British Journal of Haernatology. 1988, 69, 61-66

Immunochemical specificity of autoanti-Gerbich from two patients with autoimmune haemolytic anaemia and concomitant alteration in the red cell membrane sialoglycoprotein

MARION E. RE ID,^ VIRGINIA VENGELEN-TYLER,~ IRA S H U L M A N ~ AND MARILYN V. REYNOLDS Department of Laboratory Medicine, San Francisco General Hospital Medical Center, San Francisco, California, ’Reference Laboratory, American Red Cross Blood Services, Los Angeles and Orange Counties Region, Us Angeles, California, 3Department of Health Services, Los Angeles County-University of Southern CaliJornia Medical Center, Los Angeles, CaliJornia, and *Blood Bank, Tucson Medical Center, Tucson, Arizona, U.S.A.

Received 30 June 198 7; accepted for publication 15 October 198 7

Summary. Blood samples from two patients with autoim- mune haemolytic anaemia, whose autoantibodies failed to agglutinate certain examples of red cells that lack Gerbich blood group antigens, were studied using immunochemical analyses. One of these autoantibodies differed from all other anti-Ge in that it showed a unique fi sialoglycoprotein (SGP) specificity. It reacted with normal /? but not with the abnormal /?-related SGPs associated with Gerbich-negative red cells of the Gerbich and Yus types. Red cells from this patient had an alteration of fl SGP, while the a, y and 6 SGPs

appeared to be normal. The autoantibody from the other patient did not show this unique characteristic. Its immuno- chemical specificity was similar to alloanti-Ge3 in that it reacted with both /? and y SGPs from normal red cell membranes and with the abnormal /?-related SGPs found in red cell membranes from individuals with Gerbich-negative red cells of the Yus type. Red cells from this patient could not be analysed because she had recently received a massive transfusion of red cells.

Autoantibodies in the serum or bound to the red cells from some patients with autoimmune haemolytic anaemia (AIHA) may have blood group antigen specificity. Blood group specificities most commonly involve Kell and Rh blood group antigens: however, other specificities have been de- scribed (Issitt, 1985: Petz & Garratty. 1980). Autoanti- Gerbich (Ge) has been described in two cases of AIHA (Reynolds et al. 1981: Shulman et al, 1985) and in one patient with aplasia (Beattie & Sigmund, 1987). Since it is possible to determine which red cell membrane SGP(s) are recognized by alloanti-Ge (Reid et al, 1987a). sera from the two patients with AIHA were tested to establish the immuno- chemical specificity of their autoantibodies.

In rare cases of AIHA, a concomitant selective loss or depression of the blood group antigens corresponding to the autoantibodies has been reported involving Kell, Rh and Ena blood group antigens (Issitt, 1985: Petz & Garratty, 1980:

Correspondence: Dr M. E. Reid. Clinical Laboratories, San Francisco General Hospital Medical Center, 1001 Potrero Avenue, San Fran- cisco. CA 941 10. U.S.A.

Garratty et al, 1983). The mechanism for this phenomenon is not known. Beattie & Sigmund (1987) reported a similar finding in a patient with aplasia whose red cells showed an altered /? SGP (synonyms: PAS-2: glycophorin C). We tested red cells from one of the two cases of AIHA due to autoanti-Ge (Reynolds et al, 1981: Shulman et al. 1985) to determine if they had an altered fl SGP. (For a review of the Ge blood group antigens see Reid, 1986.)

CASE HISTORIES

Patient 1. A 14-year-old male was admitted to hospital with severe anaemia. A detailed medical history and serologi- cal study have been reported elsewhere (Reynolds et al, 1981). This patient’s haemoglobin level was 5.8 g/dl. haematocrit 18.2% and reticulocyte count 12.4%. His total serum bilirubin level was 4.1 mg/dl and his lactate dehydro- genase (LDH) value was 3 times normal. During hospital admission his haemogloblin and haematocrit reached nadirs of 5.0 g/dl and 16.2% respectively and the reticulocyte count peaked at 42.9%: microscopic examination of blood films

61

62 revealed nucleated red cells, macrocytosis, polychromasia, anisocytosis, poikilocytosis and spherocytosis but no ellipto- cytes. Direct and indirect antiglobulin tests were strongly positive. His red cells were Ge-positive.

