Neutrophil Antigens and Antibodies in the Diagnosis of Immune Neutropenias *f

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1 ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 19, No. 3 Copyright 1989, Institute for Clinical Science, Inc. Neutrophil Antigens and Antibodies in the Diagnosis of Immune Neutropenias *f PR EM A R. MADYASTHA, Ph.D.* and ARM AND B. GLASSMAN, M.D. Department o f Pathology and Laboratory Medicine,t Medical University o f South Carolina, Charleston, SC and Montefiore Medical Center, Bronx, NY ABSTRACT Neutrophil specific antigens (NA) are expressed exclusively on human neutrophils and were identified using alloantibodies. N eutrophil specific antigens are polymorphic, and several of them (NA1, NA2, NB1, NB2, N C I, ND1, NE1, and 9A), are thought to define genes at different loci. Feto-m aternal incompatibility of NA has resulted in alloimmune neonatal neutropenia. Also, NA are the target antigens for autoantibody production in infants and young children with autoimmune neutropenia of infancy and chronic idiopathic neutropenia in adults. Autoimmune neutropenia can occur secondary to several other diseases, including AIDS. Numerous assays are useful in detecting granulocyte antibodies in patients with neutropenia. Among these assays, granulocyte agglutination (GA) and granulocyte im m unofluorescence (GIF) are available in some clinical laboratories. Both IgG and IgM agglutinins are detected by GA: in addition, IgG, IgM, and IgA are detected by GIF. Im m une neutropenia (IN) occurs in all age groups. Originally thought to be rare, IN is being increasingly recognized in recent years. F urther investigations should lead to a greater understanding of the role of NA in immune neutropenias and to identify as yet unknown NA specificities. W ith the availability of reproducible and sensitive assays to d etect granulocyte antibodies and the increasing knowledge and understanding of various disease aspects of IN, proper diagnosis and appropriate clinical m anagem ent are being applied. * Presented at the Applied Seminar on Diagnostic Hematology Sponsored by the Association of Clinical Scientists, November 1988, Hartford, CT. t R ep rint requests to: Prem a R. M adyastha, Ph.D., D epartm ent of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC Introduction N e u t r o p h il A n t ig e n s a n d A n t ib o d ie s : H is t o r ic a l P e r s p e c t iv e s a n d N e w H o r iz o n s H um an granulocyte antigens are h e t erogenous with diverse properties. They /89/ $01.50 Institute for Clinical Science, Inc.

2 IMMUNE NEUTROPENIA 147 may be shared with other blood and tissu e cells (system ic) or d ev elo p exclusively on neutrophils (tissue specific). C lear-cu t exam ples of tissue-specific alloantigens lim ited to a single cell line a g e -th e neutrophils, w ere described by Lalezari et al while investigating the causes of neonatal neutropenia, and neutrophil specific agglutinating antibodies w ere successfully docum ented in the m aternal sera. 16 These antibodies, being IgG, cross the placenta and react with fetal neutrophils causing neonatal n eu tropenia. Feto-m aternal incompatibility in n eu tro p h il antigen system (NA) is responsible for this disorder w hich is known as alloim m une neonatal n eu tro p enia (ANN). 12 A lloim m une neonatal neutropenia is the neutrophil analog of hemolytic disease of the newborn. The paternally derived fetal leukocyte antigens actively cross the placenta during pregnancy35 and, when the m other lacks the antigens, cause alloim m unization. Forty percent of ANN is observed in the first born. 40 Lalezari et al investigated several fam ilies w ith infants afflicted with ANN, characterized the antibodies involved, and identified the NA that caused th e feto-m aternal incom patibility. 16 N eutrophil antigens are present in m a tu re g ra n u lo c y te s an d a re fu lly expressed in cord neutrophils. 