the minus-minus phenotype in the kidd system

The Minus-Minus Phenotype in the Kidd System DOROTHY DAY, HERBERT A. PERKINS,

AND BRUCE SAMS From the Irwin Memorial Blood Bank of the San Francisco Medical Society and

the Kaiser Foundation Hospital, San Francisco, Calijornia

A delayed hemolytic reaction following transfusion occurred in a 28-year-old Chinese mother of six. Her blood was Jk(a-b-), and her serum contained a crocwreacting auti-JkaJkb as well as a q a r a b l e anti-Jk.. Her husband and children were all Jk(a-b+). Although genetic translnieeiQn of Jk(a-b-) blood h a not been proven ~LI yet, it appears m a t probably to be the homozypus expression of a third allele or of gene deletion. To date, all instances of t h i s type of blood have been found in populations of Asian origin.

THE phenotypic expressions of the Kidd blood group system have been identified by two antisera, anti-Jka and anti-Jkb. The corresponding antigens (Jka and Jkb) are inherited as codominant alleles which are not sex-linked. In 1959 Pinkerton et al.5 reported studies on the blood of a patient who was brought to their attention by a delayed hemolytic transfusion reaction. The red blood cells of this patient failed to react with either anti-Jka or anti-Jkb, and the serum contained a cross-reacting anti-JkaJkb. Seven other examples of Jk (a-b-) blood have been discovered since then, two in Hawaii and five in South America3 This report will present data on another example of Jk(a-b-) blood, the second to contain cross-reacting anti- JkaJkb. It was brought to our attention, as in the Hawaiian case, by a delayed hemolytic reaction following transfusion.

Case Report Mrs. N.L.. a 32-yedr-old Chinese housewife, was

admitted to the Kaiser Foundation Hospital in San Francisco November 30, 1963 for elective removal

Presented in part at the 17th Annual Meeting of the American Association of Blood Banks, Au- gust 1964.

Received for publication October 22, 1964.

of a stone in the right kidney. Prior to this hos- pitalization she had been tiansfused with only a single unit of blood, given 14 years previously at the time of delivery of her first child. No reaction to this blood is remembered. She had six pregnancies, all terminating with healthy newborns devoid of signs of hemolytic disease. On routine physical examinations twice in the two years prior to admission, copper sulfate screening tests revealed hemoglobin levels over 15 grams per 100 ml. Her personal history revealed no jaundice nor anemia; her family history was also free of these complaints, and there was no consanguinity.

Physical examination on this admission revealed mild pallor, but no hepatosplenomegaly. Urinalysis showed occasional red and white blood cells; the blood urea nitrogen was 33 mg per 100 ml. The renal calculus was removed uneventfully several days later. Her hematocrit was low prior to surgery (Fig.. 1). and three units of blood were transfused during the procedure. These had been found compatible with the .serum of the patient by saline (room temperature and 37 C) , high prottin and antiglobulin technics.

The postoperative period was complicated by persistent fever with spikes to 4OC daily. On the sixth day the urine was reported to be amber colored, and on the following day scleral icterus was noted. -4n abrupt drop in the hematocrit (Fig. 1) and the absence of detectable blood loss+ suggested that hemolysis was responsible for the jaundice. This possibility was corroborated by demonstration of an antibody in the patient’s Serum which reacted with the red blood cells of the units which had been transfused (see “SpeciJ Studies” below). Liver function tests suggest4 hepatocellular insufficiency. The bone marrow was cellular with increased erythroid activity and plenti- ful iron. The peripheral blood revealed hypo- chromic red cells, target cells and poikilocytes. A tentative diagnosis of thalassemia minor was con- firmed by finding the hemoglobin A2 level a t twice normal values.

Jaundice rapidly subsided, but fever persisted, and the hematocrit slowly dropped despite reticulo- cyte counts averaging 3 to 4 per cent. Knowing the propensity of Kidd antibodies for inducing delayed hemolytic reactions, and with evidence that

Stool guaiac tests were negative.

