IgA plasma cell myeloma presenting as cold agglutinin-induced haemolytic transfusion reaction

Abstract

Cold agglutinins produced in the setting of B cell neoplasms, such as lymphoplasmacytic lymphoma and plasma cell myeloma, can mediate autoimmune haemolytic anemia. Transfusion of these patients can exacerbate cold agglutinin-mediated haemolysis. Moreover, the workup for these reactions represents a diagnostic challenge due in part to false negative direct antiglobulin tests (DATs). Here, we report an anaemic patient who after a red blood cell (RBC) transfusion performed without blood warming, experienced a DAT-negative haemolytic transfusion reaction, and was later diagnosed with IgA-multiple myeloma, which showed an uncommon granular pattern by CD138 immunohistochemistry. Extensive workup excluded other diagnostic possibilities, including the presence of Donath-Landsteiner antibodies and cryoglobulins. Successful treatment with CyBorD (cyclophosphamide, bortezomib and dexamethasone) achieved complete remission, and additional RBC transfusions using warmers were completed uneventfully.

Background

Cold agglutinin disease (CAD) is a rare type of primary autoimmune haemolytic anaemia, caused by immunoglobulins (usually IgM but also IgG or IgA) that react with erythrocytic surface antigens (most commonly I/i or H proteins) as the temperature approaches 0°C.¹

When cold agglutinin production is secondary to an underlying disorder, such as autoimmune disease, viral infection or B cell malignancies, the term cold agglutinin syndrome (CAS) is used. Most commonly, CAS is associated with CD5-negative/CD10-negative low-grade B-cell lymphomas with IgM monoclonal gammopathies, such as lymphoplasmacytic lymphoma. However, other non-Hodgkin’s lymphomas and more rarely plasma cell neoplasms can also be involved.^{2 3}

Recognition of CAS can be challenging due to variable clinical presentation, agglutinin’s thermal amplitude and low diagnostic test sensitivity. A high index of suspicion is necessary when the ‘rule of three’ (correlation between red blood cell count (RBC count), haemoglobin (Hb) and haematocrit values) is violated in the setting of haemolytic anaemia,⁴ and when associated disorders, such as infections, autoimmunity or haematolymphoid malignancies are present.

Case presentation

A woman in her 70s with a medical history of essential hypertension, Hashimoto thyroiditis, primary endometrial carcinoma in remission status post-total abdominal hysterectomy/bilateral salpingo-ophorectomy presented at our hospital for progressive dyspnoea on exertion and fatigue for 6 weeks. Dizziness, presyncope/syncope, restless legs or B symptoms, including fevers, chills/night sweats or weight loss were denied. Medical history of bleeding disorders, anaemia or prior transfusions was absent. Family and personal medical history were unremarkable to haematological malignancy.

No urinary abnormalities (anuria, oliguria or colour changes), exposure to new drugs, kidney/muscle injury or events of intermittent anaemia suggestive of enzymatic deficiencies, such as glucose-6-phosphate dehydrogenase were identified. Physical examination revealed pale conjunctiva, normal vital signs, and no organomegaly.

Investigations

The patient was found to have pancytopenia. Complete blood count revealed normocytic anaemia (RBC of 2.3×10ˆ12/L, Hb of 6.7 g/dL, haematocrit of 20.3% and mean corpuscular volume of 87.7 fl), leucopenia of 2300/µL with mild lymphopenia (700/µL and thrombocytopenia of 48 000/µL. Borderline low reticulocyte count of 21.5×10 ³/µL, normal reticulocyte percentage (0.94), increased ferritin (2209 ng/mL), decreased total iron binding capacity (221 ug/dL), decreased transferrin (189 mg/dL); and normal iron (81 µg/dL), transferrin saturation (30.6%) and vitamin B12 (452 pg/mL), allowed the diagnosis of anaemia of chronic disease. Other laboratory tests were unremarkable (ESR, CHEM7, PT, PTT, troponin I, TSH, free T3, free T4, and Parvovirus B19 DNA, ANA, ANCA, Complement C4), except for liver function tests showing hypoalbuminaemia (3.6 g/dL), mildly increased SGOT (59 units/L) and elevated C reactive protein (2.7 mg/dL). Pulmonary embolism was ruled out by CT angiography. She received 2 units of packed red blood cells (pRBCs) with an appropriate increase in Hb to 9.1 g/dL. The patient was discharged and was readmitted to the hospital 2 weeks later for dyspnoea on exertion. Due to symptomatic anaemia (Hb of 7.1 g/dL) another unit of pRBCs was transfused, after which she reported overnight dark bloody urine and an episode of inability to swallow characterised by globus sensation with food (liquids/solids). A transfusion reaction workup was initiated detecting no significant change in blood pressure, temperature, pulse or respiratory frequency. A clerical check revealed no error, and the bag and tubing were intact.