27 The first neutrophil antigen identified is desig n ated as NA1, 12 th e le tte r N relating to neutrophils and the independent genetic loci being further described by alphabetic letters A, B, C, etc. Arabic num bers are used to define individual a lle le s. O th e r n e u tr o p h il a n tig e n s NA2, 13 NB1, 15 NB2, 17 and N C I14 were subsequently identified using sera of m others of infants with ANN. Several investigators have shown that NA are the targets for autoantibody production.24 Infants and young children less than two years of age have been shown to develop neutrophil specific autoantibodies. This disorder, known as a u to im m u n e n e u tro p e n ia of infancy (AINI), 19 is analogous to autoim m une hem olytic anemia. Although the m echanism of the autoantibody production is n ot well u nderstood, the clinical re le vances of these antibodies and th e antigens involved have been investigated. N eutrophil antigens NA1 and NA2 are frequently shown to be the target antigens in AINI. 5 6,7,31 Antigens ND139 and N E l 3 have b e e n id e n tifie d less fre q u en tly in th e sera of p a tien ts w ith autoim m une neutropenia. The routine screening of the sera of m ultiparous wom en for leukocyte antibodies has led to the identification of two antigens, 9A38 and M art. 11 The antigen 9A appears to be granulocyte specific and related to or identical with NB2. 17 M art is present not only on granulocytes b u t also on monocytes and T-lymphocytes. These antigens are described in tab le I. T h e ir gen e fre q u e n c ie s are reported in m any publications.5 N ew Specificities N e u t r o p h il S p e c i f i c A n t ig e n CN1 It is clear that in the 28 years since the discovery of neutrophil antigens few new neutrophil specific antigens have been detected. Technical problem s w ere a m ajor difficulty; b u t it was speculated that there might be only a few polymorphic genes that code for such antigens. 8 The majority of the NA were identified using m aternal sera of ANN infants. A lloim m une neonatal n e u tro p e n ia is considered to be a rare disease, although it is likely that mild cases go unnoticed. Since the examination of the blood of the neonate is not done routinely, th e diagnosis of n e u tro p e n ia is m ade usually b e c au se of in fe c tio n s in th e ch ild. A lloim m unization against neutrophils has been estim ated to be in the order of 0.1 percent4 to three percent40 in multi-

3 148 MADYATHSA AND GLASSMAN TABLE I Currently Recognized Neutrophil Antigens the Method of Identification, Source of the Antisera and Their Clinical Implications Antigens Source of Sera Clinical Implications Method of Identification N A1,NA2,NBl Maternal, ANN, AINI, GA, GIF infant AIN (A), TR NB2 Maternal ANN, TR GA NCI Maternal ANN GA, GIF NDl Adult AIN (A) GA, GIF NE1 Infant AINI GA, GIF 9A Multiparous - GA HGA-3 Multiparous, TR, IN GC, GIF multitransfused. AIN CNl Maternal ANN GA, GIF ANN = Alloirranune neonatal neutropenia AINI = Auto-immune neutropenia of infancy AIN(A) = Auto-immune neutropenia in adults TR = Transfusion reactions IN = Immune neutropenia GA = Granulocyte agglutination GIF = Granulocyte inununo-fluorescence GC = Granulocyte cytoxity CN1 = Tentative designation of a new antigen identified in Charleston parous women. In our investigations of newborns over a six m onth period, 20,28 ANN was found in 16 percent of neonatal patients with infection or sepsis. This represented two in live births (0.2 percent) and 1.5 percent of all adm issions to the neonatal special care unit. Incidentally, th e specificity d eterm in a tion of one of the m aternal sera led to the identification of a u nique n eu tro p h il antigen tentatively designated as CN This antigen is present in 31 percent of blacks and 1.5 percent in whites in data from o u r study.* As p o in te d o u t by Engelfriet et al, 8 the genes coding for NA all seem to have one allele with a very high and one allele with a very low frequency, su g g estin g ra th e r re c e n t mutation. Recently four additional and as yet unnam ed specificities belonging to th e N -series have been recognized in Lalezari s laboratory. 18 The CN1 would have gone unnoticed and unidentified if this infant w ith n e u tro p e n ia had not b e e n in v e s tig a te d by th e p r e s e n t * Submitted for publication. authors. M ore investigations of ANN cases will reveal o ther unidentified specificities and should lead to the expansion of NA polymorphism. M ethods Em ployed to D etect N eutrophil Antibodies B e n e f i t s, P it f a l l s and C l in ic a l A p p l ic a t io n s Several m ethods are useful in d etecting granulocyte antibodies. 