table 4. The typical presentation is that of a child who, during the preceding 12 weeks, had suffered from an upper respiratory tract infection.74,75,77,78 Usually the onset of hemolysis is signaled by a recurrence of fever and then the passage of red-brown urine.75 Hemoglobinuria, jaundice and pallor were the most common clinical ndings in 42 cases of acute PCH reported by Heddle.74 Of particular note is that hemoglobinuria was found in 41 of the 42 cases in that report. Abdominal pain and fever were also common ndings. Approximately 25 percent of cases presented with palpable liver and spleen. Hemoglobinuria following exposure to cold is rare in acute PCH, and was seen in only one of 52 patients reported by Sokol et al.77 Nevertheless, hemoglobinuria may be induced by exposure to cold.75,77 There are many descriptions in the older medical literature of Raynauds phenomenon occurring in

8
Table 4 Paroxysmal Cold Hemoglobinuria Characteristic Clinical Manifestations.

L.D. Petz

Symptoms and Signs Patient Usually a Child History of a recent upper respiratory or u-like illness. Acute onset of Illness Fever Malaise Abdominal Pain Red or Red-Brown Urine is Frequently Present Jaundice Pallor Laboratory Findings Anemia Often Severe May be Rapidly Progressive Reticulocytosis (Reticulocytopenia in Some Patients) Abnormal RBC Morphology Spherocytosis, anisocytosis, poikilocytosis, autoagglutination, Polychromatophilia. Erythrophagocytosis by neutrophils is commonly present Hemoglobinuria Erythroid Hyperplasia or Normal Results in Bone Marrow Leukocytosis (Leukopenia in Some Patients) Platelet Count usually Normal or Elevated

association with attacks of hemoglobinuria in probable cases of PCH.75 The ngers, toes, tip of the nose, lips, or ears may be blanched or become deeply cyanotic, and even gangrene has been described.

Red blood cell morphology

The presence of spherocytes, anisocytosis, poikilocytosis, fragmented red cells, basophilic stippling, polychromatophilia, autoagglutination, erythrophagocytosis, and nucleated erythrocytes have all been reported.15,57,67,77,78 Spherocytosis is particularly common in the peripheral blood of severely affected patients.67,68,75,77,78,80 Osmotic fragility is increased commensurate with the spherocytosis present,75 but has seldom been recorded.

Hematologic ndings
Typical laboratory ndings in PCH are indicated in Table 4.

Hemoglobin
Severe and rapidly progressive anemia frequently occurs. Levels of hemoglobin as low as 5 gm/dl or even lower, have been quite commonly reported.7882 Sokol et al77 reported that the minimum hemoglobin level had been less than 5 gm/dl in 17 of 51 cases (33%), and Heddle74 reviewed 42 published cases and found that the admission hemoglobin ranged from 2.5 to 12.5 gm/dl with a mean value of 6.8 gm/dl. Gottsche et al 70 re ported hemoglobin levels less than 5 gm/dl in six out of 22 cases, the minimum being 4.4 gm/dl.

Bone marrow examination

Bone marrow aspiration is not often performed, but, when carried out, generally reveals normal results or erythroid hyperplasia.77

Leukocytes
Leukopenia followed by a neutrophil leukocytosis with a shift to the left may occur during a paroxysm induced by cold, but usually normal white blood cell counts or leukocytosis are reported during acute attacks.

Reticulocytes
A relative reticulocytopenia occurs in some patients with PCH at the time of presentation.71,72,77,78,80 Heddle74 reported that reticulocytopenia was prominent feature in four of the ve cases seen in her medical center.

Platelets
Platelet counts seem to have been carried out infrequently in cases of acute PCH.75 Where reported, the counts usually were normal, although

Cold antibody autoimmune hemolytic anemias increased numbers have been reported in a few patients.

