May, 1972 T h e ]ournal of P E D I A T R I C S
Isoimmune neonatal neutroDenia The third child of a 34-year-old Caucasian woman presented at two weeks of age with pyoderma of five days" duration and severe neutropeuia. The neutropenia persisted for at least 28 days. A neutrophiI-speeific antibody was demonstrated in both the infant's and mother's blood. Muramidase determinations on the infant's serum revealed an initial level of 3.8 #g per milliliter at four weeks of age with a gradual fall to 2.2 #g per milliliter at eight weeks; this decrease possibly reflected neutrophil degradation. This infant represents a case of immune-induced neutropenia following maternal isoimmunization.
Laurence A. Boxer, M.D.,* Mitsuo Yokoyama, M.D., Honolulu, Hawaii, and Parviz Lalezari, M.D., N e w York, N. Y.
ISOI~MVNB N E O N A T A L neutropenia (INN) due to maternal isoimmunization has been reported in nine well-documented cases?-" In these cases the disease has been characterized by a severe but transient neutropenia associated with cutaneous and occasionally with systemic infection. Agglutinating leukocyte antibodies have been found in the maternal sera which do not react with the mother's own cells but react with the paternal leukocytes. In several additional cases, either the neutropenia has been too mild 1~ or the From the Department of Pediatrics, Tripler Army Medical Center, Kuakini Medical Research Foundation, and Blood Bank, Montefiore Hospital and Medical Center. Supported by Grant HE 10036-05 from the National Heart Institute, National Institutes o/ Health, United States Public Health Service, and Contract 69-2071, Transplantation Immunology Branch, National Institute o[ Allergy and Infectious Diseases, United States Public Health Service. Presented in part at the X I I I International Congres of Hematology, Munich, Germany, August 2 to 8, 1970. ~Reprint address: Trlpler Army Medical Center, APO San Francisco 96438.
association with the antibody has not been established? 1-13 Leukocyte antibodies have been demonstrated in 20 per cent of randomly selected multiparous women, but retrospective studies on these families have not revealed evidence for clinical involvement of the newborn infants. The absence of neutropenia in several neonates whose mothers had antibody led Payne 14 to suggest that infection rather than leukoagglutinins may haize been the cause of neutropenia in the reported cases of INN. The association of leukocyte agglutinins with neonatal neutropenia was established when it was demonstrated that the leukocyte antibodies involved in INN, in contrast to those studied by Payne, reacted with antigens that are specific for blood neutrophils2 -7 Recently, Lalezari and associates 8 reported a case in which, in addition to a neutrophilspecific antibody, the maternal blood contained a "broad-spectrum" antibody with lymphocytotoxicity and reactivity with nonneutrophilic granulocytes. Herein is described Vol. 80, No. 5, pp. 783-787
Boxer, Yokoyama, and Lalezari
The Journal of Pediatrics May 1972
Table I. Peripheral blood of the affected newborn infant at different stages of the disease Age White (days) blood cells 14 12,100
1 2 2 87 2 4 2
Differential Myeloblast Myelocytes Metamyelocytes Lymphocytes Monocytes Eosinophils Basophils Myelocytes Metamyelocyte Bands Lymphoeytes Monocytes Basophil
3 1 6 80 9 1
5 Polymorphs 91 Lymphoeytes 4 Monocytes
4 73 20 2 1
Polymorphs Lymphocytes Monocytes Eosinophils Basophil
18 2 65 11 3 1
Polymorphs Bands Lymphocytes Monocytes Eosinophils Basophil
Antibody titer 64
a case of I N N in which a leukoagglutinating antibody specific for neutrophils was found in both the mother's and the infant's blood. Recovery of the infan': was associated with the disappearance of the antibody. During the neutropenic phase, the infant had elevated blood muramidase activity which decreased when the neutrophil count returned to normal. CASE REPORT
Patient C. B., a boy, was the third child of a 34-year-old Caucasian mother who was seronegative and had never received a blood transfusion. The baby was first seen at two weeks of age with pyoderma of five days' duration, from which a group A beta hemolytic streptococcus was cultured. Initial hemogram revealed a hematocrit of 42 per cent and a white blood ceil count of 12,100 per cubic miliimeter. In