5 They were developed based on the type of antigen and antibody interactions, the various m echanism s of antibody m ediated cell destruction, and the visible pattern by which the antigen-antibody interactions can be m easured. They fall into three major catagories. The first and second group of assays involve a prim ary recognition of antigen by antibody and their interactions result eith er in m icroscopically visible secondary reactions, as in the first group [granulocyte agglutination (GA), an d g ra n u lo c y te c y to to x ic ity (GC)], or in the alterations of th e functional capacity of normal neutrophils, as in the second group (opsonization, and inhibition of hexose m onophosphate shunt). The third group of assay systems are dependent only on prim ary recognition of antibody to th e cell surface antig e n s. T he b in d in g of a n tib o d y is m e a s u re d by a se co n d p ro b e (im - m u n o f l u o r e s c e n t e n z y m e - lin k e d im m unosorbent assays [ELISA] enzymelin k e d or ra d io -la b e le d a n tih u m a n im m unoglobulin assays). G r a n u l o c y t e A g g l u t in a t io n Macro, micro, and capillary leukoagglutination tech n iq u es w ere initially em ployed to d etect granulocyte antibodies. Among these three assays, the micro technique known as G A is sim ple, inexpensive, highly rep ro d u cib le, and is u se d in m any clin ical la b o ra to rie s.

4 IMMUNE NEUTROPENIA 149 Unlike red cell agglutination, G A is an active biphasic process involving p rimary interaction of antigen-antibody and cell m ovem ent which is tim e and tem perature dependent. This necessitates the use of viable cells and longer incubatio n c o n d itio n s. T h e a v a ila b ility o f Ficoll-H ypaque density gradients to isolate viable cells23 and th e use of disodium e th y le n e d iam in e te tra actate (EDTA) to prevent non-specific aggregation of granulocytes, greatly facilitate the w ider applicability of GA. T he IgG n eu tro p h il agglutinins in A N N, IgG an d IgM in A IN I, and autoimmune neutropenia (AIN) in adults are d etected by GA. G ranulocyte agglutination is not only useful in detecting the alio- and auto-antibodies, but also extrem ely sensitive in identifying the antibody specificity directed against the N-series antigens that are involved in these disorder. G ranulocyte agglutination is the only technique available to determ ine the neutrophil antigen p h e notype of patients or norm al donors. McCullough has shown that antibodies d etected by GA correlate b e tte r w ith clinical findings than those detected by G C G r a n u l o c y t e C y t o t o x ic it y Granulocyte cytotoxicity assays utilizin g c o m p le m e n t w e re su c c e ssfu lly employed by Thompson et al to detect granulocytotoxins in several p atien ts w ith granulocytopenia. 36 G ranulocyte cytotoxicity assays involve th e in teraction of cells and serum containing antibody capable of fixing complement, and, thus, activate the com plem ent cascade resulting in m em brane dam age. This allows the penetration of vital dyes like trypan blue. Live cells exclude the dye. By determ ining the ratio of live and dead cells, the strength of the antibody is m easured. A modification of this assay is the fluorochrom asia granulocytotoxicity involving double staining. T he utilization of diacetyl fluorescein to give green fluorescence to viable cells and ethidium brom ide to give red fluorescence to dead cells further enhances the reproducibility of this assay. This technique employs micro quantities of serum and cell suspensions and is less tim e consum ing. M ccullough et al have observed that IgM cytotoxins may not be clinically significant in A IN I G r a n u l o c y t e I m m u n o f l u o r e s c e n c e Granulocyte antibodies that bind to cell surface antigens without resulting in visible reaction patterns are detectable by immunofluorescent techniques (GIF) with the help of a fluorescent labelled second antibody, usually produced in anim als against h u m an im m unoglobulins. Granulocyte im m unofluorescence has been modified for the use of micro quantities of th e serum and is b eing em ployed in clinical la b o ra to rie s. 32 D esp ite th e g reat success of G IF, it involves the m anual reading of fluorescent positive cells u n d er a fluorescent microscope, and, thus, is tim e consum ing, tedious, and subjective. In recent years, the availability of flow cytometry has red u ced th e above draw backs of GIF.33 Recently, a granulocyte flow cytom etric micro m ethod (GFCy) has been developed by us that utilizes five to 1 0 jjli of the seru m. 29 O ur newly developed GFCy is extrem ely sim ple and highly reproducible in detecting IgG granulocyte antibody. It should be useful in detecting IgM and IgA antibodies.* U s e o f M u l t ip l e M e t h o d s Antibodies directed against N-series antigens are detected by GA and GIF, since th e m ajority of th e agglutinating * Manuscript is in preparation.