9 young children with acute transient PCH. Three reports devoted to this phenomenon are accompanied by photographs of erythrophagocytosis.8486

Diagnostic serologic tests for paroxysmal cold hemoglobinuria

The autoantibody associated with PCH is termed a biphasic hemolysin, that is to say, it sensitizes RBCs in the cold but only hemolyzes them when the RBCs reach 37 C. The diagnostic test is the Donath Landsteiner (DL) test where normal RBCs are incubated with the patients serum at 0 C (e.g., melting ice) and then moved to 37 C for a further incubation. No lysis occurs following the incubation at 0 C, and no lysis occurs if the incubation is carried out only at 37 C. The thermal amplitude of this antibody is usually less than 20 C, that is to say, the antibody will give a positive DL test only when the initial incubation is <20 C; stronger results will occur as the temperature of the initial incubation is lowered. Rare patients have been described when the DL test is positive when the rst incubation phase is as high as 32 C, or their DL antibody would sensitize RBCs up to 37 C, as detected by the IAT. If positive results are obtained in the DL test, determination of the specicity of the autoantibody is indicated. Almost all cases of PCH are associated with anti-P specicity, although rare reports of other specicities said to be associated with PCH have been reported. RBCs necessary for determining anti-P specicity are rare but, with the assistance of reference laboratories, specicity testing can be carried out. It should be noted that the DL test must be used to determine specicity. A comparison of the typical characteristics of the antibody in CAS with the Donath-Landsteiner antibody of PCH is indicated in Table 5. Erythrophagocytosis. Red cell adherence and erythrophagocytosis by neutrophils, rather than monocytes, is a prominent nding in acute transient PCH,75,83 but is seen rarely in other forms of immune-mediated hemolytic anemia74 including WAIHA.83 Heddle74 reported the phenomenon to be present in 20% of all PCH cases and in 80% of

Treatment and prognosis

The prognosis of acute PCH is excellent, although deaths have been reported.76 Acute attacks are frequently severe but the acute illness characteristically resolves spontaneously within a few days to several weeks after onset and rarely recurs.78 Accordingly, the patients require only supportive care. During the acute phase of the disease, severe intravascular hemolysis is usual, and transfusion of RBC may be necessary. Cortiocosteroids are often given although their effectiveness is difcult to evaluate because of the transient nature of the hemolysis. The Donath-Landsteiner antibody may persist in low titer and with low thermal amplitude for longer period of time. Indeed, Dacie reported the persistence of the Donath-Landsteiner antibody for eight years after a single episode of hemoglobinuria.58 More commonly, the titer and thermal range of the antibody fall rapidly, so that two to three months later it is no longer detectable.87

Combined cold and warm AIHA or Mixed AIHA

In unusual instances, a patient will be found who has serologic ndings characteristic of warm antibody AIHA while also having a cold agglutinin of high titer and thermal amplitude, thus satisfying the criteria for both warm antibody AIHA and cold agglutinin syndrome.2 Such cases are frequently referred to as combined cold and warm AIHA or mixed AIHA, and reviews of AIHA generally include such a category.8893

Clinical characteristics
Sokol et al94 described 25 patients with mixed AIHA, and indicated that such patients accounted

Table 5 Comparison of typical characteristics of the antibody in cold agglutinin syndrome with the DonathLandsteiner antibody of paroxysmal cold hemoglobinuria. Cold agglutinin Syndrome Titer (4 C) Thermal range Bithermic lysis (Donath-Landsteiner test) Immunoglobulin class Specicity high (>500) high (>30 C) negative IgM anti-I or i Donath-Landsteiner Antibody moderate (<64) moderate (< 20 C) positive IgG anti-P

10 for about 7% of their cases of AIHA. The patients tended to have severe hemolysis which although usually responding well to initial treatment, ran a chronic course with intermittent exacerbations, thus making overall management difcult. Shulman et al95 reported that 12 of their 144 patients (8.3%) with AIHA satised diagnostic criteria for both warm antibody AIHA and cold agglutinin syndrome. All patients had cold agglutinins that reacted up to 37 C, although in none of the patients was the cold agglutinin titer at 4 C greater than 64. All 12 patients had severe hemolytic anemia that responded dramatically to corticosteroid therapy, with the mean hemoglobin level increasing from 6.3 gm/dl to 12.9 gm/dl. However, similar to the patients reported by Sokol et al94 most of the patients reported by Shulman et al95 were found to develop chronic hemolysis. Only in 2 patients was corticosteroid therapy tapered off completely without a relapse of AIHA. Of the seven patients followed up who had chronic hemolysis, ve showed persistence of both IgG warm autoantibodies and high-thermal amplitude cold autohemagglutinins, while two lost their cold autohemagglutinins and showed serologic ndings indistinguishable from those of warm antibody AIHA.