examination of blood smear, a selective and complete absence of neutrophils was noted (Table I). Bone marrow examination showed some development through the entire myeloid series with a preponderance of early myeloid forms indicative of marrow arrest at the myelocyte stage. Ratio of the myeloid forms to erythroid cells was 2:1. Pyoderma cleared in three days with oral penicillin therapy, and subsequently the infant remained asymptomatic. During the third week of life neutrophils reappeared in the peripheral blood, and a blood examination on the forty-second day of life was normal. Leukocyte counts and differentials, performed on maternal pre- and postpartum blood samples, have been consistently normal. The infant has remained healthy up to the present time (16 months of age). MATERIAL
T h e agglutination test and preparation of stained slides were performed by a previously described method in which ethylenediaminetetra-acetate ( E D T A ) is used as an anticoagulant. 6 Lymphocytotoxicity tests were performed by the method of Terasaki and McClelland? 5 Immunofluorescein antibody test was performed according to the technique described by K a w a m u r a ? 6 Bone marrow smears of the infant and his father were prepared on glass slides and fixed by 95 per cent ethanol. T h e preparations were flooded with the sera from the mother or the infant and incubated at 37 ~ C. for one hour. T h e slides were washed twice with phosphatebuffered saline ( p H 7.4) and then flooded with fluorescein-conjugated anti-IgG, antiIgA, or a n t i - I g M (Hyland Laboratories, Los Angeles, Calif.) for an additional hour. The slides were washed again and then examined. Control samples were prepared by exposing 9the bone m a r r o w smears to normal h u m a n sera. Absorption test. Leukocytes, containing primarily neutrophils, were prepared by an oil bottle technique. 17 Lymphocytes were isolated by the method of Walford and associates. is Platelets were separated by differential centrifugation. T h e mother's plasma was absorbed with platelets and lymphocytes isolated from the father, other members of the ram-
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ily, or from random blood donors. Three volumes of test plasma were incubated for two hours at 37 ~ C. against one-third volume of absorbing packed platelets or lymphocytes. Subsequently, platelets or lymphocytes were sedimented by centrifugation for ten minutes in Serofuge (Clay Adams, CT-1660, Standard Scientific Company, Edison, N. J.), and the supernates were titrated against the leukocytes obtained from each blood donor. Similar absorption procedures were followed by the use of leukocytes that were prepared from two unrelated donors.
Isoimraune neonatal neutropenia
Table II. Reactivity of the leukocyte antibody in the maternal serum results of absorption with donors' own platelets and lymphocytes Titer after absorption aftgr Titer absorption with before with lymphoabsorption platelets cytes Titer
Father Mother First child Second child Proposltus (third child)
128 0 64 64
64 0 32 32
64 0 32 32
D E A E cellulose column chromatography.
Ten milliliters of the mother's serum were dialyzed for 16 hours against 0.01M sodium phosphate buffer p H 8.0 and then applied to a diethytaminoethyl cellulose (DAEA) cellulose column equilibrated with the same buffer. Molarity was increased to 0.02M and then by 0.02M increments until 0.10M was reached. Fractions at different molarities were collected and characterized for the content of immunoglobulins using ring diffusion in antibody-agar plates. 19 The characterized fractions were concentrated and, after equilibration to isotinicity, were tested for leukoagglutinins. Immunoglobulins were also determined in the infant's serum. Muramidase. Muramidase determinations on different serum samples obtained from the infant were performed according to the method of Smolelis and Hartsell. 2~ The sera were kept frozen at -20 ~ C. before use. The muramidase activity of serum from normal infants aged two weeks to eight weeks were used as controls. RESULTS
A leukoagglutinin was demonstrated in both the maternal and the infant's plasma against the paternal leukocytes and those of the siblings; no reaction was obtained when the mother's own leukocytes were used (Table I I ) . Among unrelated donors, the antibody titer varied from 1 to 128. After recovery, the infant's plasma sample, collected during the neutropenic period, and the maternal plasmas were tested against the child's leukocytes; in each instance there was
