Original Paper

Vox Sang 1092:63:227-231

Vicente Pinto Fernando Fernandez-Fuertes Carlos Cecchini Inrnaculada Soto Abelardo Barez

Department of Haematology and Haemotherapy, Hospital Central de Asturias. Oviedo, Spain

I I -0xycorticosteroid-Dependent Antibody in the Serum of a Patient with Bladder Neoplasm

................................................................................................. Abstract An IgM 11-oxycorticosteroid-dependent antibody was identified in the serum of a patient with bladder malignancy, which at 4-37°C reacted with all the 11-oxycorticosteroids tested but not with desoxycorticosterone, testosterone, nortestosterone and progesterone. The resulting drug-antidrug antibody com- plex combines nonspecifically with red blood cells causing agglutination. This reactivity is enhanced both by acid pH and by high drug solution concentra- tions as well as by ficin test. The antibody is complement-independent and has no blood group specificity. No in vivo or in vitro hemolysis was observed. .....................

Introduction Material and Methods

One of the rarer causes of positive pretransfusion tests is the presence in the patient’s serum of antidrug antibod- ies or antibodies enhanced by drugs present in the diluent of commercial reagent red cells, commercial antisera or reagent additives [l, 21. We report an ll-oxycorticoste- roid-dependent antibody detected from positive antibody screen and identification test using manufacturer A’s re- agent red blood cells (RBCs) but negative using manu- facturers B and C’s reagent RBCs panels. Our hypothesis was the serum reacted against some drug added to diluent A and absent in the diluents B and C. According to the manufacturers’ specifications only gentamicin and steroid met this requirement.

Cusr History. A 68-year-old male patient with bladder malignancy and anemia secondary to persistent hematuria was admitted to the hospital. There was no history of transfusion or corticosteroid therapy and no clinical o r laboratory data of hemolytic anemia o r adrenal insufficiency. The most important laboratory data were: groupOI+,CcDee,cortisola.m. 16 pg,cortisolp.m. 8 &ml, ACTH 30 pg/ml. Reticulocytes, haptoglobin, total bilirubin and LDH were within normal limits. No hemoglobinemia o r hemoglobinuria were observed.

Pretrunsfusimz Test. Reagent RBCs were obtained from Ortho Diagnostics, Raritan, N.J. , Gamma Biologicals, Houston, Tex. and BCA (Organon-Teknika), Durham, N.C. Cross-matching, antibody detection and identification, specificity of cold-reactive autoaggluti- nins, titers and scores, and direct antiglobulin tests (DAT) were per- formed as described previously [3]. Unless otherwise stated the serial dilutions of drugs solution and patient’s serum in phosphate-buffered salinc (PBS) p H 7.3, drug solutions at 1 mg/ml in PBS pH 7.3, RBCs suspended to 5% in PBS p H 7.3 and 2: 1 serum-to-cell ratio were used in the following serological studies.

Received: May 23. I Y Y I Vicente Pinto Garci:i 0 1992 S . K q e I AG. Basel Revised manuscript Dcpartment of Hacmatology a id I-iaemotherapy 0042-‘~(X17/Y?/Oh~~~l227 received: January 6 . 1092 Hospital Centi-al cle Asturias $?.75/0 Accepted: February 4, I092 Celestiiio Villnmil hi

E-330(15 Ovietlo (Spain)

Table 1. Initial study RBCs Diluents Saline tests ' Specificity

4°C 22°C 37°C IAT

Manufacturer A manufacturer A 64 64 128 32 no Manufacturer A PBS pH 7.3 16 0 0 0 anti-I Group 0 PBS pH 7.3 16 0 0 0 anti-I Group 0 manufacturer A 64 64 128 32 no Group 0 gentamicin solution 16 0 0 0 anti-I Group 0 hydrocortisone solution 64 64 128 32 no

' Titers.

Confirmution of Hypothesis. The serum was tested as shown in table 1 with reagent RBCs A suspended in the original diluent, re- agent RBCs A washed and resuspended in PBS, and group 0 RBCs suspended in PBS, diluent A, and gentamicin and hydrocortisone solutions.

Drug Adsorption Test. 1 ml of group 0 RBCs was incubated with 15 ml of hydrocortisone solutions (100, 10, 5 and 1 mg/ml in PBS pH 6.5), at 37"Cfor 120 min and then washed and suspended to 5% in PBS pH 6.5. These RBCs were mixed with the patient's serum and fresh AB serum (as negative control), and the same RBCs not in- cubated with the drug were mixed with the patient's serum. No posi- tive control was performed due to lack of known serum. Reactivity was examined after incubating at 22°C for 15 min, and again at 37°C and in indirect antiglobulin test (IAT) after incubating at 37°C for 120 min [3].

Immune Complex Test. This was performed in duplicate (saline and ficin tests) as described previously [3] and as shown in table 2, using group 0 RBCs and equal volumes of patient's serum, fresh AB serum as source of complement, PBS pH 7.3 and drug solutions (3:l test mixture:cell ratio). Reactivity was examined at 37°C and in IAT after incubating for 120 min at 37°C.