L.D. Petz with the clinical course of patients with warm or cold AIHAs has not been made.

Secondary autoimmune hemolytic anemias

Autoimmune hemolytic anemias are classied as secondary for any of several reasons. One reason is the association of AIHA with an underlying disease with a frequency greater than can be explained by chance alone. For example, all authors agree that the incidence of warm antibody AIHA is higher in patients with chronic lymphocytic leukemia (CLL) and systemic lupus erythematosus (SLE) than in the general population. Another criterion for categorizing a given case of AIHA as secondary is the reversal of the hemolytic anemia simultaneously with the correction of the associated disease. Ovarian tumors are a good example; well-documented cases of cure of the AIHA after surgical removal of the tumor have been reported.98101 Still another reason for suspecting a relationship between the occurrence of AIHA and an associated disease consists of evidence of immunologic aberration as part of the underlying disorder, especially if the associated disease is thought to have an autoimmune pathogenesis. However, when the coexistence of two immunologic disorders in a single patient is infrequent, the pathogenetic signicance is conjectural and interpretations vary. For example, rheumatoid arthritis is an extremely common disorder and reports of an associated AIHA are infrequent, so the combination may well be coincidental.99 Most cases of secondary CAS are associated with infectious agents or hematologic malignancies.

Cautions concerning the diagnosis of cold and warm AIHA

The diagnosis of mixed cold and warm antibody AIHA is sometimes made on the basis of inadequate serologic studies.96,97 Appropriate characterization of the serum antibodies must be made prior to determining the specic type of AIHA present. An important source of potential error is suggested by our data2 which indicate that 35% of patients with WAIHA have cold agglutinins reactive at 20 C. However, a large majority of these are cold agglutinins are clinically insignicant, and we found that only 5% reacted at 37 C. Unless one can document a cold autoantibody with a high thermal amplitude (>30 C) in association with a warm autoantibody, a diagnosis of cold and warm (mixed) AIHA is not warranted.

CAS and infectious diseases

Mycoplasma pneumonia infection and infectious mononucleosis are the most common infectious diseases associated with CAS. Less commonly, cold agglutinin syndrome may be associated with rubella, varicella, cytomegalovirus, Legionnaires Disease and HIV infection.1

Clinical course Lymphoproliferative disorders

Some authors suggest that patients with cold and warm AIHA have a more severe onset and more chronic course than patients with other categories of AIHA. However, a comprehensive comparison The records of 637 patients with lymphoproliferative disorders and 346 patients with myeloproliferative disorders were retrospectively analyzed for

Cold antibody autoimmune hemolytic anemias the presence of co-existent autoimmune derangements.102 The frequency of autoimmune perturbations in lymphoproliferative diseases (51 cases; 8.0%) was signicantly higher than in myeloproliferative diseases (six cases; 1.7%; p < 0.0001). Rheumatic disorders, autoallergic hematological manifestations and other organ-specic autoimmune derangements were responsible for about one third each of the observed disturbances. These data are similar to those of Miller103 who observed diffuse connective tissue diseases and autoimmune hemolytic anemias in 6.8% of his lymphoma patients. AIHA is the most common autoimmune disease associated with CLL and CLL is the most common of the known causes of AIHA.104,105 Various reports indicate that AIHA occurs in 537% of CLL patients.105111 The gures are highly dependent on the duration and extent of disease.105 In the great majority of cases of CLL complicated by AIHA the responsible autoantibodies have been of the warm type but there have been some reports of cold antibody AIHA in association with CLL.112114 Ruzickova et al115 reported a patient with a 7-year history of idiopathic CAS in whom B-CLL subsequently developed. They determined that the B-CLL had developed from the patients cold agglutinin-producing B-cell population. AIHA is an uncommon occurrence in patients with Hodgkins disease. In 1967 Eisner et al116 reported the incidence of AIHA in a series of 219 patients was 2.7%. With one exception, all of the patients had advanced disease at the time of detection of the positive DAT. Levine et al117 reviewed 71 cases of Hodgkins disease and found three who had AIHA (4.2%). Only a minority of cases of secondary AIHA in non-Hodgkins lymphoma are associated with cold antibodies although a number of reports have emphasized this association.118124 Economopoulos et al118 reviewed 370 patients with NHL and found that 23 (6.2%) had AIHA, 4 of whom (1.1%) had cold reacting antibodies.