5 150 MADÏATHSA AND GLASSMAN antibodies have cell surface binding properties. O ur own data and th at of others have shown that in some cases of ANN, th e m aternal antibodies w ere detected only by G FC y,t GFCy is much m ore sensitive than GA, p articularly when GA antibodies have a low titer. It rem ains to be seen w hether of not antibodies detectable by GFCy are different from those detectable by GA. It has also been observed in several infants w ith A INI th at autoantibodies d etected by GFCy were stronger than those detected by GA. In some cases the autoantibodies w ere d e te c te d only by G F C y. 29 It is im portant that both GA and G IF (GFCy) are em ployed simultaneously to detect granulocyte antibodies. locytes, differentiation antigens, and those present in precursor cells are all know n to induce antibodies and are implicated in patients with im m une neutropenias. It is not known w hether the antibodies detected by different assays have similar specificity, have the same m echanism of cell destruction, or have sim ilar clinical relevance. 21 No single tech nique has thus far been found to d e te c t all clinically significant granuloc y te a n tib o d ie s. In v e stig a tio n s a re needed to employ various assays sim ultaneously to detect granulocyte antibodies in granulocytopenic disorders. E x p en sive, cumbersome, or those assays that can not be used widely should be eliminated. O t h e r N e u t r o p h il A n t ib o d y A ssays O ther less commonly used assays are Staphylococcus protein A (SPA) assays, ELISA, solid phase radioimmunoassays (RIA), Fab-anti-Fab assays, Avidin-Biotin assay, and the functional assays such as opsonization and hexose m onophosphate shunt inhibition.5 These assays require larger volumes of sera and cells and are technically more difficult to perform. The micro serological assays (GA, GIF, GFCy, and GC) have proved to be more useful, particularly for large scale population and family studies. Discussion A lth o u g h im m u n e n e u tro p e n ia is being increasingly recognized in recent years, the role of different granulocyte antigen system s involved in the granulocytopenic disorders are poorly u n d e r stood. N -se rie s a n tig e n s b e lo n g in g exclusively to neutrophils, G-series antigens common to all granulocytes, GMseries shared w ith monocytes and granut Unpublished data. D ia g n o s is, N a t u r a l H ist o r y a n d T r e a t m e n t In table II are given the details of the testing protocol to detect and identify th e g ran u lo cy te antib o d y in various D is o r d e r TABLE II Granulocyte Antibody Testing of Immune Neutropenias by Granulocyte Agglutination and Granulocyte Immuno-Fluorescence T e s t S era T e s t C e lls (G r a n u lo c y te s ) R e a c tio n s ANN Infant Father Positive Mother Infant* Positive Mother Negative Donorst Positive Transitory Infant Mother Positive Neonatal Mother Infant* Positive Neutropenia Father Pos/Neg AINI Infant Father Pos/Neg Mother Pos/Neg Infant* Positive Donorst Positive AIN Autologous Autologous* Positive (Adults) Donorst Positive Sera (maternal or from patients with history of transfusions)/should be tested for HLA antibodies and# if present, should be absorbed and then retested for granulocyte antibody. * If available t Selective ANN = Alloimmune neonatal neutropenia AINI =* Auto-immune neutropenia of infancy AIN = Auto-immune neutropenia