Red discolouration of plasma was observed in the post-transfusion sample (figure 1A) indicating haemolysis, supported by elevated LDH (1194 IU/L) and total bilirubin (3.5 mg/dL) at the expense of indirect bilirubin, decreased haptoglobin (8 mg/dL), elevated plasma free Hb (17.4 mg/dL, normal range <8.4 mg/dL) and haemoglobinuria. A routine direct antiglobulin test (DAT) (IgG and C3, Immunocore, Houston, TX) was negative. To rule out the possibility of an immune haemolytic transfusion reaction due to low antibody titres (below the sensitivity of our assay), a 3% RBC suspension of the pretransfusion sample was incubated for 10 min at 4°C followed by microscopic examination (figure 1B). This pretransfusion sample showed RBC agglutination, which disappeared after incubation for 1 min at 37°C, demonstrating cold agglutination. As expected, a sample from a normal patient used as a control did not show agglutination. In an attempt to detect antiglobulins, peripheral blood samples collected 4 days post-transfusion were sent to reference laboratories (Quest Diagnostics, Chantilly, VA, for cold haemagglutinin titre testing based on macroscopic visual inspection; and The American Red Cross, Baltimore, MD, for complete direct antiglobulin investigation, including low-level IgA/IgM/IgG and eluate testing). However, these analyses did not detect an immune basis for the observed haemolytic anaemia, which could be explained as the patient was not haemolysing at the time of this specialised testing. Furthermore, paroxysmal nocturnal haemoglobinuria flow cytometry (QuestDiagnostics, Chantilly, VA) and infectious work-up were also negative.

Figure 1.

(A) Pretransfusion peripheral blood sample (tube on the left) showing normal light gold-coloured serum, next to a post-transfusion sample (tube on the right) demonstrating red-coloured serum indicative of haemolysis. (B) Microscopic agglutination of pretransfusion red blood cells after incubation at 4°C (400×). (C) Peripheral blood smear with arrows showing mild rouleaux formation but no agglutination (modified Giemsa stain, 400×). (D) Light and heavy chain immunoglobulin immunofixation (Sebia Hydragel) demonstrating IgA kappa (k) monoclonal bands (arrows).

A peripheral blood smear (figure 1C) showed minimal left shift, thrombocytopenia, anisopoikilocytosis and occasional rouleaux formation, raising the possibility of a B-cell lymphoproliferative disorder. Therefore, additional work-up for anaemia of chronic disease was continued with a focus on haematolymphoid neoplasms.

Interestingly, serum protein electrophoresis showed increased beta globulins (2.4 g/dL) with a possible monoclonal band, proven to be IgA-kappa monoclonal protein by immunofixation (IF) (figure 1D). Serum IgA was elevated (2219 mg/dL), while IgG (544 mg/dL) and IgM (<25 mg/dL) were normal. A kappa Bence-Jones protein was also identified by urine IF. Bone marrow biopsy revealed kappa-restricted plasma cell myeloma with a 50% tumour burden (figure 2A,B). Interestingly an unusual cytoplasmic pattern was noted by CD138 immunohistochemistry (figure 2C).

Figure 2.

Bone marrow sections showing infiltration by plasma cells myeloma (H&E stain at 100× in (A) and 400× in (B)). Positive for CD138 (C, 200×) and negative for Pax5 (D, 200×) by immunohistochemistry, which rules out the possibility of lymphoplasmacytic lymphoma.

Cytogenetic analysis demonstrated an unremarkable 46XX karyotype with normal patterns of chromosomal hybridisation using fluorescent probes for IGH, TP53, 1q21, 13q, 9, 11 and 15.

Treatment

Eighteen days after the transfusion reaction another RBC transfusion was required due to low Hb (6.7 g/dL), which was performed uneventfully using a blood warmer. The patient received chemotherapy (CyBorD (cyclophosphamide, bortezomib and dexamethasone)) for plasma cell myeloma achieving complete remission. Of interest, 2 months after initiating chemotherapy a new nadir of Hb (6.7 g/dL) required one more RBC transfusion, which was also completed without adverse events or haemolysis using a blood warmer. Taken together, the data support the diagnosis of cold agglutinin-induced haemolysis, as the best explanation for this clinical presentation.

