British Journal of Naen~atology. 1982, 50, 345-350

Autoimmune haemolytic anaemia with monoclonal IgM ( K ) anti-P cold autohaemolysins

A. E. G. KR. VON DEM BORNE, J . J . MOL, N. JOUSTRA-MAAS, J . G. PEGELS, M. M. A. C. L A N G E N H U I J S E N A N D ~ . 1’. ENGELFRIET, Department of Iininunohnematolog~, Central Laboratory of the Netherland Red Cross Blood Transfiision Service and Division of Haematologg, Department of lnternal Medicine, Academic Hospital of the Free University, Amsterdam, The Netherlands

Received 28 August 1980; accepted for publication 3 0 J u l y 1981

SUMMARY. A patient is described with imrnunoblastic non-Hodgkin’s lymphoma and autoimmune haemolytic anaemia of the cold autoantibody type. The autoantibodies were monoclonal IgM-k- cold haemagglutinins/haemolysins with blood-group specificity, anti-P. Red-cell autoantibodies directed against blood-group-P antigen have until now only been detected, as polyclonal IgG antibodies, in paroxysmal nocturnal haemoglobinuria.

Chronic autoimmune haemolytic anaemia of the cold autoantibody type is usually a form of monoclonal macroglobulinaemia in which the IgM paraproteins are cold auto-haemagglu- tinins and auto-haemolysins (Dacie, 1962; Wintrobe, 1974). The monoclonal autoanti- bodies often show blood-group specificity. Mostly they are anti4 (Wiener et al, 1956; Worlledge & Blajchman, 19721, sometimes anti-i (Marsh & Jenkins, 1960; Worlledge & Blajchman, 1972) and rarelyanti-Prl, anti-Pr2 and anti-Pr3 (Roelcke, 1973,1974; Roelcke et al, 1976) or anti-Gd (Roelcke et al, 1977). Until now, autoantibodies with the specificity anti-P have only been described as polyclonal biphasic haemolysins of the IgG class in acute paroxysmal cold haemoglobulinaemia. secondary to syphilis or viral infections (Levine et al, 1963; Bird, 1977).

In this report we describe a patient with non-Hodgkin’s lymphoma complicated by chronic haemolytic anaemia, due to monoclonal IgM-x cold agglutinins/haemolysins, directed against the blood-group-P antigen.

CASH HISTORY

A 54-year-old man was seen in 1975 because of an enlarged left tonsil. On biopsy the

Correspondence: Dr A. E. G. Kr. von dem Borne, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9190, 1004 AD Amsterdam, The Netherlands. 0007-1048/82/0200-0345$02.00 @ 1982 Blackwell Scientific Publications

345

346 A. E. G. Kr. von dem Borne et a1

diagnosis of non-Hodgkin's lymphoma, immunoblastic type, was made. The lymph nodes at the left side of his neck were affected, but on staging, which included laparotomy and splenectomy, no other site of involvement was found. He was treated with 4000 rads of mantle-field irradiation and 12 courses of cyclophosphamide, oncovin and prednisone (COP).

In 19 77 a relapse occurred in the left inguinal lymph nodes and 4000 rads of inverted Y-irradiation were administered. In 1978 COP treatment was given, because of further relapses involving cervical and axillary lymph-nodes. Shortly afterwards evidence of a severe haemolytic anaemia appeared (haemoglobin 4.7 g/dl, haematocrit 0.120/,, reticulocytes 7.1%, haptoglobin 10 mg%) due to cold haemolysins. Treatment with red-cell transfusions, plasmapheresis, prednisone and later with chlorambucil were of little value. He died in 1978.

MATERIALS AND METHODS

Patient's blood. Blood clotted at room temperature and at 37°C was used for the preparation of serum and red cell suspensions. Plasma was obtained by plasmapheresis, and was converted into serum by recalcification.

Antiglobulin sera. The following antisera were used: monospecific anti-IgG (KH 16-123-A1), anti-IgM (KH 15-119-A1), anti-IgA (KH 14-116-A1) and polyspecific anticomplement (KH 13-1 13-A1). All were products from the Central Laboratory.