Patient 2. A 26-year-old female was admitted to the hospital with severe anaemia (Shulman et al, 1985). On admission, the patient's haemoglobin level was 4-6 g/dl. haematocrit 12.5 and reticulocyte count 1.9%. Her total serum bilirubin level was 6.8 mg/dl and her LDH value was elevated. Microscopic examination of blood films revealed anisocytosis but no elliptocytes. Direct and indirect antiglo- bulin tests were positive. Her red cells were Ge-positive.

Marion E. Reid et a1

MATERIALS AND METHODS

Patient red cell samples were collected during the haemolytic episodes and frozen in liquid nitrogen. Red cells were tested or membranes prepared within 24 h of thawing. Plasma samples containing the autoanti-Ge had also been collected during the haemolytic episodes and stored frozen.

Anti-Ge were purified by absorption onto and elution from Ge-positive red cells (Kochwa & Rosenfield, 1964). A rabbit antibody, rendered specific for the cytoplasmic portions of b and y SGPs, was prepared as described previously (Reid et al, 198 7a).

In the indirect antiglobulin tests, anti-human globulin reagents were used for auto- and alloantibody detection and anti-mouse IgG for murine monoclonal antibody detection. Monoclonal anti-fl SGP. NBTS/BRIC-10 (BRIC-10). recog- nizes an epitope on the N-terminal portion of normal fl and abnormal fl-related SGPs associated with Gerbich and Yus type Ge-negative red cells (Anstee et al, 1984a, b). LICR/LON R1.3 (R1.3) monoclonal antibody detects an epitope on the N-terminal portion of both a and 6 SGPs (Anstee & Edwards, 1982).

Table I. Specificity of purified autoanti-Ge

Red cells were treated with sialidase or trypsin as described previously (Anstee et al. 1979; Tanner et al, 1980). Mem- branes were prepared (Dodge et al, 1963) from Ge-positive red cells that had been treated with an Endo-b-N-acetylglucosa- minidase F preparation (Endo F) (Tanner et al, 1987). Tn- activated and Cad 1 red cells were used to study the extent of 0-linked oligosaccharide involvement in the antigenic deter- minants of these autoantibodies. The population of Tn- activated red cells from a blood sample from CL (Beck et ul, 1977) was enriched using anti-Al, following a procedure described by Beattie (1972). Enrichment was confirmed by tests with anti-Al and BRIC-10. The enriched population of Tn-activated red cells was strongly agglutinated by anti-Al but was not agglutinated by BRIC-10. Tn-activated red cells lack galactose and sialic acid residues which are usually attached to membrane SGPs as 0-linked tetrasaccharides (Dahr et al, 1974). Red cells from a Cad 1 donor were tested because they have an additional N-acetylgalactosamine residue on a majority of the 0-linked oligosaccharides (Blanchard et al. 1983).

f i SGP was purified from normal blood as described by Reid et ul (1987a). Equal volumes of this preparation (reconsti- tuted in PBS containing 10% bovine serum albumin) and BRIC-10 or purified anti-Ge were incubated at RT for 20 min. Ge-positive red cells were added, the mixture incubated for 45 min, and indirect antiglobulin testing performed to determine whether inhibition of the antibody activity had occurred.

SDS-polyacrylamide gel electrophoresis of red cell mem- branes was carried out on gels containing 10% acrylamide with a 3% acrylamide stacking gel (Laemmli. 1970). Immu- noblotting was performed as previously described (Merry et al. 1986). except that 5% (w/v) powdered milk in PBS at pH 7.4 was used as the blocking agent. Binding of antibodies to nitrocellulose paper was detected using horseradish peroxi- dase-conjugated goat anti-human, anti-rabbit or anti-mouse IgG (BioRad, Richmond, Calif.).