Inhibition Test. Equal volumes of serum and twofold dilutions of hydrocortisone solution (range 250-0.01 mglml) were incubated at 37°C for 120 min, subsequently adding 5% ficin-treated group 0 RBCs. Reactivity was examined at 37°C and IAT after incubating at 37°C for 30 min.

Immiinoglobuliri Class. Equal volumes of twofold dilution of se- rum incubated in 2-mercaptoethanol and serum incubated in PBS pH 7.3 (as control), and hydrocortisone solution were incubated at 22°C for 15 rnin with 5% ficin-treated group 0 RBCs [3].

Study of Complement Role. Equal volume of twofold dilution of serum preheated at 56°C for 30 rnin and nonheated serum (as con- trol), and hydrocortisone solution were incubated at 22°C for 15 rnin with 5% ficin-treated group 0 RBCs [4].

Effect of the p H and Drug Solution Concentration on Antibody Activity. This test was performed using hydrocortisone solutions in PBS with a pH range of 6-8 and a concentration range of 10mg/ ml-0.005 pg/ml. Antibody activity was tested by titer analysis after incubating at 22°C for 15 rnin equal volumes of each drug solution, twofold serum dilutions (in the same drug solution), and 5 % suspen- sion in the same drug solution of ficin-treated group 0 RBCs.

Results

Initial Study. Compatibility tests were negative. Pan- agglutination at 4-37"C, titer 128, score 74, without recog- nizable blood group specificity and negative autologous control were observed using the panel of reagent RBCs A, while anti-I at 4"C, titer 16, score 51 was found using pan- els of reagent RBCs B and C. The DAT test was negative. The serum did not react with reagent RBCs A resuspend- ed in PBS or with group 0 RBCs suspended in gentamicin solution, but did react with group 0 RBCs suspended both in hydrocortisone solution and in diluent A (table 1).

Drug Adsorption Test. The serum did not react with hydrocortisone-incubated RBCs. We cannot claim to have achieved the sensitization of RBCs by the drug, al- though the pH and concentrations of drug solutions used were optimum.

Immune Complex Test. The serum reacted with RBCs in the presence of the 11-oxycorticosteroid solutions used and did not react with the solutions of desoxycorticoste- rone, progesterone, testosterone or nortestosterone (ta- ble 2). Complement addition was not necessary for agglu- tination and hemolysis was not observed.

Inhibition Test. The agglutination observed in the im- mune complex test was not inhibited by preincubating serum with hydrocortisone solutions and subsequent ad- dition of RBCs.

2-Mercaptoethanol Test. The 2-mercaptoethanol-incu- bated serum did not react with RBCs in the presence of hydrocortisone solution, while PBS-incubated serum did (titer 64, score 80).

Study of Role of the Complement. Both the preheated and the nonpreheated serum reacted with RBCs in the presence of hydrocortisone solution (titer 64, score 77, and titer 64, score 75, respectively).

228 Pinto/Fernandez-Fuertes/Cecchini/Soto/ 11-Oxycorticosteroid-Dependent Antibody Barez

Table 2. Study of antibody reactivity: complex immune mecha- nism

Serum Fresh Drug solution PBS Ficin tests of AB (lmg/ml) patient serum

37°C IAT

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

hydrocortisone 6-m-prednisolone prednisone dexamethasone paramethasone deflazacort desoxycorticosterone testosterone nortestosterone progesterone hydrocortisone 6-rn-prednisolone prednisone dexamethasone paramethasone deflazacort desoxycorticosterone testosterone nortestosterone progesterone

hydrocortisone 6-m-prednisolone prednisone dexamethasone paramethasone deflazacort desoxycorticosterone testosterone nortestosterone progesterone

Effect of the p H and Drug Solution Concentration on Antibody Activity. Using drug solutions at a concentration of 2.5 mg/ml, agglutination varies titer 256 at pH 6 to titer 16 at pH 8; and using drug solutions at pH 6, it varies from titer 128 at a concentration of 10 mg/ml to titer 1 at a con- centration of 0.02 pg/ml (table 3 ) .

Table 3. The effect of the pH of the hydrocortisone solution and concentration on antibody reactivity

mg/ml PH 6.0 6.5 7.0 7.25 7.5 8.0

10

2.5

0.62

0.1.5

0.02

0.004

0.0003

0.00002

0.000005

T 128 S 89 T 256 s 101 T 128 S 94 T 64 S 73 T 16 S 53 T 8 S 48 T 4 S 18 T 1 S 5 T 0 S 0

128 89

2.56 101 64 75 16 51 16 53 8

48 4

18 1 5 0 0

64 64 32 16 74 77 72 51

128 64 64 16 84 77 70 48 16 16 16 8 58 58 51 48 16 16 16 4 48 46 43 34 8 8 8 2

48 46 34 7 8 8 8 1

48 46 34 2 2 2 2 0

13 10 7 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0

T = Titer: S = score.

