Hemolytic Anemia Complicating Infectious Mononucleosis Due to the Interaction of an IgG Cold Anti-i and an IgM

Cold Rheumatoid Factor

P. GRONEMEYER, H. CHAPLIN, V. GHAZARIAN, F. TUSCANY, A N D G. D. WILNER

From the Departments of Pathology and Preventive Medicine. The Jewish Hospiial and Washington University School of Medicine. St. Louis. Missouri

This is the case of a 25-year-old man who presented with infectious mononucleosis complicated by hemolytic anemia and hepatitis. Antibody evaluation revealed a cold-reactive IgC anti-i and an IgM cold rheumatoid factor. The patient's hemolytic anemia improved fol- lowing treatment with corticosteroids.

HEMOLYTIC A N E M I A is a well-known, though rare, complication of infectious mononucle- osis (IM). Reports have implicated the in- volvement of high thermal amplitude cold agglutinins of anti-i specificity. 1,2,4~6,7,9-1 I The determination of the immunoglobulin class of the involved antibodies has been described in numerous report^.'.^.^,',^'^'^ Troxel et a1.I' found an IgM autoagglutinin of anti-i spe- cificity. Burkart and Hsul implicated an IgM cold-warm hemolysin with anti-i specificity in the production of hemolytic anemia in a 23- year-old with IM. Goldberg and Barnett4 demonstrated interaction of an IgG directed against a red blood cell antigen and an IgM active at 4 C against the IgG. Capra et aL2 found a similar interaction, with the IgG directed against fetal (i) cells, and implied that this was the most common serologic pattern in 1M patients with autoimmune hemolytic anemia. Wilkinson et al." were unable to document this interaction. The case that we report documents the interaction of an IgM cold anti-IgG and an IgG cold anti-i in the

Received for publication July 14, 1980; accepted August 25. 1980.

production of hemolytic anemia in a patient with IM.

Case Report This is a 25-year-old man with a two-week

history of fever, myalgias, and cervical lymphad- enopathy. He was seen in his private physician's office, where the diagnosis of infectious mono- nucleosis was confirmed by a positive heterophil test. Two days prior to admission he noted the development of jaundice, dark urine, nausea, and vomiting. Laboratory tests revealed an LDH of 956 units, SGOT of 116 units, SGPT of 122 units, total bilirubin of 13.8 mg/ dl with a direct bilirubin of 10.6 mg/dl. The hemoglobin and hematocrit were 8.9 g / d l and 25.3 per cent, respectively. The reticulocyte count was 4.2 per cent on admission. The patient had a positive heterophil of I / I 1 2, a cold agglutinin titer of I / 256 against adult group 0 red blood cells, and haptoglobin of less than 50 mg/dl. The direct antiglobulin test was strongly positive for complement and doubtful to negative for IgG. His hepatitis B surface antigen and antibody were negative. A serum protein electro- phoresis was normal. Oral prednisone, 100 mg per day, was administered. Liver function tests im- proved with treatment. Hematocrit and hemo- globin rose to 35.1 per cent and 11.4 g / d l on the ninth hospital day, with a reticulocyte count of 15.1 per cent. The steroids were tapered to 50 mg per day, and he was discharged. He has remained well since discharge with no evidence ofjaundice or anemia.

Methods A fraction of the patient's serum containing

predominantly IgM and only traces of IgG was prepared from the exclusion volume peak off a Sephadex (3-200 column (3 mi of serum on a 2.5 X 89-cm column). The fraction was concentrated to a

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final volume of 2 ml. A fraction containing only IgG was prepared by DEAE-cellulose chromato- graphy of 3 ml of serum equilibrated in 0.0175 M phosphate buffer pH 6.7. The IgG effluent was concentrated to a final volume of 2 ml. Agglutina- tion and indirect antiglobulin reactions employed 0.2 ml of serum or Ig fraction and 0.04 ml of 1% suspension of pooled group 0 red blood cells from three normal adult donors or three normal umbilical cord samples. Tests at 24 and 37 C were incubated for five hours; tests at 4 C were incubated for 16 hours. After incubation, the samples were centrifuged for one minute and examined for agglutination at the temperature of incubation. For the 4 C indirect antiglobulin reactions, the red blood cells were washed with ice- cold saline, and the antiglobulin reaction was carried out at 4 C. For the 24 and 37 C indirect reactions, washing and antiglobulin testing were carried out a t room temperature. The anti-y antiglobulin serum was prepared in this laboratory and absorbed with kappa and lambda Bence Jones proteins, rendering it specific for the y antigen.