11 ential counts in 14 patients with CAS. The number of lymphoid cells was between 10 and 24 percent in 5 cases, between 26 and 33 percent in 3 cases and between 41 and 95 percent in 6 cases. In several patients, the lymphoid cells increased during the course of the disease. On the basis of such ndings some authors feel that chronic CAS is probably a variant of Waldenstroms macroglobu linemia126 in which the IgM M-component has cold agglutinin activity.12,127,128

Non-hematologic malignancies and cold agglutinin syndrome

The association of nonhematologic malignant disorders with CAS is uncommon.88,129131 Wortman et al129 reported seven patients who had a variety of nonhematologic malignancies including squamous cell carcinoma of the lung, metastatic adenocarcinoma of the adrenal, metastatic adenocarcinoma of the colon, a mixed parotid tumor, a carcinoid tumor of the ampulla of Vater, carcinoma of the larynx, and a thoracic neurinoma. The high frequency of carcinomas in patients in the age group in which cold agglutinin syndrome occurs suggests that the relationship may be fortuitous in at least some instances. That this is not true in all cases is indicated by the patient reported by Lands and Foust130 in whom resolution of the AIHA occurred after excision of a renal cell carcinoma.

Secondary paroxysmal cold hemoglobinuria

In the 19th and early 20th centuries PCH was frequently found in association with latent syphilis, but with the development of effective antibiotic therapy for syphilis, this association has become rare,75,77 although not unknown.77,132138 Recent reports generally describe an acute transient hemolytic anemia often associated with a upper respiratory infection or a u-like illness of undened etiology. However, PCH has been associated with a very wide range of specic infectious agents including measles, mumps, chicken pox, Coxsackie A9, parvovirus, cytomegalovirus, infectious mononucleosis, Haemophilus inuenzae, Mycoplasma pneumoniae, Klebsiella pneumonia, adenovirus and inuenza virus A.1 Also, PCH has rarely been associated with other disorders, including non-Hodgkins lymphoma139,140 and oat cell carcinoma.141 Some patients have no history of an antecedent illness, and they appear healthy until the onset of the acute paroxysm.68

The relationship of CAS and Waldenstroms macroglobulinemia

A number of authors have noted similarities of laboratory ndings in patients with CAS and patients who have monoclonal IgM proteins without cold agglutinin activity. In both instances, bone marrow ndings may consist of increased numbers of abnormal lymphoid and plasma cells.125 Schubothe12 performed serial examinations of the bone marrow with quantitative differ-

12 Practice points  Immune hemolytic anemias are divided into distinctive categories which have differing clinical manifestations, prognosis and therapy.  Cold antibody autoimmune hemolytic anemias are comprised of cold agglutinin syndrome (CAS) and paroxysmal cold hemoglobinuria (PCH).  CAS occurs in middle-aged or elderly patients and is caused by an IgM antibody that is maximally reactive in the cold but which reacts to at least 30 C in vitro.  PCH occurs primarily in children and is caused by an IgG biphasic hemolysin that is demonstrated by incubation in the cold followed by incubation at 37 C in the presence of complement.  Both CAS and PCH may be primary (idiopathic) or secondary to other disorders.  Therapy of CAS primarily consists of avoidance of cold, immunosuppressive drugs and rituximab.  Therapy of PCH consists of supportive care since the disease is generally self-limiting, albeit after a frequently stormy course.

L.D. Petz
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