a positive agglutination reaction. The specificity of the maternal antibody for neutrophils was demonstrated by failure of platelets and lymphocytes obtained from the father and the siblings to remove the antibody (Table II). In contrast, leukocytes from two donors whose cells reacted positively with the antiserum completely absorbed the antibody, whereas platelets and lymphocytes from the same donors failed to absorb the agglutinin. This neutrophil specificity was further demonstrated on stained slides2 Preparation of stained slides from leukocytes of two different donors with high ratio of peripheral blood eosinophils revealed selective aggregation of neutrophils with eosinophils not participating in the agglutination. By means of immunofluorescent technique performed on the bone marrow smears, it was demonstrated that of various cells only myelocytes, metamyelocytes~ bands, and neutrophils became fluorescent after having been exposed to maternal serum. Fig. 1 demonstrates the cytoplasm fluorescing in a band and neutrophil. Serum from the father and normal sera showed no activity. The fluorescent reaction in the immunofluorescein antibody test were produced only by anti-IgG conjugates but not by anti-IgA or anti-IgM sera. Chromatographic fractionation of the maternal serum on a DEAE cellulose column demonstrated that the leukoagglutinin activity was restricted to the IgG fraction. Lymphocytotoxic antibodies were initially
7 8 6 Boxer, Yokoyama, and Lalezari
The Journal of Pediatrics May 1972
Table III. Muramidase levels (micrograms per milliliter)
Age (wk.) 2 4 6 8 16
Fig. I. Immunofluorescent staining with IgG of a neutrophil and a band form. found in the mother's serum at a titer of 1 : 16 and in the infant's serum at 1:2. This antibody activity was transient, however, and could not be found in the serum samples collected several months later. The maternal lymphocytotoxic antibodies were absorbed by paternal lyinphocytes; whereas the leukoagglutinin was not. Serum muramidase activity in the infant was mildly increased initially. As hematologic remission occurred, serum muramidase activity returned to normal (Table I I I ) . Immunoglobulins at three weeks of age revealed an IgG of 325 my. per 100 nal. (normal, 280 to 950), IgA 19 rag. per 100 ml. (normal, 4 to 36), and IgM 61 mg. per 100 ml. (normal, 15 to 86). DISCUSSION Isoimmune neonatal neutropenia due to fetal-maternal incompatibility has been observed previously. Antigens confined to the neutrophils have been implicated as the immunogenic factors derived from fetal neutrophils which, following passage across the placenta, provoke maternal antibody response. The maternal antibody thus produced crosses the placenta and results in selective destruction of neutrophils in the fetus without having any effect on other cells. 6 The disease is thus analogous to erythroblastosis fetalis and is characterized by a transient absence of segmented neutrophilie leukocytes in the blood and the bone marrow of the afflicted newborn infant.
[ Control subjects Patient I (term infants) -1.3; 1.6; 1.95; 2.0 3.8 0.9; 1.3; 1.6; 1.6; 1.6; 1.8; 2.0 3.3 I; 1; 2.3 2.2 1.3 ; 2.0 1.4
Tile present report concerns another example of this disorder. An antibody has been found in the blood of the mother and shown to be a 7S immunoglobulin. Its transmission across the placenta has been established by the demonstration of the antibody in blood of the neonate. Neutrophil specificity was demonstrated by specific absorption of the antibody by leukocyte preparations composed mainly of neutrophils, by failure of absorption with platelets, by lack of cytotoxicity against lymphocytes, and by preparation of stained slides from the aggregates which showed selective participation of the polymorphonuclear myeloid series; eosinophils did not agglutinate. The antigen reacting with this antibody has further been characterized; the preliminary data zl indicate that it is an allele to NA1, the first neutrophil antigen identified. NA1 antibody reacted with the neutrophils of the mother and three children but not with those of the father; whereas, the antibody in question reacted solely with neutrophils of the father and children hut not with those of the mother. Muramidase is found in neutrophils and monocytes but not in lymphocytes, erythrocytes, or platelets. Moderately elevated levels of muramidase have been reported in patients with leukopenia associated with megaloblastic anemia secondary to vitamin B12 deficiency. Perillie and associates 22 believed that elevation of muramidase was secondary to increased turnover of granulocyte precursors in the marrow. Muramidase is generally elevated in chronic granulocytic leukemia and acute granulocytic and monomyeloblastic leukemias, although some low values have been reportedY 3, 24 In the present case, the