Serological techniques. The direct and indirect antiglobulin test, the saline agglutination test at 4, 16 and 3 7"C, the two-stage bromelin test at 4.16 and 3 7"C, the test for monophasic haemolysins with normal and bromelinized red cells at 16 and 3 7°C and the test for biphasic haemolysins were performed as previously described (Engelfriet et al, 1968). IgM antibodies were inactivated by reduction with dithiothreitol (DTT) according to Olson et al(1976).

lrnniunochemical techniques. Levels of the patient's serum-IgG, -1gM. -IgA, -Clq. -C4 and -C3 were determined by standard radial immunodiffusion with precipitating monospecific antisera from the Central Laboratory. Normal values are: for IgG, 6 5-200 IU/ml (I IU = 80.4 p g ) ; TgM, 45-335 IU/ml(l IU=8.5 p g ) ; IgA, 40-225 IU/ml(l IU= 14.2 p g ) ; Clq, 69-128%; C4, 37-161%; C3,66-1240/, (xf2SD). For the complement-component assays, the value of a pool of 50 fresh frozen sera from normal donors was taken as 100%. These determinations were kindly performed in the Central Laboratory by T. Out and A. Hannema.

Simple electrophoresis in agar and immunoelectrophoresis of the patient's serum, before and after absorption with normal donor red cells, and of donor red cell eluates (see later) were done by standard methods. The sera were fractionated by precipitation with half saturated (NH4)2S04 at 0"C, followed by chromotography on G-200 Sephadex according to Killander & Hogman (1963).

Absorption and elution. In order to absorb all cold autoantibodies out of the serum, a hundred-fold dilution of the serum in phosphate-buffered saline (PBS) was absorbed twice, at 4"C, with an equal part of washed packed, P2 donor red cells. The diluted serum was then concentrated back to its original volume by pressure dialysis. No autoantibody activity was detectable in the absorbed serum after this procedure. Control absorptions, with donor pp red cells, were done in the same way.

Cold autoantibodies against red cells were purified from the serum of the patient in the

Monoclonal 1gM Anti-P in AIHA 34 7 following way: One part of serum was incubated with one part of washed packed normal P2 or pp donor red cells. After 1 h at 4°C the red cells were spun down and the supernatant was removed. The sensitized red cells were then resuspended in 9 parts of PBS, incubated for 1 5 min at 37"C, followed by an hour at 4°C. Again the red cells were spun down. The supernatant was discarded and substituted by 9 parts of fresh PBS, etc. This procedure was repeated five times. The last step consisted of the elution of the autoantibodies, from the sensitized donor cells, in an equal part of PBS at 3 7°C. This eluate was used for serological and immunochemical analysis.

RESULTS

The direct antiglobulin test was only positive with anticomplement serum and not with anti IgG, IgA or IgM. Absence of antibodies on the patient's washed red cells, was confirmed by an ether-eluate of these cells, which did not react in any of the tests for red cell antibodies. There was no autoagglutination at room temperature and there was weak autoagglutination at 4°C.

The results of the serological investigations of the patient's serum are shown in Table I. The serum contained cold agglutinins, detectable only in the presence of albumin, against the patient's own cells and normal donor red cells or after treatment of the red cells with bromelin. The optimal temperature was 0-4°C. These antibodies also showed strong monophasic haemolytic activity, especially with bromelin-treated red cells, and at 16°C. Treatment with neuraminidase (cholera filtrate) did not enhance agglutination nor haemolytic activity. The haemolysin titre was higher at pH 6.5 than at pH 7.5. The Donath-Landsteiner test with normal red cells was negative. All agglutinating and haemolytic activity was destroyed by treatment of the serum with DTT.

Immunochemical analysis of the serum showed normal immunoglobulin levels: IgG 2 10

Table I. Serological results

Incubation temperature (OOC,

Test 4°C 16°C 37°C

Saline agglutination 1/1 0 0 Albumin agglutination 1/128 1/64 0 Bromelin agglutination 1/4000 1/2 56 0 Monophasic haemolysis*

Normal red cells 0 1/512 0 Bromelinized red cells 0 1/2000 1/128

Biphasic haemolysis Normal red cells (4-37"C): 0

*At pH 6.5.

