Blut, Band 34, Seite 39-47 (1977) Institut ftir Klinische Immunologie und Transfusionsmedizin der Universit~it GieBen

Reappraisal of the Clinical and Etiologic Significance of Immunoglobulin Deviations in Autoimmune Hemolytic Anemia ('Warm Type') Christian Mueller-Eckhardt, Friederike M6hring, Volker Kretschmer, Walter HSbel and Helmut L6ffler

Summary 56 patients with autoimmune hemolytic anemia ('warm type') (AIHA) were investigated for immunoglobulin deviations. Of these, 43 were repeatedly analyzed (mean 4 times). The mean observation time was 20 months. The immunoglobnlin values were correlated with clinical (degree of hemolysis) and serological (immunoglobulin class of autoantibodies; strength of antiglobulin reaction) parameters and statistically evaluated by variance analysis. Although no significant deviations of immunoglobulins in AIHA were found as compared to a normal control group, the immunoglobulin disturbance most frequently seen was an elevation of IgM. This is interpreted as a possible lack or functional impairment of immnnoregulatory T ceils in AIHA. Zusammenfassung 56 Patienten mit autoimmunh~imolytischer An~imie vom Wfirmetyp (AIHA)wurden auf Abweichungen der Immunglobulinspiegel des Serums untersucht. Von diesen konnten 43 Patienten wiederholt (im Mittel 4real) analysiert werden. Die mittlere Beobachtungsdauer betrug 20 Monate. Die Immunglobulinwerte w-arden mit klinischen (Grad der H~imolyse) und serologischen (Immunglobulinklasse der Autoantik6rper; St/trke der Antiglobulinreaktion) Parametern korreliert und statistisch mit Hilfe der Varianzanalyse ausgewertet. Obwohl keine signifikanten Abweichungen der Immunglobnlinwerte bei AIHA im Vergleich zu einer normalen Kontrollgruppe nachgewiesen werden konnten, bestand die Mufigste Immunglobulinst6rung in einer Erh6hung des IgM. Dies wurde als Hinweis auf ein m/Sgliches Fehlen oder eine funktionelle Beeintr~ichtigung yon immunoregulatorischen T-Zellen bei der AIHA gedeutet. Key words: Autoimmune hemolytic anemia; immunoglobulins; autoantibodies.

Although autoimmune hemolytic anemia of warm type (AIHA) is established as a well-characterized clinical and serological entity with autoantibodies thought to be directly responsible for the shortened survival of red blood ceils, its etiology remains unknown. Eingegangen am 1.6, 1976

40

C. Mueller-Eckhardt, t v. M#hring, V. Krelschmer, W. I-I#bel and H. Lb'fjqer

Several reports have emphasized the occurrence of dysimmunoglobulinemias in A I H A [4,5,6,13, 18]. In 1969, B]ajchman et al. [2] investigating 88 patients with A I H A of all clinical groups found low levels of one or more classes of immunoglobulins in approximately half of their cases. Only the decreased values of I g A differed statistically significant from normal controls. Based on previous reports describing the combined occurrence of antibody defidency syndromes and A I H A [7, 12] they suggested that the development or persistence of clones of cells elaborating antibodies might be a consequence of low levels of immunoglobulins. Moreover, the hypothesis was put forward that an autoantibody against I g A might account for the reduction of I g A levels which did not correlate with the serologically detectable autoantibody type on the erythrocytes. This anti-IgA was believed to be just another manifestation of the aberrant immune system in these patients. In an earlier study [8] on a limited group of patients with A I H A we were not able to confirm the results of B]ajchman et al. [2]. Because of the potential importance of dysimmunoglobulinemias for the etiologic interpretation of A I H A we decided to reappraise this problem in a larger group of patients with particular emphasis on long-term results. We, therefore, report the statistical analysis of immunoglobulin determinations in 56 patients with A I H A , 43 of w h o m were repeatedly investigated in the course of their disease.

Material and methods 56 patients (36 females, 20 males) with AIHA were investigated. 33 patients belonged to the idiopathic, 23 to the symptomatic type of the disease. In the symptomatic group of AIHA the underlying diseases were: chronic lymphatic leukemia (8 cases); Hodgkin's disease (3 cases); reticulo- or lymphosarcoma (3 cases) ; immunocytoma (2 eases) ; lupus erythematosus disseminatns (3 cases); IgA-myeloma (1 case); drug-allergic AIHA due to s-methyl dopa (1 case) ; viral infections (2 cases). The mean observation time was 20 months. Thirteen out of 56 patients were investigated only once at the time of diagnosis, the remainder (N = 43) were studied 2 to 12 times (mean: 4). Each investigation included a serological analysis (see below) and a physical, hematological (hemoglobin, red blood cells, reticulocytes, white blood ceils, platelets, differential blood smears), and chemical status (bilirubin, LDH, SGOT, SGPT, serum iron). Other clinical investigations were carried out if necessary. The diagnosis AIHA was established if unequivocal signs of hemolysis with or without anemia were present and if antierythrocytic autoantibodies (positive Ig-type of direct antiglobulin test (DAT) with or without bound complement (C) ; C-type of DAT with warm~ hemolysins; elntable autoantibodies only) were detectable.