Outcome and follow-up

Cold agglutinins demonstrate an increased affinity for polysaccharide epitopes on membranous erythrocytic glycoproteins (usually I, i or Pr antigens) leading to RBC aggregation and activation of the classical complement pathway resulting in haemolysis. Depending on the magnitude of complement activation, this haemolysis can be intravascular (mediated by the formation of membrane attack complex on RBCs) or extravascular (when RBCs opsonised by immunoglobulins, C3b and C4b, are removed by the reticuloendothelial system). The diagnostic criteria for CAD would be a positive DAT for anti-C3d, negative or weakly positive for IgG and a cold agglutination titre of 64 or greater at 4°C.⁵ Specialised testing for non-IgM anti-antibodies (eg, anti-IgG or anti-IgA) is not widely available and could have been useful in our case. However, because DAT was negative, we could not diagnose CAD in this case of documented cold agglutin-induced haemolysis.

Discussion

We considered in the differential diagnosis two other major types of cold-sensitive antibody-related haemolytic disorders, paroxysmal cold haeoglobinuria (associated with Donath-Landsteiner antibodies and autoimmune haemolytic anaemia, can mimic CAS. However, both were clinicopathologically incompatible with our case. Donath-Landsteiner antibodies are usually IgG molecules that recognise the P antigen on RBCs, causing paroxysmal cold haemoglobinuria. Cryoglobulins (IgG, IgM or IgA either polyclonal or monoclonal), form antigen-antibody complexes that do not generally interact with RBC but instead precipitate and bind to small vessels producing systemic vasculitis or vascular occlusion.⁶

Therefore, since our patient was diagnosed with IgA-kappa restricted plasma cell myeloma and agglutination of RBC was observed microscopically after cold incubation, the best explanation for the observed haemolytic transfusion reaction is low titre mediated cold agglutination. However, we could not directly demonstrate agglutinins, and interpret the DAT as a false negative result. False negativity for DAT has been reported in up to 10% of CAD due to various factors, including low sensitivity, non-IgG type agglutinins (undetectable by the routine IgG-based tests used in our hospital), and delayed testing (as was probably the case of the samples we sent to reference laboratories). Low-affinity antibodies may frequently detach from the RBC membrane and remain unrecognised. Moreover, due to haemolysis, only a few target molecules may be attached to intact erythrocytes at the time of analysis,⁷ which probably explains our observed negativity for C3b. In this patient, additional attempts to detect a low-level immunoglobulin (IgA, IgG and IgM) associated with RBCs or in eluates also failed at a reference laboratory, which could be similarly explained because of the timing of testing. Even with optimal timing and sophisticated high-sensitivity diagnostics, detecting low titre cold agglutinins may be challenging, and we hypothesise that sample/antigen degradation in combination with low titres played a role in the observed DAT-negativity of our case. Specifically, The American Red Cross test was done 1 week after the transfusion reaction, and the QuestDiagnostics cold agglutination titre test may have been insufficiently sensitive for adequate detection of agglutination since it is tailored to detect higher-titre Mycoplasma pneumoniae agglutinins based on visual inspection (naked eye) of serial dilutions against a lightbox background. Therefore, the microscopic examination that we performed after cold incubation of a pretransfusion sample, may be more sensitive to detect agglutination than the aforementioned reference laboratory test.

Although cold agglutination is more frequently associated with IgM autoantibodies, it has also been described in IgA-myeloma and IgG-myeloma.^8–12 However, cold agglutinin-induced haemolytic reaction associated with IgA-myeloma has not been reported in PubMed.

Another rare feature of the case presented here pertains to the granular pattern noted by CD138 immunohistochemistry, which has been reported in only approximately 2% of myeloma cases in a series.¹³

Of interest, studies correlating the immunohistochemical pattern of CD138 expression in plasma cell myeloma with different immunoglobulin isotypes have not been published, to the best of our knowledge.

In summary, we present a unique haemolytic transfusion reaction induced by cold agglutinins associated with IgA-plasma cell myeloma (including a rare CD138 granular pattern by immunohistochemistry), which may facilitate the diagnoses of other cases requiring thorough clinicopathological correlation.

Learning points.

• Cold agglutinin syndrome (CAS) is a rare type of autoimmune haemolytic anaemia, caused by immunoglobulins produced in the course of autoimmune disease, viral infection or B cell neoplasms, which react with erythrocytic surface antigens at low temperatures.

• CAS is most commonly associated with CD5-negative/CD10-negative low-grade B-cell lymphomas producing IgM monoclonal gammopathies, such as lymphoplasmacytic lymphoma.

• Cold agglutinin-mediated haemolysis triggered by unwarmed transfusion in association with plasma cell myeloma is rare and may represent a diagnostic challenge due in part to false negative direct antiglobulin tests (DATs).

• DAT false negativity has been reported in up to 10% of cold agglutin-induced haemolysis due to various factors, including low sensitivity, non-IgG type agglutinins (undetectable by the routine IgG-based tests), and delayed testing, highlighting the need for a high index of suspicion and specialised testing in CAS.

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