348 A. E. G. Kr. von dem Borne et al

IU/ml, IgM 260 IU/ml, IgA 230 IU/ml. Ofthe complement factors measured, only C4 was significantly reduced to a level of 17%; C l q was 100%, C 3 90%. A test for cryoglobulins (24 h at 0°C) was negative. On agar electrophoresis, a monoclonal immunoglobulin component was found which appeared to be IgM-ti on immunoelectrophoresis. Upon (NH4)2S04 precipitation of the serum followed by Sephadex G-200 gel-filtration of the precipitated proteins, both the cold agglutinin/haemolysin activity and the monoclonal immunoglobulin were detected in 19s high-molecular-weight IgM containing fraction.

The antibodies were tested by agglutination and haemolysis for bloodgroup specificity with a panel of typed red cells. No bloodgroup-I-related specificity could be detected. However (Table 11), the antibodies were anti-P. Unfortunately, at the time of these investigations, the patient had died and his red cells were no longer available. Since the antibodies were autoreactive antibodies, it is likely that the patient’s red cells were positive for the P antigen.

After absorption of the serum with positively reacting P2 donor red cells, both cold agglutinin/haemolysin activity and the monoclonal IgM-K immunoglobulin disappeared from the serum, while they remained present after absorption with negatively reacting pp donor cells (Table 11). Cold autoantibodies were purified from the serum of the patient by repeated absorptions and elutions with P2 donor red cells. The eluate contained group P antibodies and IgM-ic paraprotein. Negative results were obtained when pp donor red cells were used.

Table 11. Blood-group specificity of the cold haemolysins*

Donor red cells PI P2 P1k pp

Serum 113200 1/6400 - - Serum, absorbed with P2 red cells - - _ - Serum, absorbed with pp red cells 1/1600 1/1600 - -

* Bromelin haemolysis test at 16°C.

DISCUSSION

Autoimmune haemolytic anaemia of the cold antibody type is a well-known, though rare. complication of non-Hodgkin’s lymphoma (NHL) and of other malignant lymphoproliferative diseases (Dacie, 2962, 1967; Pruzanski & Shumack, 1977). The responsible cold autoanti- bodies are mostly monoclonal IgM and directed against antigens of the bloodgroup Ii system.

In this report we describe a patient with immunoblastic NHL, autoimmune haemolytic anaemia, and monoclonal IgM-lc cold haemolysins, with anti-P specificity. A case such as this. to our knowledge, has not previously been described.

Anti-P cold autoantibodies, which are regularly found in paroxymal cold haemoglobu- linaemia, are always polyclonal IgG antibodies. They are incomplete antibodies, which cause hardly any agglutination of normal red cells, even at 0-4OC. With these antibodies and complement, optimal lysis of red cells only takes place upon biphasic incubation (0OC

Monoclonal IgM Anti-P in AIHA 349

followed by 3 7°C) and at pH 7.5. Enzymic treatment of the red cells produces only marginal enhancement of lysis (Davie, 1962; Schubothe, 1958). IgM-anti-I/i cold autoantibodies, on the contrary, are strong cold agglutinins, which lyse red cells optimally after enzymic treatment upon monophasic incubation at 16% and at a slightly acid pH of 6.5 (Schubothe, 1958; Dacie, 1962).

It is therefore of interest that the cold antibodies, present in the blood of our patient, showed properties of both antibody types. At 4°C normal red cells suspended in saline were not, or only weakly, agglutinated and there was hardly any autoagglutination at this temperature. Agglutination occurred only in the presence of albumin or after treatment with bromelin. Haemolysis of red cells took place only during monophasic incubation at 16°C and was markedly enhanced by lowering the pH to 6.5 and/or by bromelin treatment of the red cells.

It is unusual of 19s IgM red cell antibodies to haemolyse non-enzyme-treated cells in saline more strongly than they agglutinate them. It is aIso unusual for IgM cold autoantibodies to have anti-P specificity. Whether these two unusual characteristics of antibodies are related remains hypothetical.

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