• haemolytic anaemia;
  • cold agglutinin;
  • lymphoproliferative;
  • therapy;
  • rituximab


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The ‘primary’ form of chronic cold agglutinin disease is a clonal B-cell lymphoproliferative disorder that is notoriously difficult to treat with drugs, including corticosteroids, alkylating agents, alpha-interferon and purine analogues. We performed a small, open, uncontrolled, prospective study to evaluate the effect of therapy with the monoclonal anti-CD20 antibody rituximab. Six patients with clonal CD20+κ+ B-cell proliferation received seven courses of rituximab 375 mg/m2, d 1, 8, 15, and 22. One patient achieved a complete response. Four partial responses were observed, including a response to re-treatment in one patient. Two patients were categorized as non-responders. Haemoglobin levels increased by a median of 4·1 g/dl in the total group and 4·7 g/dl in the responders, who also experienced a substantial improvement of clinical symptoms. The treatment was well tolerated. We discuss the effect of rituximab therapy compared with other treatment options, and try to explain why two individual patients did not respond. Despite the small numbers, the results are very encouraging. Further studies of rituximab therapy for chronic cold agglutinin disease are warranted.

Chronic cold agglutinin disease (CAD) is an uncommon autoimmune haemolytic anaemia (AIHA) characterized by the production of cold agglutinins (CAs). The CAs in CAD are typically monoclonal IgMκ autoantibodies against carbohydrate antigen I, characteristically agglutinating erythrocytes with increasing affinity at decreasing temperatures (Dacie, 1992a; Ulvestad et al, 1999). Because of the haemagglutination most patients experience pallor, acrocyanosis and Raynaud's phenomenon during slight to moderate cold exposure (Berentsen et al, 1997). All patients have cold-induced haemolysis caused by concomitant complement activation during CA binding (Kirschfink et al, 1994; Ulvestad et al, 1999).

The traditional distinction between primary (idiopathic) and secondary forms of CAD may be questioned. Contrary to the conclusions from earlier investigations (Crisp & Pruzanski, 1982) nearly all patients with ‘primary’ CAD have a lymphoproliferative bone marrow disease, and the monoclonal CAs are produced directly by the neoplastic B cells (Silberstein et al, 1986; Silberstein, 1994; Berentsen et al, 1997). Clonal lymphoproliferation is usually demonstrated by flowcytometric immunophenotyping (Silberstein et al, 1986; Berentsen et al, 1997). Diagnostic histological features are identified in at least half of the patients, most often the features of lymphoplasmacytic lymphoma (Berentsen et al, 1997). The clonal B cells nearly always express the CD20+κ+ immunophenotype (Berentsen et al, 1997), and trisomy of the q arm of chromosome 3 has been shown in some cases (Silberstein et al, 1986; Michaux et al, 1998).

In patients traditionally classified as having ‘secondary’ CAD, the lymphoproliferative disease is usually of intermediate or high-grade malignancy, is easily recognized, affects predominantly lymphoid organs other than the bone marrow and tends to produce IgMλ rather than IgMκ (Crisp & Pruzanski, 1982; Dacie, 1995). It may be convenient to keep using the term ‘secondary’ for these relatively rare cases.

Corticosteroids are of little value in treating ‘primary’ CAD, and other therapeutic approaches have included alkylating agents, α-interferon and cladribine. Although most publications describe single case reports or investigations of a few patients, these drugs have failed to demonstrate a convincing clinical effect (Worlledge et al, 1968; Hippe et al, 1970; Hillen & Bakker, 1994; Berentsen et al, 1997, 2000). The low proliferative activity of the clonal B cells and the high biological activity of the IgM autoantibodies may explain the poor response to cytotoxic chemotherapy (Berentsen et al, 1997; Ulvestad et al, 1999).

The monoclonal chimaeric human-murine anti-CD20 antibody rituximab (MabThera, Rituxan) has been used successfully for the treatment of several other CD20+ low-grade lymphoproliferative B-cell diseases (Maloney et al, 1997; McLaughlin et al, 1998). Compared with most cytotoxic drugs, the adverse effects of rituximab are different and less severe. The total numbers of clonal B cells are assumed to be substantially lower in primary CAD than in other lymphomas in need of therapy (Berentsen et al, 1997).