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316 DAY, E T AL.

f r o y 0

8 I I

I I

10 2 0 30 40 S O 60 70 80 I10 Hospital days

the patient’s own red blood cells were Jk (a-b-) , it appeared likely that only blood of this unusual type would be compatible. A unit of fresh blood was obtained from one of the known Jk(a-b-) donors through the courtesy of Dr. Mermod (Blood Bank of Hawaii). It proved compatible; and its transfusion produced an appropriate rise in the patient’s hematoait. Prior to the transfusion the patient was weak, confused, had a persistent tachy- cardia and appeared extremely ill. The transfusion resulted in considerable symptomatic improvement, and there was no evidence of a reaction. Fever and a progrcssive fall in hematocrit persisted, however. A unit of blood from one of the Hawaiian donors was obtained from the American Association of Blaod Banks Rare Blood Frozen Storage Depot at the Chelsea Naval Hospital. With the availability of this blood, the patient’s wound was explored more deeply than had been previously attempted. A deep perinephric abscess was found and drained. The unit of frozen blood was given without reaction, although the rise in hematocrit was less than had been obtained with the f~esh blood. There was no grolu hemoglobinemia nor hemoglobinuria, and it may be that the failure to elevate the hematocrit more was due to blood loss during the operative procedure. With drainage of the abscess. improvement was spontaneous and continuous.

. .. Special Studies’

Special immunohematologic studies were carried out at the Irwin Memorial Blood Bank of the San Francisco Medical Society. The patient’s preoperative sample had been discarded at the hospital on the seventh postoperative day just prior to the a p

FIG. I . Hospital Course for Patient N.L.

pearance of jaundice, so it was not possible to confirm the absence of atypical antibodies from that specimen. At the time of the presumed hemolytic episode the patient’s red blood cells had a positive direct antiglobulin test. Her serum contained a saline agglutinin active at 37 C only (+). Antibody activity was more marked with the indirect antiglobulin procedure (+++), with trypsinized cells (++++) , and with trypsin plus antiglobulin (++++) . Partial hemolysis occurred when trypsinized cells were incubated with the patient’s serum. Reactions were stronger with a commercial “non-gamma” antiglobulin serum** than with a “broad spectrum” reagent,++* and were negative using a commercial “anti-gamma’‘ reagent.++ No reaction occurred with an antiC’3a.t Testing the serum against several panels of group 0 red cells and numerous random donors demonstrated no variability of its reactivity. Blood grouping tests on the patient’s red cells revealed her to be group 0, NsNs, P--, R,R, V- VS- Wiel-, Lu (a-b+), K-k+Kp (a-b+),

+ + Hyland Laboratories, Los Angeles, California. +++ Ortho Pharmaceutical Corporation, Raritan,

t Provided by Dr. H. Hugh Fudenberg, University New Jersey.

of California Medical Center, San Francisco.

MINUS-MINUS PHENOTYPE IN KIDD SYSTEM 317

TABLE 1. Absorption and Elution Studies

Results Experiment Step Source of Absorbing Supernatant solution vs. Eluate vs.

No. No. Antibody Cells Jk (a+b-) Jk (a-b+) Jk (a+b-) Jk (a-b+) ~

I 1 . Serum Jk(a+b-) +++ +++ +++ ++ of step 1 Jk(a+b-) +++ +++ +++ -k

of step 2 Jk (a+b-) I1 1. Serum Jk(a-b+) +++ +++ ++ ++

of step 1 Jk (a-b+) ++ + ++ ++ of step 2 Jk(a-b+) +++ -

2. Supernatant solution

3. Supernatant solution - - - -



(Elutions not done. Antiglobulin test was negative.)

Le (a-b+), Fy (a+b-), Jk (a-b-), Js (a-), Di (a-) , Xg (a+), Ve (a+).??

The unusual results of the Kidd typing suggested that the serum antibody might be directed against both Jka and Jkb, as in the first identified case of Jk(a-b-) bl0od.5 Support for this idea was obtained when tests demonstrated compatibility with two separate units of Jk(a-b-) blood from the Hawaiian donors, one fresh and one from frozen storage. These units were successfully transfused.

Although no antibody had been detected in the patient’s serum by compatibility tests on entry to the hospital, seven days after transfusion Kidd antibody was evi- dent at a titer of 1:64 and reached a peak of 1 :256 four days later. With characteristic lability, it was clearly decreasing in vivo several weeks later. The rapid appearance of the antibody suggests a secondary type of immune response.