Ge (+) red cells Ge (-) red cells Specificity

Sourceof: Un T S Tn Cad Yus Ge Leach Anti- SGP'

Anti-Ge Patient1 l l t 0 0 0 4 0 0 0 -Ge2 f l Patient2 10 0 8 8 9 7 0 0 -Ge3 /3+y+

fi-related (Yus)

Control sera WHA 9 0 3 5 9 0 0 0 -Ge2 y 44017 9 0 8 9 9 9 0 0 -Ge3 f l + y +

j3-related (Yus)

Un =untreated: T = trypsin treated S = sialidase treated Tn =enriched population of Tn activated red cells: Cad =red cells from Cad 1 donor: 'immunoblot results: thaemagglutination scoring system of Marsh (1972).

Autoanti-Ge and Membrane SGPs 63 Ge-positive red cells, the patient’s purified autoantibody reacted with the residual /3 SGP and with the 8 SGP after removal of the asparagine-linked oligosaccharide. These results indicate that this autoantibody is reacting with a trypsin-sensitive, sialic acid-dependent antigenic determi- nant present on the N-terminal portion of normal /3 SGP and that its binding does not require the presence of the asparagine-linked oligosaccharide.

Table 11. Effect of purified p SGP on indirect haemagglutination reactivity of autoanti-Ge

Plus PBS Plus j3 SGP

Autoanti-Ge: patient 1 patient 2

9* 0 9 8

Anti-j3 (BRIC-10) 12 0 Alloanti-y (WHA) 7 7 Alloanti-P + y + p-related (Yus) (4401 7) 9 8

* Haemagglutination scores (Marsh, 19 72) against Ge-positive indicator red cells.

RESULTS

Tests on the autoantibody of patient 1 The autoantibody had the serological specificity of anti-Ge2 (Reynolds et al. 1981). The purified autoantibody did not agglutinate trypsin- or sialidase-treated Ge-positive red cells or Tn-activated red cells, and only weakly agglutinated Cad 1 red cells in indirect antiglobulin tests (Table I). The reactivity of this autoantibody was inhibited by purified /3 SGP (Table 11). On immunoblots, the purified autoantibody bound only to j SGP from normal red cell membranes (Fig 1); it did not bind to membranes from Yus, Gerbich or Leach type red cells. When tested against membranes derived from Endo F-treated

Tests on the autoantibody of patient 2 This autoantibody. with the serological specificity of anti-Ge3 (Shulman et al, 198 5). did not agglutinate trypsin-treated Ge- positive or trypsin-treated Yus type Ge-negative red cells in direct or indirect antiglobulin tests. The purified autoanti- body agglutinated Cad 1, Tn-activated and sialidase-treated Ge-positive red cells more weakly than untreated Ge-positive red cells (Table I), suggesting that the antigenic determinant with which it reacts is partially dependent on sialic acid. This autoantibody was not inhibited by the purified b SGP preparation (Table 11). On immunoblots, the purified auto- antibody reacted with b and y SGPs from normal red cell membranes and with the abnormal 8-related SGP from Yus type red cell membranes (Fig 1). It did not react with membranes from Gerbich or Leach type red cells. The autoantibody reacted with residual and cleaved /I SGP components of membranes from Endo F-treated Ge-positive red cells indicating that its reactivity with /3 SGP was not dependent on the asparagine-linked oligosaccharide.

Fig 1. Immunoblot reactivity of autoanti-Ge with normal and Ge-negative red cells. Immunoblots with the purified anti-Ge against red cell membranes from Ge-positive (lanes 1, 3, 6 and 9), Ge-negative the Yus type (lanes 4 and 7 ) , and Ge-negative of the Leach type (lanes 2. 5,s and 10) donors. Lanes 1-2 show the anti$ specificity of the autoanti-Ge from patient 1 while lanes 3-5 show the anti-P+y+j3-related (Yus) specificity of autoanti-& from patient 2. Lanes 6-8 show the results using monoclonal anti$ (BRIC-10) and lanes 9-10 show the anti-y specificity of the alloanti-Ge2 (WHA).

