Reduction of High-Titer Cold Agglutinins by Plasmapheresis to Resolve Serological Problem in a Patient

Background: Cold agglutinins are auto-antibodies that can be a nuisance in cross matching and in blood grouping. Here we report an unusual case of a high titer and wide amplitude cold agglutinin reduced by plasmapheresis. Methods and Materials: A 56-year-old man with severe anemia requested a transfusion of red blood cells. However, there was a problem in blood for blood grouping. The discrepancy of blood typing was subsequently resolved using group O absorbed plasma along with repetition of forward grouping with warm-washed red blood cells. The presence of high-thermal-amplitude and a high-titer anti-I cold agglutinin were detected in further serologic investigation. It revealed reactivity against autologous and adult O red blood cells at 37˚C by the thermal amplitude screening test, and demonstrated a very high titer of 65,536 against adult O cells by titration studies at 4˚C. The patient received two plasma exchange sessions of 1.5 plasma volumes each. There was a significant reduction of the titer of cold agglutinins and of the thermal amplitude by plasmapheresis as well (p < 0.01). Results: After suc-cessful cross-matching with post plasma exchanges, four units of red blood cells were infused to the patient without any hemolysis symptoms or signs. Conclusions: We now reported a patient with abnormally ascended titer of cold agglutinins and wide-thermal-amplitude, but we also successfully performed ABO typing and cross matching after 2 plasma exchange sessions of plasma volumes each.


Introduction
Cold agglutinin, which was first described in 1903, and about approximately 25% of autoimmune hemolytic anemias (AIHA) was led by it [1] [2]. Cold agglutinins are antibodies that bind to erythrocyte surface antigens at low temperatures, and then can cause agglutination and complement-mediated hemolysis [2]. The low titer cold agglutinins are often detected in healthy individuals, and most of them are benign [3]. Pathological cold autoantibodies of high antibody titers and/or broad thermal amplitude, may be important clinical significance and cause acute or chronic hemolysis [4]. The immunoglobulin M antibodies usually specificity to carbohydrate antigens, are most commonly in blood group Ii [5].
The VH4-34 gene segment can encode the specific for the I/i RBCs membrane systems [6]. The same gene segment also can encode bacterial anti-lipopolysaccharide activity and other specificities. B cells containing VH4-34 gene sequence constitute part of the innate immune system, which were participated in the first line of defense against pathogens. Patients with cold agglutinins frequently accompany acute infectious diseases, such as Epstein-Barr viral infection, Mycoplasma pneumoniae pneumonia and lymphoproliferative disorders [1] [6]. The mechanism may be that the pathogen modifies the I active receptor on sialic acid to make it immunogenic, so that the body produces antibodies to this modified structure [6].
Cold agglutinins react the strongest at 2˚C -5˚C, however, its binding may occur at temperatures approaching normal body temperature of mammals, which depends on the thermal amplitude of the antibody [7]. High titer cold antibodies can agglutinate red cells from the most human adults, including autologous red cells from the patient [7] [8]. However, the presence of cold agglutinins may cause problems in blood ABO/Rh typing and cross matching for patients requiring transfusion [8].
We now reported a patient with abnormally ascended titer of cold agglutinins and wide-thermal-amplitude, but we also successfully performed ABO typing and cross matching after 2 plasma exchange sessions of 1.5 plasma volumes each.