~~ ~~

tt We are grateful to Dr. Byron Myhre (Milwau- kee Blood Center, Wisconsin), Miss Marion Lewis (Rh Laboratory, Winnipeg, Canada) and Dr. Ruth Sanger (Lister Institute, London, England) for confirmation and supplementation of. our blood group studies on the patient’s red cells. ‘Dr. Sanger reported that the patient’s cells failed to react with four samples of anti-Jka, three of anti-Jkb, and the original example of anti-JkaJkb.

With each sample of the patient’s serum, the titer was equally high against Jk(a+ b-) and Jk (a-b+) cells, suggesting that a cross-reacting antibody might be present, as in the case of Yinkerton et al. Absorp tion and elution studies (Table 1) con- firmed this, since eluates from Jk(a+b-) cells reacted with Jk (a-b+) cells and vice versa. Evidence for the existence of sepa- rable anti-Jka is apparent in the same table at various points. In Experiment I, anti-Jka was completely absorbed by exposure to three successive portions of Jk (a+b-) cells. Eluates from the first two portions of cells thus sensitized showed more anti-Jka than anti-Jkb activity. In Experiment 11, successive absorptions with Jk (a-b+) cells left in the absorbed serum clear evidence of anti-Jka (supernatant solution of step 3). Anti-Jkb was not evi- dent as a separate entity at any point. In contrast to these findings, the patient’s serum caused some hemolysis of Jk (a-b+) cells during absorptions, but never of Jk (a+b-) cells. We have no explanation for this paradox. Blood grouping studies on the patient’s

husband, children, mother and the donors used for the first series of transfusions are reported in Table 2.

318 DAY, ET AL.

TABLE 2. I.hmily Sludy

Propositus 0, RhlRh1, Jk (a-b-) Husband U, RhIRh,, Jk(a-b+) Children:

Clifton 0, RhlRh,, Jk (a-b+) James B. RhlRh?. Jk(a-b+) Arthur B, RhlRhl, Jk(a-b+) Richard B. RhIRhl, Jk (a-b+) Bernice 0, RhlRhz, Jk (a-b+) Dianne 0, RhlRhl, Jk (a-b+)

Mother of propositus A, Rhlrh, Jk (a+b-)

Blood Donors 34263 0. RhIRhl, Jk (a-b+) 34701 0. RhlRhl, Jk (a+b+) 3 4 m 0, RhlRhl, Jk (a-b+)

Discussion

Minus-minus phenotypes,1 the absence of any blood factors known to be produced by a group of allelic genes, can be explained in one of three ways: 1) There may be one or more as yet unrecognized alleles occupy- ing that position on the chromosome and producing an undetectable factor. (There may be an amorph at the Kidd locus. There may be a third allele that produces an antigen not yet detectable by a specific antibody.) 2) There may be homozygous absence of the genes with resulting absence of the factors (gene deletion). 3) The usual genes may be present, but may be s u p pressed; i.e., prevented from expressing themselves in the phenotype by other genes or environmental action. The genes might not be operative because of absence of pre- cursor substance. No other studies have been reported in

families of Jk (a- b-) propositi, and it is unfortunate that the present large family does not prove genetic transmission. No blood relatives other than those mentioned are available for study. The family does provide additional evidence, however, favoring the presence of a third allele (or gene deletion) at the Kidd locus.

Proof of suppression requires family studies demonstrating that the patient transmits the suppressed gene but not the suppressor to some of his children, or that factors present in his parents which had to be inherited by the patient fail to express themselves. It is most unlikely that a dominant suppressor gene is involved in the present case. If it were present, there would be only a one in 64 chance that none of her six children would inherit it and thus demonstrate the Jk(a-b-) phenotype. The dominant characteristics of the Lu (a-b-) phenotype of the Craw- ford family4 is demonstrated by the fact that three of the four children of that propositus typed as Lu (a-b-) .

A recessive suppressor gene seems equally unlikely. If this were present, any Kidd gene inherited through Mrs. L. should be able to express itself in her children. If we assume that her mother is a homozygous Jk (a+) in a straightforward two allele system, then Jka must be suppressed in Mrs. L., and should be detectable in at least 50 per cent of her children. Again the chance that all children would be Jk(a-b+) is only one in 64.