64 Marion E. Reid et al Tests on red cells ofpatient 1 The strength of haemagglutination of this patient's red cells with human alloanti-Ge reagents (which contain anti-y SGP specificity) was indistinguishable from normal control sam- ples. However, haemagglutination was weaker with BRIC-10 than with control Ge-positive red cells. When equivalent loadings of red cell membranes were subjected to electrophor- esis on SDS-polyacrylamide gels and subsequently stained with PAS, there was reduced staining of f i SGP. The relative molecular weight of j SGP was the same as in control membranes. The intensity of staining of bands corresponding to a. S and y SGPs was comparable to controls. The reduction in p SGP was again apparent on immunoblots with BRIC-10 and human anti-Ge containing anti-fi SGP reactivity. Because both PAS staining and anti-fi reagents are depen- dent on the presence of sialic acid on f i SGP for reactivity (Anstee et al, 1984b), it is possible that the reduction of BSGP was the result of a glycosylation abnormality rather than a reduction in the content of p SGP molecules. For this reason, immunoblots were carried out using a rabbit antiserum that had been rendered specific for the cytoplasmic portions of p and y SGPs. The staining intensities in p and y SGP regions of the patient's red cell membranes were indistinguishable from control membranes (data not shown).

When red cells from this patient were tested with dilutions of R1.3, the haemagglutination titration scores were the same as for control Ge-positive red cells. Coomassie brilliant blue staining of gels showed the membranes apparently had a normal complement of skeletal proteins.

Collectively. these data suggest that although the f l SGP content of the red cells from this patient (collected at the time of his haemolytic episode) was normal, the extracellular domain had undergone a modification that was specific to fi SGP.

Tests on red cells ofpatient 2 Since this patient had been transfused prior to collection of the blood samples and no prestransfusion sample was available, red cells were not extensively tested.

Tests on other red cells To rule out the possibility that weakened expression of p SGP is a general phenomenon associated with anaemia, we tested 20 blood samples from patients with acute and chronic anaemias with haemoglobin values of less than 6.9 g/dl. We also tested pretransfusion blood samples from six patients with AIHA but whose autoantibodies did not have anti-Ge specificity. Haemagglutination of red cells from these 26 samples by BRIC-10 (used at a dilution which weakly agglutinated red cells from patient 1) was indistinguishable from control samples, indicating that SGP was not altered. Red cells from one of these patients, who had a haemoglobin of 2.5 g/dl and marked reticulocytosis, were tested further. Immunoblotting with BRIC-10 and PAS staining of a SDS- polyacrylamide gel showed p SGP to be present in amounts that were comparable to normal control membranes.

DISCUSSION

Our results show that, like alloanti-Ge, autoanti-Ge can have

different serological and immunochemical specificities. Both examples of autoanti-Ge were associated with AIHA accom- panied by dramatic haemolysis. The autoanti-Ge2 produced by patient 1 recognized a sialic acid-dependent, trypsin- sensitive determinant located on j SGP. This antibody is unlike any other example of allo- or autoanti-Ge with f i SGP specificity (Reid et al, 1987a: Beattie & Sigmund, 1987) in that it did not react with the abnormal /3-related SGPs associated with Gerbich and Yus types of Ge-negative red cells nor did it react with y SGP. The irnmunochemical specificity of the autoanti-Ge3 produced by patient 2 was apparently similar to alloanti-Ge3 (Reid et al, 1987a). It recognized an antigenic determinant located on the trypsin sensitive, N- terminal portions of f i , y and abnormal j-related SGPs associated with Yus type Ge-negative red cells. Binding of this autoantibody to its antigenic determinant was partially dependent on the presence of sialic acid but not on the presence of the asparagine-linked oligosaccharide. Thus, it is now apparent that human anti-Ge2 may have one of three immunochemical specificities: anti-y SGP, anti-y + f i SGP and anti-fi SGP.