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increased m u r a m i d a s e activity is p r e s u m e d to represent n e u t r o p h i l destruction by the a n t i n e u t r o p h i l antibody. We are indebted to Stuart C. Finch, M.D., Professor of Hematology, Yale University, for performing the muramidase assays. REFERENCES
1. Luhby, A. L., and Slobody, L. B.: Transient neonatal agranulocytosis in two siblings: Transplacental isoimmunization to a leukocyte factor? Quart. Rev. Pediatr. 11: 163, 1956. 2. Lalezari, P., Nussbaum, M., Gelman, S., et al.: Neonatal neutropenia due to maternal isoimmunization, Blood 15: 236, 1960. 3. Braun, E. H., Buckwold, A. E., Emson, H. E., and Russell, A. V.: Familial neonatal neutropenla with maternal leukocyte antibodies, Blood 16: 1745, 1960. 4. Hitzig, W. H., and Gitzelmann, R.: Transplacental transfer of leukocyte agglutinins, Vox Sang. 4: 445, 1959. 5. Rossl, J. P., and Brandt, I. K.: Transient granulocy~openia of the newborn associated with sepsis due to Shigella alkalescens and maternal leukocyte agglutinins, J. PEmATR. 56: 639, 1960. 6. Lalezari, P., and Bernard, G. E.: An isologous antigen-antibody reaction with human neutrophiles, related to neonatal neutropenia, J . Clin. Invest. 45: 1741, 1966. 7. Lalezari, P., Murphy, G. B., and Alien, F. H., Jr.: NB1, a new neutrophil-specific antigen involved in the pathogenesis of neonatal neutropenia, J. Clin. Invest. 50: 1108, 1971. 8. Lalezari, P., Thalenfeld, B., and Weinstein, W. J.: The third neutrophil antigen. Histocompatibility testing 1970, Copenhagen, 1970, Ejnar Munksgaards Forlag, p. 319. 9. van der Weerdt, Ch. M., and Lalezari, P.: Another example of isoimmune neonatal neutropenia due to anti-NA1, Vox Sang. In press. 10. Halvorsen, K.: Neonatal leukopenla due to fetomaternal leiakocyte incompatibility, Acta Paediatr. Scan& 54: 86, 1965.
Isoimmune neonatal neutropenia
11. Schumer, L.: Agranulozytose im Neugeborenenalter, Arch. Kinderheilkd. 147: 164, 1953. 12. Jensen, K. G.: Transplacental passage of leukocyte agglutinins occurring on account of pregnancy, Dan. Med. Bull. 7: 55, 1960. 13. Tygstrup, I., Ghristensen, H. E., Sorensen, B., and Zachau Christiansen, B.: Congenital sepsis presumably llsteriosis associated with neutropenia, Acta Pathol. Microbiol. Scand. 74: 223, 1968. 14. Payne, R.: Neonatal neutropenia and leukoagglutinins, Pediatrics 33: 194, 1964. 15. Terasaki, P. I., and McClelland, J. D.: Microdroplet assay of human serum cytotoxins, Nature 204: 998, 1964. 16. Kawamura, A., Jr.: Fluorescent antibody techniques and their applications, Baltimore and Manchester, 1969, University of Tokyo Press, Tokyo University Park Press. 17. Green, I., and Solomon, W.: Separation of human lymphocytes and monocytes using an "oil bottle," J. Clin. Pathol. 16: 180, 1963. 18. Walford, R. L., Gallagher, R., and Troup, G. M.: Human lymphocyte typing with isologous antlsera: Technical considerations and a preliminary study of the cytotoxlc reaction system, TranspIantation 3: 387, 1965. 19. Fahey, J. L., and McKelvey, E. M.: Quantitative determination of serum immunoglobulins in antibody-agar plates, J. Immunol. 94: 84, 1965. 20. Smolells, A. N., and Hartsell, S. E.: Determination of lysozyme, J. Bacteriol. 58: 731, 1949. 21. Lalezari, P., Boxer, L., and Yokoyama, M.: NA2, an allele to neutrophile-specific antigen, NA1. In preparation. 22. Perillie, P., Kaplan, S. S., and Finch, S. C.: Serum muramidase activity in megaloblastic anemia, N. Engl. J. Med. 277: 10, 1967. 23. Wiernik, P. H., and Serpick, A. A.: Clinical significance of serum and urinary muramldase activity in leukemia and other hematological malignancies, Am. J. Med. 46: 330, 1969. 24. Youman, J. D., Saarni, M. I., and Linman, J. W.: Diagnostic value of muramidase (lysozyme) in acute leukemia and preleukemia, Mayo Glin. Proc. 45: 219, 1970.