Case Report
A 56-year-old male of Han nationality presented to the Hematology Department of our hospital with symptoms of extreme asthenia. At admission, he was very pale with evidently undernourished and the BP (blood pressure) was 80/50 mmHg. In addition, he was dyspnoeic and tachycardiac (110 bpm) at rest. Heart sounds soft, but there is no other pathologic sign concerning his lungs and abdomen. In the past months, he had complained about cough. Routine blood test revealed a white blood cell count of 8.28 (4 -10) × 10 9 /L, platelet count of 134 (100 -300) × 10 9 /L, hemoglobin of 48 (120 -170) g/L, however, the hematocrit, mean corpuscular volume, and the number of red blood cells could not be measured because of the interference by agglutinated erythrocytes. A lactic de- Hematologist decided to reduce autoantibody titer by plasma exchange when the wide-thermal-amplitude and high titer cold agglutinins were discovered in the patient. One session of plasmapheresis was performed every other day, and two sessions of plasmapheresis were finally performed in two days.
A COBE Spectra Blood separator was used to involve continuous flow plasma exchange, 1.5 plasma volumes were replaced each time with a combination of 5% albumin in 0.9% saline. After commencing plasmapheresis, cold autoantibody levels rapidly decreased to half or less and cold agglutinin titers at 4˚C dropped from 65,536 to 2048 ( Table 2). The blood was retaken 12 h after the second plasmapheresis and performed cross-matched test, which showed a compatible cross match with 4 units RBCs (type B, D+) were safely infusion using a blood warmer. Two days later, the transfusion had an expectedly results and his Hb levels had been increased by 19 g/L. Furthermore, the patient's posttransfusion indirect bilirubin level had decreased to 36.60 μmol/L and the LDH was 280 U/L.

Discussion
Usually, the cold agglutinin is considered to have no clinically significant because of its low thermal amplitude. The thermal amplitude refers to the highest temperature at which the cold agglutinin reacts with the antigen. When the thermal amplitude exceeds 28˚C to 31˚C, erythrocytes may agglutinate in acral parts of the body even at mild temperatures, and often leading to complement binding and complement-mediated hemolysis [1] [7]. Interestingly, when the patient's blood sample was first identified, the anti-I autoantibody react at IAT phases, 37˚C, against O-type red blood cells and/or autologous, which was consistent with the evidence of hemolytic in the laboratory. Cold autoantibodies of low titer are common and often found in normal individuals. However, they can be a nuisance in blood grouping and cross-matching [5] [8], as in this case. Reverse grouping was not concordant with forward typing, and the 3 panel cells were reactive with a positive auto-control which may suggest the possible presence of alloantibodies. Subsequent serological examination revealed the exact presence of a high-titer and high-thermal-amplitude cold agglutinin. Forward typing was performed after washing patient's blood cells with warm water and reverse typing was performed with pre-warmed plasma, however, the results of forward and reverse typing were still conflicting. Therefore, the blood grouping when repeated using group O absorbed plasma and warm-washed RBC, resolved blood typing discrepancy and the patient was group B.
Unfortunately, reactivity against autologous and adult O red blood cells at 37˚C were revealed by the thermal amplitude screening test, and a very high titer of 65,536 against adult O cells at 4˚C by titration studies. In general, the pathogenicity of cold agglutinin depends more on the degree of the thermal amplitude rather than on the titer [10] [11]. However, titer levels above 512 at 4˚C are considered to be clinically significant [12]. Berentsen and colleagues described a median titer of 2048 among 86 patients with cold agglutinin disease, in a study from Norway [13]. Similarly, Stone and associates observed titers ranging from 512 to 65,536 among 172 patients with the same disease [12]. In this case, the titer level of cold agglutinin was 65,536 at 4˚C, and 128 at 30˚C, the results suggested the exist of the potential antibody which could disturb blood grouping and cross matching, might the risk of in vivo hemolysis. Laboratory data from patients and subsequent serological test confirmed this concern.
The cold agglutinins involved are usually IgM class, less common are IgA or

Conclusion
Pathological cold agglutinin disease may be related to different degrees of thermal amplitude and titer. In summary, although it is not necessary to detect examples of cold agglutinins that only react below body temperatures, they are deserve considering in the context of appropriate serological when there are broad thermal amplitudes and high titers. Importantly, the case described by Koike [14] demonstrated, therapeutic plasma exchange may result in a significant decrease of the titer of cold agglutinins and the thermal amplitude, which is benefit to the solution of serological problems. The dramatic case provides further evidence on the clinical relevance of high-thermal-amplitude, a high-titer cold ag-Y. H. Cheng et al. glutinin. In addition, therapeutic plasma exchange may be a significant reduction of the thermal amplitude and the titer of cold agglutinins which facilitate the solution of serological problems.