Immunohemalolog#al siudies On each occasion, 10 ml of citrated (1 vol. 3.8% sodium citrate q- 9 vol. of blood) and 10 ml of native blood were drawn. For investigations of in vivo sensitization, red blood cells were separated from citrated blood, washed 3 times in phosphate-buffered saline and studied within 48 h after collection. Serum specimens were stored at --25 ~ C and mostly examined en bloc. In vivo fixation of immunoglobulins IgG, lgM, IgA and complement components C 3 d and C 4d on erythrocytes were determined by a quantitative modification of the DAT. The antisera specifically prepared for use in the DAT (anti-IgG, -IgA, -IgM, -}IA/1E q- ~2D, -~IA, -}IE) were purchased from Organon (sera of the Netherlands Red Cross Blood Transfusion Service, Amsterdam). Details of methods have earlier been published [11]. Elution of autoantibodies was performed by the ether method [13].

Dysimmunoglobulinemia in autoimmune hemolytic anemia ('warm lype')

41

Sera and eluates were tested for antibody activity in the enzyme and the indirect antiglobulin reaction using 7 selected panel cells of blood group 0. For the determination of warm hemolysins enzyme-treated ('incomplete hemolysins') and untreated ('complete hemolysins') e rythrocytes were used [ 11 ].

Quantitative determination of serum immunoglobulins Serum immunoglobulins IgG, IgA, and IgM were quantitated by radial immunodiffusion using Tri-Partigen| and M-Partigen| plates of Behringwerke (Marburg). The procedure recommended by the manufacturer was followed. The values are given in International Units (IU). They were calculated from the reference serum of the W H O (No. 67/87) according to the standard sera provided by Behringwerke.

Reference range (mg%) (IU/ml)

(mg%) IgG

(IU/m~ S

[1] N = 150

[2] N = 107

Authors N = 30

700-1900 80.5-218.5

800-1800 92-207

500-1600

780-1840 90-212

1200 138

1250 143

950

1313 151

(mg%) (IU/m~

range (mg%) (IU/ml)

(rag%) IgM

[3] N = 200

(IU/ml)

280 32.2 m f m f

60-250 70-280 69-287.5 80.5-322

m f m f

60-250 70-280 69-287.5 80.5-322

45-170

m f m f

125 160 143 184

m f m f

125 160 143 184

94

m f m f

53 62 61 71

(mg%) (IU/ml)

range (mg%) (IU/ml) IgA .~

(mg%)

(iu/~) s

(mg%) (IU/ml)

262 30.2 102-273 117-314

188 216

85 98

90-450 53.5-268

90-450 53.5-268

125-425

141-395 84-235

210 125

210 125

248

267 159

82 • 49

126 • 75

Tab. 1 : Range, means and standard deviations of normal immunoglobulin values in human serum. m = male; f = female

C. Mueller-Eckhardt, F. MO'hring, V. Kretschmer, IV. Hb'bel and H. L@fer

42

Clinical and statistical evaluation Deviations of immunoglobulin values from the normal range (limits taken from [1,3]) (Tab. 1) for each clinical group (idiopathic and symptomatic) were compared with a normal control group (age but not sex matched) and their statistical significance evaluated by variance analysis [14]. The degree of in vivo hemolysis was arbitrarily graded from 0 to 2 according to the following scheme: Hemolysis (Grade)

Hemoglobin (g/100 ml)

Reticulocytes (0/0o)

Lactic Dehydrogenase (U/ml)

Bilirubin (total) (mg/100 ml)