Results The patient's serum had a cold agglutinin titer of

1 / 2,560 and I / 256 against cord (i) and adult (1) red blood cells, respectively, at 4 C. Titers were 1 / 128 and zero a t 24 C. respectively, and zero at 37 C (Table I ) . These results are typical for moderately high thermal amplitude anti-i cold agglutination. A Donath-Landsteiner test (with fresh AB serum as a source of complement) was negative against cord and adult red blood cells.

The Sephadex G-200 exclusion fraction of the patient's serum (containing predominantly IgM) was compared with the IgG fraction off DEAE- cellulose for cold agglutinin reactivity a t 4 C. The results summarized in Table 2 indicate no reac- tivity of either fraction against adult cells; the IgG fraction failed to agglutinate cord cells, but the IgM fraction was weakly reactive to a titer of 1 / 20.

Table 1. Evidence for Anti-i Cold Agglutinin in Patient's Serum

Temp. of Agglutination' Incubation Cord RBC Adult RBC

37 c 0 0 24 C 1 /128 0 4 c 1 /2,560 1 /256

Agglutination examined after centrifugation at the temperature of incubation.

Table 2. Cold Agglutinin Reactivities of Patient's Serum Fractions

Fraction Cells Agglutination.

IgG (DEAE) Adult 0 Cord 0

IgM (SephadexG-200) Adult 0 Cord 1/20

IgG plus IgM Adult 0 Cord 1/160

Mixtures were incubated overnight, centri- fuged, and examined for agglutination-all at 4 c.

Note that when the fractions were combined, there was an eightfold increase in anti-i agglutination.

Agglutination and indirect antiglobulin reac- tions with the patient's IgG fraction against cord cells are illustrated in Table 3. Agglutination was not observed at any temperature. IgG was bound to cord cells at 4 C, but not a t 24 or 37 C. These results are consistent with the presence of an incomplete IgG cold anti-i antibody of low thermal amplitude. Studies for the presence of rheumatoid factor in the IgM serum fraction (Table 4) indicate strongest reactivity a t 4 C, with distinctly weaker but nonetheless significant activity a t 24 and 37 C. The IgM fraction contains a high thermal ampli- tude cold rheumatoid factor.

Attempts t o perform immunoelectrophoresis on digitonin and heat eluates from red blood cells incubated in the patient's serum a t 4 C were unsuccessful. The eluates presumably did not contain a sufficiently large concentration of im- munoglobulins to detect electrophoretically.

Discussion The association of acquired hemolytic

anemia and infectious mononucleosis has

Table 3. Studies on i Erythrocytes Incubated with the Patient's IgG Serum Fraction

Anti-y Incubation Antiglobulin

Temperature Agglutination' React ion

37 c 0 o t 24 C 0 o t 4 c 0 1-2+$

Agglutination examined after centrifugation at

t Washing and antiglobulin test performed at

$ Washing and antiglobulin test performed a t 4 C.

temperature of incubation.

24 C.

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Table 4. Rheumatoid Factor Activity of Patient's Sephadex G-200 IgM Fraction

Incubation Temperature Titer End Point

37 c 1 /160 RT 1 /a0 4c 1 /640

been well documented. Green and Golden- berg' described a case of a 45-year-old man who developed severe hemolytic anemia and massive hemoglobinuria. The patient was treated with steroids, The patient had a cold agglutinin titer of 1/256 with the range of activity limited to 3 C. These authors con- cluded that the etiology of the hemolytic anemia was autoimmune. No further docu- mentation of this hypothesis was given. Rosenfeld and co-workers9 reported the pres- ence of cold agglutinins with anti-i specificity in the serum of patients with uncomplicated infectious mononucleosis. Jenkins et a1.' reported hemolytic anemia as a complication of infectious mononucleosis and identified anti-i as the cold agglutinin. Troxel et a1." identified an IgM autoagglutinin of anti-i specificity reactive to a titer of 1 / 128 at 37 C. Burkart and Hsul described a patient with infectious mononucleosis complicated by hemolytic anemia and identified an IgM anti-i with cold-warm hemolytic activity. The patient had a rheumatoid factor titer of 128. Adsorption with IgG-coated latex particles resulted in complete removal of rheumatoid factor reactivity, but this did not decrease the cold agglutinin or cold-warm lytic activities. These activities were lost following treatment with 2-mercaptoethanol.