The evidence in this family is thus against the possibility of a suppressor gene as the basis for the Jk(a-b-) phenotype. It seems likely that she is homozygous for a third allele. Further evidence for the third allele hypothesis is provided in the apparent discrepancy in the Kidd inheri- tance in the Crawford family4 and in the correlation between the observed and ex- pected Jk (a-b-) phenotype of the South American Indians7 when a third allele was used in the gene frequency calculations. This has been discussed at some length by Race and Sanger.6

If a third allele is present in Caucasian or Negro populations, i t must be very rare. Our patient is of Chinese ancestry, and the three instances recognized in Hawaii are Filipino-Chinese-Spanish, Filipino-Spanish,

MINUS-MINUS PHENOTYPE IN KIDD SYSTEM 319

and Hawaiian-Chinese. Silver, Haber and Kellner tested 88 Indians of Mato Grosso, Brazil and found five who were Jk(a- b-).7 A third allele, tentatively labeled Jk, may thus be of Asian origin. The most direct evidence for a third allele would be an antibody identifying its product, but family studies provide useful information. The Jk (a-b-) phenotype will occur only in homozygotes for the third allele. The much more common heterozygotes may be identifiable by anomalous segregation of Kidd phenotypes among their progeny as in the Crawford family.4

In each of the two cases of Jk (a-b-) blood with anti-Kidd antibodies, the antibody has been cross-reacting. By analogy to the ABO system, this might be regarded as an argument against a third allele with phenotypic expression on the assumption that cross-reacting antibody reacts with a basic antigenic configuration common to all factors in the group. It may well be, there- fore, that the third allele is an amorph or that the phenotype is the result of a com- plete gene deletion.

The assumption that the antibody is cross-reacting is based on evidence that it reacts equally well with cells which are Jk(a+b-) and those which are Jk(a- b+). These results would occur if the antibody was directed against a distinct new antigen of very high frequency in the Kidd system.

In Pinkerton’s case there was, in addition to the cross-reacting antibody, an anti-Jkb which fitted well with the known exposure of the patient through pregnancies to the Jkb antigen. In the present case, pregnancy stimulations were also of the Jkb type only, but anti-Jka rather than anti-JkFl was found in addition to the cross-reacting antibody. It is possible that this resulted from an initial stimulation by a tranfusion 14 years previously. One of the units received during the present admission was Jk(a+).

The Hawaiian case came to attention after a delayed transfusion reaction. The sequence of events appears similar in the present case. Evidence that hemolysis occurred seems incontrovertable, but the pic- ture was complicated by obvious signs of acute liver decompensation which were never adequately explained. There is some evidence that the anesthetic, Halothane, which the patient received during surgery, is occasionally hepatotoxic,s but there is no agreement on this subject at the mo- ment.2 Although transitory hepatic failure, wound infection and underlying thalassemia minor complicated the clinical pic- ture considerably and played a role in in- creasing the patient’s anemia, there is little doubt that a delayed hemolytic transfusion reaction had occurred.

References 1. Allen, F. H.: “Minus Minus” phenotypes (Edi-

torial). Transfusion 1: 209, 1961. 2. Allen, H. L. and D. W. Mctcalf: A search for

halothane liver complications. Anesth. Analg. 4 3 159, 1964.

3. Chadwick, D. A. and R. C. Jenning: Massive hepatic necrosis associated with halothane anaesthesia. Lancet 1: 793, 1964.

4. Crawford, M. N.. T. J. Greenwalt, T. Sasaki, P. Tippett, R. Sanger, and R. R. Race: The phenotype Lu (a-b-) together with uncon- ventional Kidd groups in one family. Trans- fusion 1:228, 1961.

5. Pinkerton, F. J., L. E. Mermod, B. A. Liles, J. A. Jack, Jr., and J. Noadec: The phenotype Jk(a-b-) in the Kidd blood group system. Vox Sang. 4: 155, 1959.

6. Race, R. R. and R. Sanger: Blood Groups in Man. Philadelphia, F. A. Davis Co., 4th Edition, 1962.

7. Silver, R. T., J. M. Haber, and A. Kellner: Evidence for a new allele in the Kidd blood group system in Indians of northern Mato Grow, Brazil. Nature 186:481, 1960.

Dorothy Day, A.B., BB (ASCP), Technical Super- visor of Investigative Studies.

Herbert A. Perkins, M.D., Director of Research, Irwin Memorial Blood Bank of the San Franciscu Medical Society, San Francisco, California.

tion Hospital, San Francisco, California. Bruce Sams, M.D., Staff Physician, Kaiser Founda-

the minus-minus phenotype in the kidd system

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