0 1 2

14-16 9-14 35

90-190 190-230 > 230

< 1.0 1.0-1.5 > 1.5

In questionable situations the decision was based on the hemoglobin value. The strength of the serological reaction was divided into 5 categories (0 to 4 q-) as judged from the macroscopic appearance of the DAT. Results With two exceptions, the D A T was positive in all cases. The eluates of the two Coombs-negative patients contained strong antibody activity. One case was kindly confirmed by Dr. C. P. Enge]friet (Amsterdam). The other patient has died from pulmonary embolism after splenectomy in 1974. Because of the clear clinical picture of hemolytic anemia they were considered to be true cases of A I H A [15]. A detailed description will be given elsewhere. The autoantibodies fixed to the erythrocytes were almost exclusively of the I g G class. In 5 cases I g M and/or I g A autoantibodies were found in addition. Complement proteins alone were b o u n d to red blood cells in 3 patients (so-called 'pure complement' types [20]). All three had, at least transiently, enzyme-reactive autoantibodies in their sera. An analysis of immunoglobulin deviations with regard to clinical groups is presented in Table 2. AIHA

N

Dysimmunoglobulinemia

total group

56

44

t +

8 7

20 8

idiopathic group

33

23

~

4

9

5

+

3

1

5

~' q,

4 4

11 6

1 4

symptomatic group

23

21

IgG

IgM

IgA

6 9

Tab. 2: Immunoglobulin deviations of sera of 56 patients with AIHA in relation to immunoglobulin class and clinical group ( ~' = increased; 4r = decreased).

Dysimmunoglobulinemia in autoimmune hemolytic anemia ('warm ty#e')

43

In 44 out of 56 patients (79%) immunoglobulin deviations from normal were noticed at the time of maximum hemolysis. The percentage of abnormalities was higher in the symptomatic (91%) than in the idiopathic group (70%). The immunoglobulin most often involved was I g M (28 times as compared to 15 times for both I g G and IgA), particularly in the symptomatic group. For IgG, no relevant differences became apparent between both groups, whereas deviations of I g A levels were more frequent in the idiopathic group. In 12 cases, all immunoglobulin determinations remained within normal limits. Deviation of immunoglobulins

Increased levels

Decreased levels

Mixed deviations

Immunoglobulins involved

Clinical group of A I H A

IgO

IgM

IgA

idiopathic

symptomatic

total

I'

n

j'

+

n

1

3

4

n

2

0

n n n

2

'1' n J'

~' J" n

0 0 6

1 4 8

1 4 14

~, ,I, ~, n

n ,1, n ~

n n ~, ~

0 1 2 0

3 1 2 2

n

n

~,

3 0 0 2 0

2

2

n

~

n

2

1

3

1

~p

~,

n

1

0

,1

1'

~

1

0

1

4 n ,l, J'

r ~ J" n

4 t n ,I,

1 1 1 0

0 0 0 1

1 1 1 1

Tab. 3: Types of dysimmunoglobulinemia in relation to clinical groups o f A I H A ( j' = increased; ~, = decreased; n = normal). I n Tab. 3 detailed information as to all types of immunoglobulin deviations observed is given. A total of 17 different combinations were seen. However, there were no pronounced differences of the types of immunoglobulin deviations in both clinical groups. Also, no correlation between immunoglobulin deviations and the types of cell-fixed autoantibodies was noticed. Variations of immunoglobulin values in the course of the disease appeared in idiopathic and in symptomatic A I H A . In the idiopathic group, 3 patients showed a concomitant normalization of the immunoglobulin values with clinical remission. However, in these cases the initial anemia was only slight. Three patients had no correction of immunoglobulins despite clinical and serological improvement. I n

C. Mueller-Eckhard*, F. Mb'hring, V. Kre~schmer, W. fqb'beland H. Lbffler

44 lc

IgM ( I,U./z] )

o

600

u

o

9

m

i3

ee e

5OO o

,oo

T

Oo

~

:T

- -

.it"

tl o

3OO

/

//

n~

/"

_o

/

~ o

"T o

:e

~T

n

:

_

0

/

. / //',~

,oo

I/ 9

///~

/ / . ,~'/Z

;~/A o

o

9

o

o o

o 0

$

o o

se

2

Z

@

3

m

v,+~§

S~rength of agglutination

Fig. 1 : Correlation of serological reactivity (strength of agglutination in DAT) and IgM levels of serum. 9 Symptomatic AIHA; [ ] Idiopathic AIHA. 2c

/gM ( J.U.l,l ) 0

600

~e

a

9 e

0

D O Oo

113

400

o

300

9

,lo

0 o o

0 0 0

"e

,0 I

200

I00

"_// / / J ~

(//~'//A/////~.//. e~ 9

O0 o n

0

I

2

Fig. 2 : Correlation of degree of hemolysis and IgM levels of serum. 9 Symptomatic AIHA; [ ] Idiopathic AIHA.