Discussion

One of the least frequent causes of positive pretrans- fusion tests is the presence in the patient’s serum of anti- bodies against chloramphenicol [5], neomycin [6], tetra- cycline [3], hydrocortisone [7], which are often used as preservatives in the preparation of reagent RBCs. It may also be produced by alloantibodies or autoantibodies whose detection requires the presence of drugs, such as caprylate (autoanti-e) [8], borate (anti-A) [9] ,chloram- phenicol (anti-A,) [lo], paraben (autoanti-Jka) [ll], thime- rosal (IgG autoantibody) [12], sodium azide (anti-I) [13], LISS (anti-Pr,) [14]. Drug-dependent antibodies without apparent group specificity have also been described [15], and some antidrug antibodies may adhere to the RBC surface by means of the Matuhasi-Ogata phenomenon

The data set out reveal the presence in the patient’s serum of an IgM 11-oxycorticosteroid-dependent anti- body which agglutinates the RBCs in the presence of the drug by immune complex mechanisms. The inhibition test confirms that agglutination is due to nonspecific adsorp- tion of drug-antidrug antibody complexes on the RBCs [17]. The antibody activity depends on p H and concentra-

[161.

229

Table 4. Relation between drug struc- ture and the reactivity with the antibody Drug Structure Reactivity

Hydrocortisone I I, 17,21-triHYDROXYpregn-4-ene-3,20-dione Yes 6-rn-Prednisolone II,17,21-triHYDROXYpregna-1,4-diene-3,20-dione yes Prednisone 17,21-dihydroxypregna-l,4-diene-3,11,20-triONE yes Dexamethasone 9-fluoro-11, 17,21-triHYDROXY-16-methylpregna- yes

Paramethasone 6-fluoro-11, 17,21-triHYDROXY-16-methylpregna- yes 1,4-diene-3,20-dione

1,4-diene-3,20-dione

dieno [17,16-d]oxazole-3,20-dione 21-acetate Deflazacort ZI, 2l-diHYDROXY-2’-methyl-SH-pregna-l, 4- Yes

Desoxycorticosterone 21-hydroxypregn-4-ene-3,20-dione no Testosterone 17-hydroxyandrost-4-ene-3-one no Nortestosterone 17-hydroxyestr-4-en-3-one no Progesterone pregn-4-ene-3,20-dione no

p = 0.0047 (Fisher’s exact test).

tion of drug solutions is enhanced by the ficin test, does not show blood group specificity, has a thermal range of 4-37”C, is complement-independent and does not cause hemolysis in vitro or in vivo. The antibody only reacts with the drugs having 11-hydroxy or 11-ketone (one) groups (11-oxycorticosteroids), and does not react with drugs lacking these groups (p = 0.0047, Fisher’s exact test; ta- ble4). The antibody does not react with the remaining chemical structures: 20-ketone and 21-hydroxy groups are present in desoxycorticosterone, which is nonreactive. The 17-hydroxy group is not present in deflazacort and the C-1 to C-2 double bond (1-ene) is not present in hydrocor- tisone, which are reactive. 10-Methyl (except nortestoste- rone), 13-methyl and 3-ketone groups and the C-4 to C-5 double bond (6ene) are present both in reactive drugs and in nonreactive drugs. The above data could indicate that the antibody reacts with endogenous and synthetic glucocorticosteroids. These contain an oxygen atom as hydroxyl or ketone group at position 11. The glucocorti- costeroids having a 11-ketone group must be reduced (pri- marily in the liver) to their corresponding 11-hydroxyl group analogs in order to be pharmacologically active on glucose metabolism or about the anti-inflammatory ef- fects [MI. We could not study the antibody’s reactivity with aldosterone due to this drug not being available.

Owing to the absence of a history of corticosteroid therapy, the possibility that the antibody might have orig- inated from immunization to exogenous steroids can be ruled out. Mann [7] reports that 64% of 25 cases of anti- hydrocortisone antibody appeared in patients with ne-

oplasm, as in this case, which would suggest an immuno- logical relationship between antibody formation and tu- mor development. The absence of signs of hemolysis suggests that the antibody does not shorten the life of RBCs in vivo. We do not know of reports of hemolytic anemia induced by corticosteroids. The absence of symp- toms of adrenal insufficiency and the fact that both corti- sol and ACTH plasma values are normal would seem to indicate that the antibody neither neutralizes nor inactiva- tes the patient’s corticosteroids, although the amount of cortisol (8 pg/ml) is within the antibody’s range of action (up to 0.3 pg/ml at pH 7.25-7.5, table 3 ) .

230 Pinto/Fernandez-Fuertes/Cecchini/Soto/ 1 I-Oxycorticosteroid-Dependent Antibody Barez

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..................... 15th Congress of the International Society of Forensic Haemogenetics - ISFH Venice, Palazzo del Cinema, Lido di Venezia, Italy October 13-15. 1993

Topics: All aspects of forensic haemogenetics, especially DNA polymorphisms, PCR, biostatistics, population genetics and data- bases, quality assurance, blood group markers, parentage testing, stain analysis, identification.

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