0 1984 S. Karger AG. Bmel 0042-9007/84/0465-0300 $2.7510 Vox Sang. 46: 306305 (1984)

Inhibition of Serological Reactions with Enzyme-Treated Red Cells by Complement Binding Alloantibodies

J. A. G. Lown, P. A. T. Holland, A. L. Barr Department of Haematology, Royal Perth Hospital, Perth, W. Australia

Abstract. Complement-binding antibodies (2 examples of antiJka and 2 anti-kell) which inhibit agglutination of enzyme-treated cells have been investigated in the sera of 4 patients. The sera containing anti-Jka also contained rhesus antibodies (c+E and D+C) which could not be readily identified by an enzyme technique due to failure of the JP+ cells of the corresponding Rh phenotype to react. The inhibition of the enzyme test reactivity was revealed using a control reagent added to each test after the initial examination for agglu- tination. Addition of ethylenediaminetetraacetic acid to the sera prior to testing resulted in identifiable reactivity with panel red cells. Failure to recognise this phenomenon in sera containing antibodies reacting only by an enzyme technique could result in misidentifica- tion, delay in providing compatible blood and transfusion of incompatible blood. Routine control of enzyme tests to detect false-negative results is recommended.

Introduction

A serum factor has previously been de- scribed by Lown et al. [l], which was found to inhibit serological reactions with enzyme- treated red cells in 5 patients. Inhibition occurred with all red cells tested, including autologous cells, and was complement me- diated. In all cases, no blood group specific- ity could be associated with the serum factor which was present in low concentration and appeared to be IgG in nature. In one of the reported cases an anti-E antibody of proven clinical significance, which reacted only by

enzyme techniques, could not be detected unless ethylenediaminetetraacetic acid (EDTA) was first added to the serum to inac- tivate complement.

The phenomenon was recognised by us- ing a control reagent added to each negative enzyme test. This reagent only agglutinated enzyme-treated red cells and a positive con- trol result therefore validated a negative test. Failure of the reagent to agglutinate the test cells revealed the presence of the inhibiting factor.

In this report we describe 4 patients whose sera were found to contain comple-

Enzyme Test Inhibition 30 I

ment binding alloantibodies with recognised specificities which inhibited enzyme test reactions only with red cells of correspond- ing phenotype. Sera from 2 of the patients had in addition, Rhesus antibodies which, while detectable, could not be readily iden- tified because the inhibiting effect of the accompanying complement binding anti- body resulted in an unrecognisable reaction pattern with red cell panels.

Case Reports

Pafientl; A 40-year-old male was admitted with 30% bums. The patient’s serum was known to contain D +C, Fyb, Jka, and S antibodies. Examination of his serum revealed anomalies in the reactivity of the anti- D + C by a two stage papain procedure which were associated with the reactivity of the anti-Jka. Debride- ment was successfully carried out without blood trans- fusion.

Patirnr2; A 45-year-old female with a 25-year his- tory of rheumatoid arthritis was first admitted in 1978 for a total knee replacement. Anti-c+E antibodies were detected in her serum. In 1981, when she was admitted for a synovectomy of the elbow and silastic radial head implant, an additional antibody, anti-Jka was found. This antibody inhibited the reactivity of the anti-c+E antibodies in the two stage papain procedure.

Patienf3: An 88-year-old female was admitted with a fractured femur. An anti-K antibody was detected in her serum. This antibody prevented agglutination of enzyme-treated K positive cells by the control re- agent.

Paf ieni l : A 34-year-old female was admitted for a ventriculoperitoneal shunt operation. Antibody screening revealed an anti-K antibody with similar serological behaviour to that in patient 3.

Materials and Methods

Antibody investigations were conducted using pub- lished techniques [2] and commercial red cell panels (Dade, Miami, Fla. and Ortho Diagnostic Systems, Rar-

itan, N.J.). Reactions were recorded using the reaction grading system recommended by the Ortho Diagnostics Consultation Service. Monospecific anti-IgG and anti- C 3 X 4 sera (Ortho Diagnostic Systems) were used in antiglobulin tests on papainised and untreated red cells exposed to sera from the 4 patients.

Enzyme tests were performed using papain (Com- monwealth Serum Laboratories, Melbourne, Australia) bromelase (Dade, Miami, Fla.) and ficin prepared and used as previously described [I]. Routine use of the enzyme test control reagent (ETCR) has been pre- viously reported [I]. A tile papain technique was per- formed according to the broadsheet supplied with the reagent.

The C3 component ofcomplement was determined by immunodiffusion [3] and C4 by nephelometry [4] using automated Technicon equipment 151.