6 IMMUNE NEUTROPENIA 151 im m une neutropenias. The diagnosis of infants with ANN is made by the docum entation of neutrophil specific antibodies in the m other and the infant that react with paternal neutrophils and not w ith m ate rn a l c e lls. 30 A n tib o d ies to h u m an leucocyte antigens (HLA a n tibodies) th at in te rfe re w ith th e assay should be tested, and, if present, should be absorbed and the m aternal serum retested. The antibody may be identified in a cell panel. W hen the infant recovers from neutropenia, follow-up testing is useful to dem onstrate the clearance of antibody. Infants with ANN are usually born with neutropenia, but neutropenia may not be seen until a few days after birth. The reason for this is unknown. N eutropenia persists from two weeks to t h r e e m o n th s, s o m e tim e s lo n g e r d e p e n d in g upon th e stre n g th of th e m atern al antibody. T he n e u tro p e n ia resolves with the simultaneous clearance of antibody. These infants may rem ain asymptomatic, but with severe and persisting neutropenia, they may becom e s u s c e p tib le to in fe c tio n, in c lu d in g sepsis. Infections docum ented in infants with ANN are various forms of skin infections, omphalitis, otitis, fever, and respiratory and u rinary in fectio n. 16 The b acteria in v o lv e d a re m ostly stap h y lo co cci, G roup A, B-hem olytic streptococcus, and Echerichia coli. Deaths have been observed in septic infants. Since steroids are ineffective, affected infants need appropriate antibiotics for serious infections. Neonatal neutrophil transfusion has been shown to be beneficial in some patients w ith sepsis.2 Cross-m atch testing should be done with infant serum to select appropriate donor neutrophils. Plasm a exchange or the use of intravenous im m une globulin may be alternative treatm ents, although they have not been tried as yet. N eonatal n e u tro p e n ia may also be caused by the transplacental passage of m aternal neutrophil autoantibodies; this disorder is known as transitory n eutropenia. 37 This is docum ented by the presence of antibody in m aternal and infant sera reacting with m aternal and infant neutrophils and not with paternal cells. N eutropenia in the m other may be dem onstrated by a com plete blood count and differential. A lth o u g h th e e x act in c id e n c e is unknown, autoim m une neutropenia of infancy is th e m ost com m on form of chronic n eutropenia in infants. 19 The clinical symptoms are described in detail in our earlier reports. 7,24 Currently AINI is recognized by severe and persisting n e u tro p e n ia b e tw e e n four to seven m onths of age. A utoim m une n e u tro p e n ia of infancy is a self-lim iting d isorder, but it may become complicated by various form s of bacterial and fungal infections. It is diagnosed by the p resence of neutrophil specific autoantibodies in th e in fa n t se ru m an d th e ir absence in the m aternal serum. Since the autoantibodies are directed against alloantigens, th e infant serum reacts w ith e ith e r one or both of the paren t neutrophils when they share the same antigens. The antibody can be further identified by a cell panel. In general, these infants generally recover within one to four years, b u t AINI m ay contin ue beyond childhood. E p in ep h rin e and steroid stim ulation fail to increase the neutrophil count. The bone marrow m ay b e n o rm a l w ith a s ig n ific a n t decrease in m ature neutrophils. Since th e se infants are su sce p tib le to infections, antibiotics are necessary w hen indicated. In severe cases, IVIg has been successful. 1 Steroids have been found to give a transient effect in some cases. A utoim m une neutropenia m ay also occur in adults with chronic idiopathic neutropenia (CIN) or secondary to other autoim m une diseases, lym phatic malignancies, im m une deficiency diseases including A IDS, and thyroid disorders

7 152 MADYATHSA AND GLASSMAN (table III). The antibodies are generally detected by G IF assays. The antigens involved in these disorders are poorly understood. However, detection of granulocyte antibodies in the sera of these patients has proved helpful in u n d e r standing the cause of granulocytopenia and in instituting appropriate therapy for th e c lin ic a l m a n a g e m e n t o f th e s e p atien ts. Various form s of secondary im m u n e n e u tr o p e n ia s h a v e b e e n recently review ed by M ccullough.22 Sum m ary and Conclusions N eutrophil specific alloantigens are polymorphic. Progress in uncovering the polym orphism and identifying