Goldberg and Barnett4 identified both IgM and IgG in eluted cold agglutinins from a patient with IM. Neither the chromato- graphically pure IgM nor the IgG exhibited cold agglutinin activity by itself, and the IgG component did not show specificity for cord cells. Capra et al.' described a cold IgG anti-i antibody that interacted with a cold IgM anti-IgG. This combination was demon-

strated in up to 66 per cent of their sera from patients with 1M. High titers oftheantibodies were seen in patients with severe hemolytic anemia and/or leukopenia. Wilkinson er al. " did not confirm the high incidence of com- bined IgG anti-i and IgM anti-IgG in IM, nor was one of the present authors (H.C.) able to demonstrate such a combination in a retro- spective study of frozen-stored sera from ten patients with IM-associated autoimmune hemolysis (unpublished observations). It was of considerable interest therefore when studies of serum from the present patient confirmed in almost every detail the findings of Capra et ul.' The only difference was the higher thermal amplitude of the IgM cold rheuma- toid factor in the present case. Capra et aL2 described the simultaneous presence of 19s anti-i in 35 per cent of their sera. The weak anti-i cold reactivity of the IgM fraction illustrated in Table 2 is not considered conclusive evidence for the presence of IgM anti-i, since IgM fractions prepared by Sephadex (3-200 filtration are known to have trace contamination by IgG and therefore could possibly contain sufficient IgG anti-i to bring about the weak agglutination that was observed.

Detection of a cold reactive IgG anti-i in patients with hemolytic anemia may be of more than theoretical interest with respect to the decision to treat with corticosteroids. It is generally acknowledged that corticosteroids are of little or no value in the treatment of IgM-mediated cold antibody hemolytic ane- ~ n i a , ~ while of great value in management of IgG-mediated warm antibody hemolytic syn- dromes. A response to fulldose corticoste- roid therapy in a patient with an IgG cold incomplete antibody has been reported by Moore and Chaplin.* The IM patient de- scribed in the present report was treated with corticosteroids (primarily for his associated hepatitis); his hemoglobin rose promptly, along with improvement in his general condi- tion. However, since 1M is generally a self- limited illness, caution is necessary in

718 GRONEMEYER ET AL. Transfusion November-December 1981

attributing apparent responses in associated hemolytic anemia to corticosteroid ad- ministration.

References I . Burkart PT. Hsu TCS. IgM cold-warm hemolysins

in infectious mononucleosis. Transfusion 1979; 19:535.

2. Capra J D , Dowling P, Cook S, Kunkel HG. An incomplete cold-reactive y G antibody with i specificity in infectious mononucleosis. Vox Sang 1969;16:10.

3. Dacie JV. The Haemolytic Anaemias. Part I I . The Autoimmune Haemolytic Anaemias, 2nd ed. New York, Grune and Stratton, 1962:689.

4. Goldberg LS, Barnett EV. Mixed yG-yM cold agglutinin. J lmmunol 1967;99:803.

5 . Green N, Goldenberg H . Acute hemolytic anemia and hemoglobinuria complicating infectious mononucleosis. Arch Intern Med 1960; 105: 108.

6. Hossaini AA. Anti-i in infectious mononucleosis. Am J Clin Pathol 1970;53:198.

7. Jenkins WJ, Koster HG, Marsh WL, Carter RL. Infectious mononucleosis: an unsuspected source of anti-i. Br J Haematol 1965;l 1:480.

8. Moore JA, Chaplin H. Autoimmune hemolytic anemia associated with an IgG cold incomplete antibody. Vox Sang 1973;24236.

9. Rosenfield RE, Schmidt PJ, Calvo RC, McGinniss

MH. Anti-i, a frequent cold agglutinin in infec- tious mononucleosis. Vox Sang 1965;10:631.

10. Troxel DB. lnnella F. Cohen RJ. Infectious mono- nucleosis complicated by hemolytic anemia due to anti-i. Am J Clin Pathol 1966;46:625.

I I . Wilkinson LS. Petz LD. Garratty G. Reappraisal of the role of anti-i in haemolytic anaemia in infectious mononucleosis. Br J Haematol 1973; 257 15.

P. Gronemeyer, M.D., Resident. St. Louis University School of Medicine, Cardinal Glennon Hospital, De- partment of Pathology, 1402 South Grand, St. Louis, Missouri 63104.

Hugh Chaplin, M.D.. Professor of Medicine and Preventive Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63 I 10.

V. Ghazarian. MT(ASCP)SBB. Assistant Supervisor, Blood Bank. The Jewish Hospital ofSt. Louis, 216South Kingshighway. St. Louis, Missouri 631 10.

Frances Tuscany. MT(ASCP)SBB, Supervisor, Blood Bank, The Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, Missouri 631 10.

George D. Wilner, M.D., Director, Laboratory Medi- cine, Associate Professor of Pathology, Assistant Pro- fessor of Medicine, The Jewish Hospital of St. Louis. Washington University School of Medicine, 2 I6 South Kingshighway, St. Louis, Missouri 631 10.