We performed a small, open, uncontrolled, prospective study to investigate the effect of rituximab therapy in primary CAD. The study was approved by the Regional Ethical Committee of Health Region 2 in Norway.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients were recruited from the haematological units of three Norwegian university hospitals and some larger central and county hospitals between 1 January and 31 October 2000.

The inclusion criteria were (i) CAD, as defined by the combination of chronic haemolytic anaemia with cold agglutinin titre > 64 and a typical pattern for the direct antiglobulin test (DAT); a positive test with polyspecific antiserum, negative or only weakly positive with anti-IgG, and strongly positive with anti-C3d (Dacie, 1992a; Berentsen et al, 1997; Ulvestad et al, 1999). (ii) The presence of a clonal lymphoproliferation of the CD20+κ+ phenotype, as defined by the combination of a monoclonal IgMκ band in serum with a clonal expansion of CD20+κ+ cells in the bone marrow, demonstrated by bone marrow histology and/or flowcytometric immunophenotyping (Isaksson et al, 1996; Berentsen et al, 1997). (iii) The presence of clinical symptoms requiring treatment, e.g. anaemia or Raynaud-like symptoms and (iv) Informed consent.

Patients with ‘secondary’ CAD (e.g. intermediate or high-grade malignant lymphoma) were not eligible for the study. Patients were also excluded if they had lymphocytosis > 50 × 109/l in peripheral blood, non-lymphocytic malignant disease, any other severe disease in addition to CAD or contraindications to rituximab therapy.

Before inclusion, all patients underwent clinical examination, chest radiograph and abdominal ultrasonography. Flowcytometric immunophenotyping was performed as described previously (Berentsen et al, 1997). The intensity of CD20 expression was estimated semiquantitatively as 0, + or ++ respectively. Bone marrow biopsies were examined by a trained lymphoma pathologist and classified according to the REAL (Revised European–American lymphoma) classification (Harris et al, 1994).

Rituximab was administered as described previously (McLaughlin et al, 1998) at a dose of 375 mg/m2 as an intravenous infusion on d 1, 8, 15 and 22. Infusion-related side-effects were recorded.

The patients were assessed monthly for 6 months. Cold-induced pallor, acrocyanosis, and Raynaud's phenomena were assessed using a 0–2 scale (0 = no improvement; 2 = complete resolution of clinical symptoms).

Blood counts and measurements of biochemical parameters and IgM levels were performed at each visit. CA titration and serum electrophoresis with immunofixation were repeated at 3 and 6 months after therapy. Bone marrow assessment by histology and flow cytometry was performed at 3 months.

Complete response (CR) was defined as absence of anaemia, no signs of haemolysis, disappearance of clinical symptoms of CAD, undetectable monoclonal serum immunoglobulin and no signs of clonal lymphoproliferation as assessed by bone marrow histology and flow cytometry. Partial response (PR) was defined as a stable increase of haemoglobin (Hb) levels by > 2 g/dl and a 50% reduction in elevated serum IgM concentrations, combined with improvement of clinical symptoms and no need for transfusions. Patients were classified as non-responders (NR) if they did not achieve a complete or partial response.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Six patients were included, two men and four women, with a mean age of 68 years (range 54–80) and mean disease duration of 12 years (range 2–18). Patient 4, who relapsed 6 months after therapy, was subsequently re-treated as patient 7 using the same treatment protocol.

Three patients were previously untreated. The pre-treated patients had received corticosteroids (two patients), chlorambucil (one), cyclophosphamide (one) or cladribine (three). Patient 4/7 had had a splenectomy 6 years before inclusion. The number of different therapy regimens before inclusion was one regimen in one patient, two regimens in one and five regimens in patient 4/7. None of the patients had achieved any improvement after previous treatment.

Therapy was completed in all patients. They were all alive after a median observation period of 11 months after therapy (range 6–14).

Table I shows some baseline characteristics of individual patients. The following median values were found: Hb 8·1 g/dl (range 6·3–9·6), serum IgM 8·3 g/l (4·1–12·5), lactate dehydrogenase (LDH) 871 U/l (499–1399) and bilirubin 53 µmol/l (34–132). CA titres at 4°C ranged from 512 to 256 000.

Table I.  Baseline characteristics.
   Serum Bone marrow aspirate
 Sex   CD20+κ+  
Patient numberAge (years)Hb g/dlIgM g/lLDH U/lBilirubin µmol/LCA titre (4°C) Bone marrow biopsycells (% of lymphocytes)CD20 expression* κ:λ-ratio
  • *

    Semi-quantitative density evaluation using a 0 to ++ scale.