m any unknown antigens has been ham pered since the first discovery by Lalezari et al 28 years ago. Technical problem s in isolating viable granulocytes and the need for suitable, sensitive assays to achieve reproducible results greatly contributed to the discouragem ent faced by the initial investigators. W ith the availability of TABLE III Granulocyte Antibodies in Primary and Secondary Immune Neutropenias Immune N e u tr o p e n ia s (A n tib o d y M e d ia te d ) Primary I. Congenital (alloimmune) (a) ANN (b) Transitory neutropenias II. Acquired (auto-immune) (а) Idiopathic (i) AINI (ii) CIN or AIN Secondary III. Associated with systemic or other diseases (1) Hematologic diseases (2) Collagen vascular diseases (3) Lymphoproliferative diseases (4) Lymphatic malignancies (5) Immunodeficiancy diseases, AIDS (б ) Thyroid diseases IV Drug induced ANN = Alloimmune neonatal neutropenia AINI = Auto-immune neutropenia of infancy CIN = Chronic idiopathic neutropenia AIN = Auto-immune neutropenia AIDS = Acquired immune deficiency syndrome Ficoll-H ypaque density gradients to isolate pure and viable granulocytes and GA and G IF techniques to d etect accura te ly th e g ran u lo cy te a n tib o d ie s, a renew ed enthusiasm has been shown in recen t years. F urtherm ore, the availability of flow cytom etric assays replacing th e m an u al G IF te c h n iq u e s sh o u ld enhance th e understanding of the granulocytopenic disorders. C urrently, GA, G IF, and G FC y have b een shown to have im portant clinical usefulness for the diagnosis of ANN, AINI, CIN, and secondary AIN. In addition, application of these assays will lead to a b etter understanding of the role of granulocyte antibodies in non-hemolytic transfusion and pulm onary reactions and may lead to th eir routine use in diagnosing these reactions. Although some serological and pathophysiological aspects of g ran u lo cytes are analogous to red cells, the antibodies detected by various techniques seem to suggest that granulocyte serology is m uch m ore com plicated. Thus, there is a need for new er approaches to define various antigens, their biological and functional roles, and their clinical im plications. 5 The N-series antigens have im portant implications in ANN, AINI, and transfusion reactions. Identification of various polymorphic N-series antigens will lead to a greater understanding of their role in neonatal alloimmune and autoimmune neutropenias. These alloantigens have been shown to be useful as biological and genetic markers to evaluate the engraftm ent of donor bone marrow in ABO and H LA com patible bone m arrow tra n s plantation. 34 Investigations are needed to support these initial observations. Although the role of m aternal antibodies in infants with ANN is known, the clinical significance of p refo rm ed antibodies in subsequent pregnancies has not been well investigated. In several instances, m alform ation, prem aturity, and deaths have been shown to be asso

8 IMMUNE NEUTROPENIA 153 ciated am ong infants of m others w ith leukocyte a n tib o d ie s. 910,16 Recently, Lalezari et al recorded a 19 percent incidence of abortion in his laboratory but indicated that his study lacked the evidence to support a cause and effect relationship. 18 In our small series of infants with ANN, prem aturity and/or low birth weight was associated in many cases.20 W ith the availability of new er m ethodologies and w ider applications, a b etter understanding of the NA and NAbs in ANN cases should result in more definitive diagnosis and therapy. Acknowledgments Thanks are extended to Ms. Kathleen Guy MT (ASCP) for technical assistance and for additional help in preparing the manuscript. References 1. B u s s e l, J., La leza r i, P., H ilg a r tner, M., Part in, J., F ik r ig, S., O M alley, J., and Baran- DERN, S.: Reversal of neutropenia with intraven o u s g a m m a g lo b u lin in a u to im m u n e neutropenia of infancy. Blood 6 2 : , C h r is t e n s e n, R. D., Ro t