In rare cases of AIHA, expression of a blood group antigen can be so reduced that the autoantibody masquerades as an alloantibody. When the haemolytic episode is over, the antigen is again expressed fully so that these red cells are agglutinated by the patient's serum stored at the time of the haemolytic crisis. The cause of this transient weakened expression of blood group antigens is not known, although several theories have been proposed (Conley et al, 1982: Garratty et al, 1983: Branch et al, 1984: Vengelen-Tyler et al, 1987). Of cases of AIHA involving autoanti-a SGP, only one has been reported to be associated with depressed target antigen (EnaTS) (Garratty et al, 1983: Dahr et al, 1986). This case is analogous to patient 1 in the present study whose serum contained an autoantibody directed against a trypsin- sensitive antigenic determinant carried on a red cell mem- brane sialoglycoprotein and a depressed expression of the antigenic determinant.

The cytoplasmic portion of fi SGP in patient 1 was present in levels comparable to control membranes, suggesting that the reduced expression of p SGP was not quantitative. Since PAS staining and anti-p SGP reagents require the presence of sialic acid, one possibility is that there was a change in glycosylation of the ,8 SGP molecules. If this is the case, a similar effect would be expected to occur with the other red cell membrane SGPs (a , 6 and y) because glycosylation occurs in the endoplasmic reticulum-Golgi complex where all newly synthesized SGPs would be exposed to the same environment (reviewed by Schachter, 1986). We have also shown that alteration of red cell membrane p SGP molecules is not a generalized phenomenon in patients with acute AIHA. The change(s) which occurred on p SGP molecules from patient 1 did not induce a detectable change in the relative molecular weight as determined by SDS-polyacrylamide gel electro- phoresis. However, since this method does not provide an accurate molecular weight for glycoproteins (Neville & Glossman, 1974). a change in molecular weight could occur and go undetected. Insufficient sample was available to test whether a similar effect could be induced on normal Ge-

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positive red cells by coating them with autoantibody from patient 1 prior to immunochemical analysis.

The only other report of a weakening of SGP. which did occur transiently, was in a patient with aplastic anaemia with a concomitant transient elliptocytosis (Beattie & Sig- mund, 1987). Red cells from individuals who lack f i and y SGP molecules (Leach type Ge-negative) (Anstee et af, 1984a: Reid et aZ, 1987a) are elliptocytic (Anstee et a!, 1984a; Daniels et al, 1986: Reid et aZ, 1987b). Since red cells from patient 1 in the present study were not elliptocytic, the modification of the /I SGP did not compromise the integrity of these cells.

We have shown that in two patients with AIHA, the autoantibodies were directed against a specific red cell sialoglycoprotein. In the patient with the autoanti-Ge of unique specificity, the SGP carrying the target antigen was altered during the haemolytic crisis while other red cell membrane SGPs were unaffected. In the other patient, the presence of transfused Ge-negative red cells prevented quan- titative analysis of f i SGP.

ACKNOWLEDGMENTS

We thank M. L. Beck from the Community Blood Center of Greater Kansas City, Mo., and M. Wood from United Blood Services, Billings, Mont., for making the Tn-activated red cells available and to J-P. Cartron from the Centre National de Transfusion Sanguine Institut, Paris, for providing the Cad 1 red cells. We also thank the following for supplying red cell samples from patients with AIHA: M. 0. Bergren, Irwin Memorial Blood Bank, San Francisco, Calif., E. Bossom, University of California, San Francisco, Calif., and T. O’Day, Central California Red Cross Blood Services, San Jose, Calif. We also are grateful to D. J. Anstee from the South Western Blood Transfusion Centre, Bristol. for gifts of monoclonal antibodies and M. J. A. Tanner from the University of Bristol, U.K., for providing the Endo F-treated red cells. We are grateful to C. J. Decker from Southern Arizona Red Cross Blood Program, Tucson, Ariz. for providing the anti-Ge 44017 and S. Cornwall from the Canadian Red Cross, Toronto, Canada, for providing the anti-Ge WHA.

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