  • Unsuccessful staining for surface light chains in aspirate;

  • κ


  • λ

    λ-ratio determined in biopsy.

1F 548·64·1592681024Lymphoplasmacytic lymphoma34++4·7
2M 699·19·097873512Lymphoplasmacytic lymphoma5Unsuccessful4·0
3F 597·04·4139941512No histomorphologi calsigns of lymphoma18++4·8
4F 676·38·339234256 000Lymphoplasmacytic lymphoma40++20
5F 807·112·58715364 000Lymphoplasmacytic lymphoma75++74
6M 779·68·489013216 384Expansion of B lymphocytes ( CD20+,IgM++). Lymphoma?28 (CD20+)++4·0†
7F 678·15·049934128 000Lymphoplasmacytic lymphoma28++28

The findings in bone marrow biopsies were classified as lymphoplasmacytic lymphoma in four of six patients, clonal lymphoproliferation (lymphoma?) in one and no histological signs of lymphoproliferative disease in one. A clonal B-cell expansion of the CD20+κ+ immunophenotype was demonstrated in all patients using flow cytometry in bone marrow aspirates, the proportion of CD20+κ+ cells averaging 33% of the cells within the lymphocyte gate (range 5–75%). The CD20 expression was found to be strong (grade ++) on the clonal B cells in six patients, whereas this parameter could not be properly assessed in the remaining one. The mean κ:λ ratio was 19 (range 4–74).

Table II shows the Hb levels in individual patients at the start and during the first 6 months after therapy. Other follow-up data are listed in Table III.

Table II.  Haemoglobin levels.*
PatientBeforePost therapy
numbertherapy1 month2 months3 months4 months6 monthsIncrease
  • *


Table III.  Response parameters.
 Serum IgM decrease 3 months after therapyPersisting mono-Cold agglutinin titre (4°C) 3 months after therapyHistomorpho- logical response (bone marrowDecrease in CD20+ cells (bone marrow)Symptom improvementOverall
Patient numberDecrease (g/l)Decrease (%)clonal IgMκTitreDecrease (%)biopsy) 3 months after therapyby flow cytometry (% of pre-treatment percentage)score (0–2) response evaluation
13·483No< 32> 96Complete> 972CR
25·966YesNot doneNot donePartial> 801PR
30·614Yes5120Not relevantUnsuccessful0NR
62·732Yes819250Partial> 960NR

The patients achieved a median increase in Hb of 4·1 g/dl (range 0·8–4·9) and a median decrease in IgM levels of 3·4 g/l (range 0·6–7·9). For the responders these figures were 4·7 g/dl (range 3·3–4·9) and 4·2 g/l (range 2·5–7·9) respectively. We observed a median decrease in LDH levels of 203 U/l for all patients and 240 U/l for the responders (range 0–466). The corresponding figures for bilirubin were 20 µmol/l (range 0–47) and 26 µmol/l (range 11–47) respectively. Clinical symptoms improved only in the responders who reported an improvement of 1–2 using a 0–2 scale.

One patient achieved CR. After treatment, she was able to tolerate Norwegian outdoor winter temperatures without any symptoms, and she enjoyed cross-country skiing for the first time in years.

PR was observed in four cases, i.e. three individual patients including the re-treated patient who responded to both courses of therapy. One of the partial responders had no corresponding histomorphological remission of the bone marrow disorder.

One partial responder had a disease progression 6 months after therapy. In four cases the response duration cannot yet be determined but was > 6 months.

The result was classified as NR in two patients. One of the non-responders had a substantial reduction of the clonal cell mass as judged by bone marrow biopsy, flow cytometry and serum IgM concentration, but did not achieve a significant increase in Hb level or improvement in the clinical symptoms.

Infusion-related side-effects did not occur. No patient asked for the treatment to be stopped. One patient developed transient neutropenia grade 3 without fever or other clinical complications. No other serious adverse effects were observed.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

It is remarkable that one patient achieved a complete response, even though the criteria for CR were very strict. To the best of our knowledge, CR to therapy for CAD has never been reported before.

The total response rate (including PR and CR) was five out of seven. It should be emphasized that to be classified as PR, patients had to achieve both a substantial improvement of laboratory parameters and a clinical improvement.

Because of the low prevalence of the disease, the small number of patients and the uncontrolled design, this study does not permit statistical calculations. However, the data should be compared with published results of other forms of therapy for CAD.