h s t e in, G., Anstall, H. B., et al.: Granulocyte transfusion in neonates with bacterial infection, neutropenia and depletion of m ature neutrophils. Pediatrics 7 0 :1-6, C laas, F. H. J., L an g er a k, J., Sa b be, L. J. M., and van R o o d, J. J.: N E 1 : A new neutrophil specific antigen. Tissue Antigens 2 3 : , C lay, M., K u n e, W., and M c C u l l o u g h, J.: Serological examination of postpartum sera for the presence of neutrophil specific antibodies. Transfusion 22:616, C lay, M. E., and K l in e, W. E.: Detection of granulocyte antigens and antibodies: C urrent perspectives and approaches. Transfusion Therapy. Garraty, G., ed. Arlington, VA, American Association of Blood Banks, 1985, pp C o nw ay, L. T., C lay, M. E., K l in e, W. E., Ra m sey, N. K. C., Kr iv it, W., and M c C u l lo u g h, J.: Natural history of primary autoimmune neutropenia in infancy. Pediatrics 79: , D ucos, R., M adyastha, P. R., Wa rrier, R. P., G lassm a n, A. B., and Shir ley, L. R.: Neutrophil agglutinins in idiopathic chronic neutropenia of early childhood. Am. J. Dis. Child. 140: , E n g e l f r ie t, C. P., T e t t e r o o, P. A. T., Van d e r Ve e n, J. P. W., W e r n e r, W. F., Van d e r p l a s-v an D a l e n, C., a n d v o n d e m B o r n e, A. E. G., Kr.: Granulocyte specific antigens and methods for their detection. Advances in Im munobiology: Blood Cell Antigens and Bone Marrow Transplantation. New York, Alan R. Liss, Inc., Prog. Clin. Biol. Res., vol. 149, 1984, pp H a l v o r sen, K.: Neonatal leucopenia due to fetom aternal leucocyte incom patibility. Acta Pediat. Scand. 54:86-90, JE N SE N, G.: Transplacental passage of leucocyte agglutinin occurring on account of pregnancy. Danish Med. Bull. 7:55-58, K l i n e, W. E., P r e s s, C., C l a y, M. E., K e a - S H E N -SC H N E L L, M., H A C K EL, E., and McCUL- LO U GH, J. J.: Three sera defining a new granulocyte-monocyte-t-lymphocyte antigen. Vox Sang. 50: , L a leza r i, P., N u sba u m, M., G e l m a n, S., etal.: Neonatal neutropenia due to m aternal isoimmunization. Blood 25: , L a leza r i, P., and Bern a r d, J. E.: An isologous antigen-antibody reaction with human neutrophils related to neonatal neutropenia. J. Clin. Invest. 45: , L a leza r i, P., T h a l e n f e l d, B., and W e in s t e in, W. J.: The third neutrophil antigen. Histocompatibility Testing. Terasaki, P. E., ed. Baltimore, Williams & Wilkins Co., 1970, pp L a l e z a r i, P., M u r ph y, G. B., and A l l e n, F. H.: NB1. A new neutrophil antigen involved in the pathogenesis of neonatal neutropenia. J. Clin. Invest. 50: , L alezari, P., and Ra d el, E.: Neutrophil specific antigens: Immunology and clinical significance. Semi. Hematol. 22: , L a leza r i, P., P etr o so v a, M., and J ia n g, A. F.: NB2, An allele of NB1 neutrophil specific antigen: Relationship to 9A. Transfusion 22:433, L a l e z a r i, P.: G ranulocyte antigen systems. Im m unohem atology. E ngelfriet, C. P., van Logherm, J. J., and von dem Borne, A. E. G., Kr., eds. Amsterdam Elsevier Scientific Publishing B. V., 1984, pp L a leza r i, P., K h o r s h id i, M., and P etroso v a, M.: A utoim m une neutropenia of infancy. J. Pediatr. 109: , L ev in e, D. H., and M adyastha, P. R.: Isoimm une neonatal neutropenia. Am J. Perinatol. 3: , M cc u l l o u g h, J., C lay, M. E., P r ie s t, J. R., J e n s e n, N. J., L a u, S., N o r e e n, H. J., Krtvit, W., and L a leza r i, P.: A comparison of methods for detecting leukocyte antibodies in autoim m une neutropenia. Transfusion 22: , M cc u l l o u g h, J. J.: The clinical significance of granulocyte antibodies and in vivo studies of the fate of granulocytes. Current perspectives and approaches. Transfusion Therapy. Garraty, G., ed. A rlington, VA: American Association of Blood Banks, 1985, pp M adyastha, P., M adyastha, K. R., W a d e, T., and L e v in e, D.: An improved method for rapid layering of Ficoll-Hypaque double density gra

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