Oegree of

hemolysis

Dysimmunoglobulinemia in auSoimmunehemolytic anemia ('warm/ype')

45

9 patients dysimmunoglobulinemia persisted. In the symptomatic group, 3 patients showed normalization of immunoglobulins, in 9 patients no change of the dysimmunoglobulinemia was observed. In an attempt to analyze possible associations between serological or clinical parameters on one hand and the degree of dysimmunoglobulinemias on the other, the degree of agglutination in the DAT or the severity of anemia was plotted against all immunoglobulin values obtained. For statistical evaluation, immunoglobulin values corresponding to serological or clinical categories (for definition see material and methods) were computed for means and standard deviations and compared between the various groups. Patients with idiopathic AIHA were regarded separately. As examples, values of IgM determinations in relation to the strength of agglutination (Fig. 1) and the degree of hemolysis (Fig. 2) are depicted. In brief, there were no significant differences between these parameters and the various clinical groups. Discussion Immunoglobulins present in serum or external secretions are products of so-called B lymphocytes, cells presumably of gut-associated lympho-epithelial origin that evolve from as yet unidentified precursor ceils [17]. Any disorder affecting this regulatory system directly or indirectly may, therefore, be expected to lead to abnormal tmmunoglobulin levels. Thus, in patients with certain autoimmune disorders, i.e. rheumatoid arthritis, Sj6gren's syndrome, systemic lupus erythematosus and chronic active hepatitis, increased serum immunoglobnlins are a rather characteristic feature of the disease thought to be causally related to the disease process. Moreover, NZB/BL strains of mice which have an inherited tendency to develop various autoimmune disorders including hemolytic anemia have also raised levels of immunoglobulins especially of the IgM class [19]. In line with these observations, we found that the most frequent deviation of immunoglobulins in human AIHA is an elevation of tgM. 9 out of 23 patients with idiopathic AIHA and 11 out of 21 patients with symptomatic AIHA showed this type of abnormality. Within the two other major immunoglobulin classes elevations and reductions were more or less evenly distributed. It must be stressed, however, that almost every possible combination of immunoglobulin deviations occurred in our material. Thus, these results are similar to those of our earlier report [8] and do not confirm the finding of a significant IgA reduction in AIHA [2]. A possible and probably the most likely explanation for this discrepancy may be sought in the different limits of the normal range accepted for statistical evaluation. Particularly the lower limit of IgA (125 mg/100 ml) as given by the English authors is considerably higher than the figures recommended by others (Tab. 1). The lower limit for IgG, on the other hand, was extraordinarly low (500 mg%). If the ranges given by Becker & Stariko [1] and Dallesandro & Topi [3] would be applied to the figures of Blajchman et al. [2] it appears doubtful whether statistical significance for the IgA deviations would still be valid. The IgM levels of the British material were, again somewhat different from our results, widely scattered with lowered levels in 15 and raised levels in 11 cases. It is rather unlikely that the minor discrepancies between the British and our material bear any significance.

46

C. Mueller-t~ckhardt, F. Mb'hring, V. Kretschmer, W. Hb'bel and H. LOftier

In the intention to analyze the influence of factors possibly operative in chronic A I H A of long duration we have performed a follow-up study of immunoglobulin alterations in the course of this disease. Although there was a tendency toward normalization of immunoglobulin values associated with clinical improvement or remission this was not regularly the case. In 12 out of 43 patients repeatedly investigated the dysimmunoglobulinemia persisted inspite of clinical improvement. Also the reverse situation (i.e. persistent normal immunoglobulins with clinical and/or serological abnormalities) was seen. If all immunoglobulin determinations in the sera of all patients were evaluated statistically, no correlation existed between the different forms of dysimmunoglobulinemia and the severity of the disease or the strength of the serological reaction. Thus, from the values of immunoglobulins in the serum of patients with AIHA, no definite conclusions as to the severity, activity, or type of A I H A can be drawn. Our present state of knowledge does not allow final clues as to the still unknown etiology of AIHA. Contrary to Blajchman et al. [2] who attempted to explain the lowered immunoglobulinlevels on the basis of genetic or inherited factors or suppression of immunoglobulin antibodies we suggest another hypothesis. It has recently been demonstrated by Waldmann et al. [18] that a population of thymus-related lymphocytes (T-cells) exert a suppressive effect on the capacity of B-cells to synthesize immunoglobulins. Therefore, it can be speculated that the overproduction of polyclonal immunoglobulins in autoimmune diseases is due to a lack or functional impairment of a subset of immunoregulatory T-cells or macrophages [10,17]. Two arguments are in favour of the hypothesis that a similar mechanism is operative in chronic A I H A : First, the most common immunoglobulin abnormality, at least in our material, is an elevation of IgM levels, a familiar trait of experimental and human autoimmnne disorders; and, secondly, our recent finding [9] that there exists in A I H A a T-cell deficiency which may represent the reduction of immunoregulatory T-cells necessary to control the immunoglobulin production in this disease. More extensive studies are required to establish the presence and role of immunoglobulin-suppressor cells (or substances) in A I H A for better understanding of the dysimmunoglobulinemias observed in this and other autoimmune disorders. Supported by the Deutsche Forschungsgemeinschaft (Mu 277/6).

