Blood, 1957, Vol. 12, No. 3, pp. 217-237.
© 1957 American Society of Hematology, Inc.
Acquired Hemolytic Anemia
Properties of Eluted Agglutinins and Their Interaction with
Red Blood Cells and Coombs’ Serum
KARL L. ROTH 1 and
ABRAHAM M. FRUMIN 1
1 Department of Research, Southern Division, Albert Einstein Medical Center, Philadelphia, Pa.
Immunologic studies were performed on 27 blood specimens obtained at various intervals from 17 patients with acquired hemolytic anemia. RBC and serum
agglutinin titers, hemolysins, and viability of coated RBC in serum and in saline
were determined. Diseased cells, freshly separated from their parent sera, were
highly resistant to incubation at 37 C, in buffered, isotonic saline. Heparin was
found a most effective inhibitor of nonimmune hemolysis, as shown by its ability
to preserve the integrity of normal and diseased RBC in serum or in saline.
Twenty-six RBC samples were eluted and the agglutinating substances compared with respect to solubility, pH, heat resistance, UV absorption spectra,
and reactivity toward heparin and trypsin. Spectrophotometric data revealed
the appearance and time-dependent increase of an absorption hand at 4100 Å
upon incubation of normal RBC with eluted agglutinins. Reasons were given to
indicate that this phenomenon was not hemolysis.
Coating of normal RBC could be inhibited by normal sera and by some patients’ sera. All fractions tested were inhibitory. In general, inhibition was independent of complement activity, but may have been due to (one of its) thermostable component(s). Eluates incubated with human sera could not be recovered,
even with trypsinized RBC. Furthermore, no changes in serum agglutinin titers
were obtained by the addition of eluates to human sera.
A critical analysis of the variables influencing the outcome of a Coombs’
reaction was made. Considering eluated agglutinins as "antibodies" with respect
to RBC and "antigens" with respect to Coombs’ serum, the observed variables
were separated into two independent groups according to their influence on:
(a) RBC-eluate interaction; (b) agglutinin (or coated RBC)-Coombs’ serum
interaction. The former was not affected by eluate concentration or "Group"
(apparent immunologic specificity of eluates for different Coombs’ serum fractions), but varied with avidity of eluates (i.e., the speed necessary for optimal
coating in saline, and the amount of agglutinin required for such coating). Avidity was, in turn, greatly influenced by the suspension media used. It was found
that optimal coating with at least 2 agglutinins ("supersensitization") could be
obtained, regardless of the type of sensitization (simultaneous or consecutive),
when a saline suspension of RBC was used. While no damage to RBC resulted
under these conditions, supersensitization in an albumin suspension could be
carried to the point of total hemolysis. Thus, the attraction of eluted agglutinins
to RBC was not (necessarily) mediated by antigenic groupings but by the surface as a whole, with theoretically no limit to the amount of agglutinin which
could be fixed under suitable conditions. The agglutinin-Coombs’ serum interaction was subject to laws applicable to antigen-antibody reactions, with the
complications common to cross-reacting, heterologous mixtures.
Examples of prozones in both eluate and Coombs' serum excess were given.
A method was described for the determination of Coombs’ serum-eluate equivalence zone proportions (C/E ratios) in the absence of a precipitin test. During
this procedure, most of the eluates were categorized into a gamma globulin and
two non-gamma globulin "Groups." Cold hemagglutinins were excluded from
the present study.
Statistical data involving eluates showed no correlation; however, direct
Coombs’ titers on patients’ RBC vs. autoagglutinin titers of the respective parent
sera gave a determination coefficient (r2) of 0.3662 with P(t) of 0.001.
The nature of hemolysins, RBC and serum agglutinins, and the possibility to
define and classify them were briefly discussed. Emphasis was placed on artifacts
and their possible overinterpretation. Three tentative conclusions were reached:
1) It is highly doubtful whether serum and RBC factors are identical or were
so prior to adsorption on RBC. Hemolysis is dependent on the serum factor. The
substance which can be eluted from RBC presumably represents a protective
complex.
2) Despite superficial similarities, the eluted substances seemed to have individual characteristics for each patient studied. Evidence of their protein nature
was overshadowed by properties common to nucleic acids.
3) While the value of a quantitative Coombs’ test is questionable since the
number of RBC antigens (?) seems unlimited, much information about eluates
can be obtained by employing identical RBC populations and the same Coombs’
serum.
Submitted on June 25, 1956
Accepted on August 18, 1956