To determine whether agglutination of untreated red cells could be inhibited by the anti-Jka in the serum of patient I , rr Jka+ cells were incubated with the patients serum at 37 ‘C for 30 min. The tube was cen- trifuged and the serum removed and the unwashed cell button tested with anti-c typing serum (RST Gamma Biologicals, Houston, Tex.) according to the manufac- turer’s instructions.

Results

The serum of patient 1 was found to con- tain anti-D+C, reacting by enzyme and anti- globulin techniques and Jk”, S and Fyb anti- bodies reacting only by the antiglobulin pro- cedure. The results of the red cell panel investigations using the two stage papain technique are shown in table I. The patient’s anti-D+C reacted with only one of the D or C positive cells. When the ETCR was added to all non-reacting test tubes only one other panel cell reacted. Both panel cells produc- ing a positive result were the only Jka- cells on the panel. The association of the anti-Jk” with the inhibition.of papainised panel cell reactivity was confirmed using 46 commer- cial panel cells. Retesting the serum after

302 Lown/Holland/Barr

Table 1. Results of enzyme tests with a red cell panel and serum from patient I

Panel cell Rh phenotype rr rr r’r r”r R,r R,,r R,R, R,R, RfvR, R,R, Jkaphenotype Jk- Jkat Jka+ Jk“ J k q Jka+ Jka+ JkL Jka+ J k a t

Patients’ serum + ETCR - - - - - - N T - - +k

addition of EDTA produced the expected cell panel reaction profile for the anti-D+C antibody. The anti-D+C could be identified by the antiglobulin test after absorption with rr Jka+, Fyb+, S+ cells. The antigobulin test showed the anti-Jk” to be strongly comple- ment binding and the amount of comple- ment bound to Jk”+cells by the antibody was markedly increased when papainised cells were used. When the serum was tested after absorption with untreated rr Jka+ cells, no inhibition of anti-D+C reactivity occurred and complement binding almost eliminated. With some serum samples from the patient dosage effect on the degree of inhibition was observed in that heterozygous Jka+ cells only partially inhibited the anti-D+C reactions.

The large amount of complement bound to papainised red cells was not responsible for the inhibition of reactivity with the anti- D+C. When Jka+ R,R, papainised cells were incubated with the patients serum and sub- sequently washed once with saline, they were then agglutinated by the ETCR, while still reacting strongly with anti-C3b. As

would be expected the ant i4 and anti-Fyb did not react with papainised cells. Neither antibody bound complement to untreated cells. The anti-Jk” did not inhibit agglutina- tion of untreated rr Jka+ red cells by saline agglutinating anti-c serum after incubation of the cells with the patients serum.

Table I1 show the results of papain tests with a red cell panel using serum from pa- tient 2. Only two of the c and/or E positive cells reacted with the previously identified anti-c+E. These two cells were the only Jka- cells on the panel. The ETCR did not agglu- tinate any of the remaining panel cells. When EDTA was added to the patients se- rum and panel tests repeated the c+E pattern was obtained. As with patient 1 the associa- tion with Jka was established by repeat tests with several red cell panels. The anti-c + E antibodies were detectable only with an en- zyme test.

The papain red cell pane1 results with the serum of patient 3 are shown in tableIII. Similar results were obtained with the serum of patient 4. Both patients had only anti-K

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Table 11. Results ofenzyme tests with a red cell panel and serum from patient 2

Panel cell Rhgenotype RjQ, R ,R , R,R, R‘r r”r rr rr rr rr rr Jkaphenotype Jka+ Jkat Jka+ Jkat Jka+ Jkr- J k L Jkat Jka+ Jka+

Patients’ serum + ETCR - - - - - - - NT N T -

Patients’ serum/EDTA - * + * * * * * * -

Table 111. Results ofenzyme tests with a red cell panel and serum from patient 3 ~

Panel cell Kell pheno- type K- K- K- K- K- K+ K+ * K- K-

Patients’ serum + ETCR * * * * * * * - -

Patients’ serum/EDTA - - - - - - - + +

antibodies reacting by the antiglobulin test, and by the papain test after the addition of EDTA to the serum. Unlike the antiJk” antibodies in patients 1 and 2 the anti-K antibodies in these 2 patients inhibited their own reactivity in the papain test. Both anti- bodies bound a large amount ofcomplement to the red cells and as with the antiJk” anti-

bodies, agglutination with the ETCR oc- curred after washing K+cells following incu- bation with the patients serum.

The inhibition of enzyme reactions in these patients occurred in tube tests using bromelase and ficin as well as papain. How- ever, when the tile procedure was used this phenomenon was not observed.