Available data do not suggest that corticosteroid therapy is an effective therapeutic approach in this disorder (Dacie, 1992b; Berentsen et al, 1997). Alkylating agents such as chlorambucil and cyclophosphamide have been used for more than three decades. Early reports demonstrated some effect on the laboratory parameters, and a clinical benefit was observed in some patients (Worlledge et al, 1968; Hippe et al, 1970). Based on more recent reports, the clinical effect seems less convincing (Dacie, 1992b). In a previous survey of 14 CAD patients, we found that four had received alkylating agents (Berentsen et al, 1997). The IgM levels decreased in three patients, but none of them achieved a clinical or haematological improvement meeting the criteria for PR used in the present study. One of the patients in our present study had previously received two different alkylating regimens without any response.

The role of splenectomy has not been investigated prospectively, but clinical experience and theoretical considerations do not support its use (Dacie, 1992b). One of our patients underwent splenectomy several years ago without any improvement. The failure of mercaptane or penicillamine therapy has been summarized by Dacie (1992b). Treatment with α-interferon was described in two publications reporting an improvement of laboratory parameters and, possibly, of clinical symptoms (O'Connor et al, 1989; Rordorf et al, 1994), but these results could not be reproduced by others (Hillen & Bakker, 1994). Previously, we have published a prospective study on cladribine therapy in five patients, but did not observe any clinical or haematological benefit (Berentsen et al, 2000).

Thus, the response to rituximab observed in the present study compares very favourably with the poor effect of all other therapies. The favourable response to a second course of rituximab therapy in patient 7 is promising, although this single observation does not permit any general statement about the effect of re-treatment.

Although LDH and bilirubin levels decreased in most responders and were normalized in the complete responder, the wide ranges suggest that these parameters are not very useful for response evaluation. This can probably be explained by the wide range of baseline levels and by intra-individual fluctuations. The CA titre decrease seems to correlate better to the degree of clinical and haematological remission, as could be expected from the high and variable biological activity of the CAs in CAD.

Two patients failed to show any response to rituximab therapy despite high levels of CD20 expression on their neoplastic cells. The mechanism of B-cell destruction by anti-CD20 monoclonal antibody is not completely understood, but in vitro experiments have demonstrated both antibody-dependent cellular cytotoxicity (ADCC), direct complement-dependent cytotoxicity (CDC) and induction of apoptosis (Reff et al, 1994; Harjunpääet al, 2000). If CDC is an important effector mechanism (Harjunpääet al, 2000), complement depletion may limit the therapeutic efficiency of rituximab in CAD. Many CAD patients have reduced levels of complement factors C3 and C4, with C4 levels frequently below the detection limit (Ulvestad et al, 1999). Previously, we have shown that complement depletion is a limiting factor for the haemolysis in some CAD patients (Ulvestad, 1998; Ulvestad et al, 2001).

The failure of two patients to respond may also possibly relate to relatively small numbers of CD20+κ+ cells, only modestly elevated κ:λ ratios and absent or atypical morphological signs of lymphoproliferative bone marrow disease. Therefore, the small size of the clonal B-cell population may have contributed to the lack of response in these two patients.

Some other individual characteristics of the response to rituximab should be noted. Patient 6 achieved a reduction of the CA producing B-cell clone, but this was not sufficient to produce a significant improvement of haemolytic anaemia or clinical symptoms. On the other hand, patient 5 achieved a substantial clinical and haematological response combined with a reduction of CD20+ cells by flow cytometry, even though histological assessment showed no regression of the lymphoproliferative bone marrow disease. The discrepancy between clinical and cellular response may be explained in part by the high and variable biological activity of the CAs, with large individual variations in both thermal amplitude and affinity for the antigen at the same temperature (Ulvestad et al, 1999).

In conclusion, the preliminary results of rituximab therapy in CAD are very encouraging. The treatment is well tolerated. More studies should be undertaken in order to confirm the results and address issues such as response duration and potential for further improvement.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We want to thank Prof. Jens Hammerstrøm, Trondheim University Hospital, Dr Kjell Sverre Galdal, Lister Hospital, Dr Terje Sindre, Sogn og Fjordane Central Hospital, and Dr Knut Fjaestad, Hedmark Central Hospital, for participating in the treatment and follow-up of patients. We are also grateful to Roche Norge AS for providing rituximab (MabThera) at a reduced price.


  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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