References 1. Becker W. & St6riko K. : Immunological determination of immunglobulins: normal values and preparation of a standard. Progr. Immunbiol. Standardization 4, 129 (1970). 2. Blajchman M. A., Dacie J. V., HobOs J. R., Pettit J. E. & Worlledge S. M. : Immunoglobulins in warm-type autoimmune haemolytic anemia. Lancet II, 344 (1969).

3. Dallesandro G. & Topi G. : Values of immunoglobulins. Boll. isf. sieroter. milan. 52, 5 (1973). 4. Fialkow P. J., Fudenberg H. & Eppstein W. V. : "Acquired" antibody hemolytic anemia and familial aberrations in gamma globulins. Amer. J. Med. 36, 188 (1964). 5. Fudenberg H. H. & Solomon A.: "Acquired agammaglobulinemia" with autoimmune hemolytic disease: graft

Dysimmunoglobulinemia in autoimmune hemolyiic anemia ('warm type') versus host reactions? Vox Sang. 6, 68 (1961). 6. Hennemann J. H. & Rentsch J.: Idiopathische autoimmunh~molytische An~mie mit Hypogammaglobulinfimie. Klin. Wschr. 46, 156 (1968). 7. Hobbs J. R., Russell A. & Worlledge S. M.: Dysgammaglobulinaemia type IV C. Clin. exp. ImmunoL 2, 589 (1967). 8. Kretschmer V. & Mueller-Eckhardt C.: Autoimmune hemolytic anemias. II. Immunoglobulins and }lA-globulin in the serum with special regard to the immunochemical type of autoantibodies and the course of disease. Blur 25, 159 (1972). 9. Krtiger J., Rahman A., Mogk K.-U. & Mueller-Eckhardt C. : T-cell deficiency in patients with autoimmune hemolytic anemia ("warm type"). Vox Sang. 31 (1976) 1. 10. Miller J. F. A. P.: T-cell control of B-cell responsiveness. Int. Arch. Allergy & AppL ImmunoL 49, 230 (1975). 11. Mueller-Eckhardt C. & Kretschmer V.: Autoimmune hemolytic anemias. I. Investigations on immunoglobulin type and complement fixation of cell-fixed and elutable autoantibodies. Blur 25, 63 (1972). 12. Pearson H. A., Robbins J. B. & Skinner R. G. : Autoimmune hemolytic anemia

47

in a patient with congenital hypogammaglobulinemia. Pedlar. Res. 1, 215 (1967). 13. Ruhin H. : Antibody elution from red blood cells.J, din. Path. 16, 17 (1963). 14. Scheffe H. : The analysis of variance. Whiley (New York 1959). 15. Schubothe It.: Autoantik6rper und h~molytische Anfimien. In: Gross H.: Immunhfimatologie, Schattauer Verlag, Stuttgart 1970, p. 109. 16. Schwartz R. S. & Costea N.: Autoimmune hemolytic anemia: clinical correlations and biological implications. Sere. Haemat. 3, 2 (1966). 17. Solomon A.: Cellular regulations of humoral immunity. New Engl. J. Med. 293, 928 (1975). 18. Waldmann T. A., Durm M., Broder S., Blackman M., Blaese R. M. & Strober W. : Role of suppressor T-cells in pathogenesis of common variable hypogammaglobulinaemia. Lancet II, 609 (1474). 19. Warner N. L. &Wistar R. : Immunoglobulins in NZB/B1 mice; I. Serum immunoglobulin levels and immunoglobulin class of erythrocyte autoantibody. jr. exp. Med. 127, 169 (1968). 20. Worlledge S. M. & Blajchman M. A. : The autoimmune haemolytic anemias. Br#. jr. Haemat. 23, 61 (1972).

Author's address:Prof. Dr. Ch. Mueller-Eckhardt, Abteilung Klinische lmmunologie & Transfusionsmedizin der Universit~it Giegen, LanghansstraBe 7, D-6300 Giegen, German Federal Republic.

Reappraisal of the clinical and etiologic significance of immunoglobulin deviations in autoimmune hemolytic anemia ('warm type').

Blut, Band 34, Seite 39-47 (1977) Institut ftir Klinische Immunologie und Transfusionsmedizin der Universit~it GieBen Reappraisal of the Clinical and...
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