Lown/Holland/Barr 304

Table IV. Serum complement levels (dl) in 4 pa- tients with alloantibody induced inhibition of reac- tions with enzyme-treated cells

Complement Patients Reference fraction range

1 2 3 4

c3 1.35 1.80 1.25 1.35 0.75-1.4 c4 >0.52 ~ 0 . 5 2 0.52 0.26 0.16-0.50

Table IV shows the C3 and C4 levels in the sera of the 4 patients. 3 of the patients had raised levels of C4. Patient 2 aIso had a raised C3 level.

Discussion

The examples of enzyme test inhibition previously described occurred with all red cells tested. The 4 cases reported here in- volve complement binding alloantibodies inhibiting the reactivity of enzyme reacting antibodies of different specificity (patients 1 and 2) and their own reactivity with enzyme- treated cells (patients 3 and 4).

The anti-Jk" antibodies produced strong anti-C3b and anti-IgG antiglobulin reac- tions with untreated cells. However saline agglutinating anti-c was not inhibited in its reactivity with untreated rr Jk"+ cells which had been incubated with serum of patient 1. It is therefore interesting that the enhanced antiglobulin reactivity with enzyme-treated cells should reflect inhibition of agglutina- tion of these cells by Rh antibodies present in the same serum. As the complement bind- ing alone was not responsible for the inhibi- tion, perhaps the bound antibody and com- plement together were able to sterically

block Rhesus antigen sites on the modified cell membrane.

The finding of raised complement levels in three of the patients may be significant. The C3 level in the fourth patient was near the upper limit ofthe normal range. Perhaps relatively high complement levels are re- quired coincident with an efficient comple- ment binding antibody to produce the inhi- bition described.

Without a control procedure aimed at detecting false-negative results, the Rh anti- bodies in the sera of patients 1 and 2 would not be readily identified. The association of the positive enzyme-test reactions with Jka- red cells may not be recognised, as only those cells of this phenotype which possess the appropriate Rh antigens will react. Misiden- tification of the enzyme reacting antibody could occur if the distribution of Jka- red cells of the corresponding Rh phenotype coincided with that ofanother antigen on the panel. Considerable delay in providing com- patible blood could result from attempts to identify the antibody. Presence of the rhesus antibodies may not be suspected if red cells used for antibody screening are all J ka+. Any of these situations could result in incompat- ible transfusion if blood is released on the basis of an apparently compatible cross- match. As with the non-specific examples of enzyme-test inhibition previously de- scribed, the specific inhibition described here was abolished by inactivating comple- ment with EDTA. Repeating the enzyme tests after adding EDTA to the patients se- rum produced a recognisable reaction pat- tern.

Association of this phenomenon with tube tests and not a tile procedure seems to be related to the red cell concentration used for the test. The tube tests are performed

Enzyme Test Inhibition 305

using 5% cells suspension while the tile pro- cedure requires a 25% concentration. The greater number of antigen sites available in the tile technique may prevent blocking by the complement binding antibody. However this finding should not be construed as an indictment on tube tests compared with tile procedures. Tube enzyme tests can be more sensitive and the test conditions easier to control than tile procedures. Aspects of con- trol of these tests should be considered more carefully in order to recognise all situations which may produce a false result.

Acknowledgements

We thank Dr. R. E. Davis for reviewing the manu- script and Miss N. Wicks for secretarial assistance.

I

References

Lown, J.A.G.; Holland, P.A.T.; Barr, A.L.: A se- rum factor which inhibits serologic reactions with enzyme-treated red blood cells. Transfusion 22: 143-146 (1982).

2 Lown, J. A.G.; Barr, A. L.; Davis, R.E.: Use of low ionic strength saline for crossmatching and anti- body screening. J. d in . Path. 32: 1019-1024 (1979).

3 Ouchterlony, 0.; Nilsson, L. A.: Immunodiffusion and immunoelectrophoresis; in Weir, Handbook of experimental immunology; 2nd ed., chap. 19 (Blackwell, Oxford 1973).

4 Killingsworth, L. M.; Savory, J.: Automated immu- nochemical procedures for measurement of im- munoglobulins IgG, IgA and IgM in human serum. Clin. Chem. 17: 936-940 (1971).

5 Larson, C.; Overstein, P.; Ritchie, R. F. : An auto- mated method for quantitation of proteins in body fluids, in advances in automated analysis. Techni- con int. Congr., vol. I, pp. 101-104 (Thurman, Lon- don 1970).

Received: December 15,1982 Accepted: July I, 1983

J. A. G. Lown, Department of Haematology, Royal Perth Hospital, Wellington Street, Perth 6000 (W. Australia)