J Clin Pathol 1992;45:431-434 431 Autoimmune of infancy

E G H Lyall, G F Lucas, 0 B Eden J Clin Pathol: first published as 10.1136/jcp.45.5.431 on 1 May 1992. Downloaded from

Abstract from 0-10 years, we have seen five children Aim: Assessment of the clinical and with serologically confirmed ANI, suggest- haematological course of autoimmune ing an annual incidence of approximately neutropenia ofinfancy (ANI) in a defined 1/1000 000 in this population. All ofthese cases childhood population in the south east of have so far followed the classic benign course of Scotland. the condition. Methods: From January 1986 to February 1991 all children presenting with persis- Methods tent neutropenia were examined From January 1986 to February 1991 all chil- serologically for evidence of anti- dren presenting outside the neonatal period granulocyte . The clinical with isolated neutropenia (absolute neutrophil course of those children found to have count (ANC) <1-5 x 109/1), were seen by anti-granulocyte antibodies was then OBE. A full history and examination were closely monitored. carried out and particular attention was paid to Results: During the study period five previous illnesses, administered, children had serologically confirmed and family history. ANI, giving an annual incidence of Screening investigations to ascertain the approximately 1/100 000 in this popula- cause ofthe neutropenia were carried out when tion. All ofthese cases followed the classic the ANC had not returned to normal within 2- benign course of the condition. The 3 weeks. These included: serial full blood presenting illnesses were mild, often with counts; bone marrow aspiration; serum superficial skin sepsis and the initial immunoglobulin (G, A, M) and complement absolute neutrophil count (ANC) ranged concentrations; lymphocyte subsets; auto- from 0-00-0-87 x 10'/I. All have remained antibodies; viral serology; B12 and folate con- well with no serious infections. Two chil- centrations; chromosomal analysis; blood and dren attained a normal ANC after 14 and urine amino acid screening; and anti- 24 months respectively, the others granulocyte antibodies. The children found to http://jcp.bmj.com/ currently remain neutropenic. have anti-granulocyte antibodies and other Conclusions: Autoimmune neutropenia features consistent with the diagnosis of ANI of infancy is a condition which rests on a form the basis of this study. serological diagnosis. It follows a chronic Two techniques were used to detect gran- benign course and all children eventually ulocyte reactive antibodies; a microplate attain a normal ANC. The level of anti- modification of the granulocyte immuno- granulocyte in the serum often fluorescence test (GIFT)'0 and the granulocyte on October 2, 2021 by guest. Protected copyright. begins to wane prior to improvement in chemiluminescence test (GCLT)."' A micro- the ANC and can give an indication of plate modification of the lymphocyte im- when recovery will begin to occur. munofluorescence test (LIFT)'2 was used to detect lymphocyte reactive (anti-HLA) anti- Autoimmune neutropenia of infancy (ANI), bodies. Results of the immunofluorescence first described by Lalezari in 1975,' is a benign tests were scored as negative (0) or as graded condition which has become increasingly positives ranging from W + (weak +) to 4+. characterised as reliable techniques for detect- The GCLT is a functional assay which Department of ing anti-granulocyte antibodies have become measures the response of human monocytes to Haematology, Royal more available. In Lalezari et al2 The results of the Hospital for Sick widely 1986, opsonised granulocytes. Children, Sciennes reported 121 infants with this disorder, 119 of GCLT were expressed as a ratio (the opsonic Rd, Edinburgh EH9 whom were demonstrated to have anti- index) between the response of monocytes to 1LF granulocyte antibodies. Smaller studies'7 have granulocytes incubated with test serum and the E G H Lyall and 0 B Eden confirmed both the clinical serological response of monocytes to granulocytes International Blood features of the condition and it seems likely incubated with serum from untransfused male Group Reference that the condition previously termed chronic (group AB) donors. The normal range of the Laboratory, South benign neutropenia is equivalent to ANI.289 GCLT is 1-0 +0 3 (3 SD). Western Regional Transfusion Centre, The exact incidence of ANI is unknown but The techniques were performed as Southmead Rd, because of its benign nature, the disorder may previously described except for the following Bristol BS10 SND be more common than is suggested by the modifications: granulocytes and mononuclear G F Lucas available literature. cells used in both techniques were isolated Correspondence to: From 1986 to February 1991, within from EDTA anticoagulated blood from normal E G H Lyall January the Lothian of Scotland, which has a donors using a double density gradient tech- Accepted for publication region 30 October 1991 childhood population of just over 96 000 aged nique. Red cells contaminating the granulocyte 432 Lyall, Lucas, Eden

Table 1 Cases of autoimmune neutropaenia of infancy in south east Scotland (1986-1991)

Age Serum Bone marrow Increased Time to at Initial Lymphocyte IgG infections normal onset ANC TWC granulocyte subsets IgM M:E Maturation during ANC Cases (months) Sex Presenting illness TWC diff (%) serology T4:T8 IgA ratio arrest ANI (months) J Clin Pathol: first published as 10.1136/jcp.45.5.431 on 1 May 1992. Downloaded from 1 13 F 0Q N-0 01-10-0 9-83:1 N 6-5:1 NO NO 24 6-2 L-86 Antig IgG 3 + no specific target Ag identified 2 8 M Fever and skin 0-19 N-4 01-12-8 4-6:1 N 8 5:1 NO NO Not yet sepsis 4-8 L-68 Antig IgG 3 + recovered M-26 antig IgM W + after myelo-I no specific 19 months meta-1 target Ag identified 3 8 M Skin sepsis, Q-8 N-10 OI-8-6 4-3:1 N 9-4:1 NO NO Not yet lymphadenitis, 8-7 L-62 Antig IgG 3 + recovered upper respiratory M-27 antig IgM 0 after tract infection E-1 target Ag NAl 11 months 4 11 F Skin sepsis QJ01 N-3 01-15-9 2-7:1 N 2-5:1 NO NO Not yet 4-4 L-80 Antig IgG 2 + recovered M-12 Antig IgM 0 after E-4 target Ag NAb 7 months BI 5 7 F Fever, vomiting 0Q0 N-o OI-1-2 2-7:1 N 1-33:1 NO NO 14 oral/anal 10-9 L-91 Antig IgG 1+ candidiasis M-9 Antig IgM NEG target Ag NAb

ANC = absolute neutrophil count x 10'/l; TWC = total white count x 109/l; TWC percentage differential: N = neutrophils; L = lymphocytes; M = monocytes; Myelo = myelocytes; Meta = metamyelocytes; E = eosinophils; B = basophils. 01 = opsonic index; Antig = antigranulocyte; Ag = antigen; N = normal.

fraction were lysed using a hypo-osmolar solu- present. None of these children has suffered tion of ammonium oxalate. Granulocytes were any severe infections or any great increase in inactivated by incubation at 52°C for 2 minutes minor infections during the course of the prior to incubation with test serum and were neutropenia. Case 1 has been closely followed not treated with paraformaldehyde. through to complete recovery and the clinical Target granulocytes and lymphocytes were course of this patient demonstrates the typical obtained from normal donors typed for the features of this disorder (figure): neutrophil specific antigens NAI, NA2, and (1) The total white blood count always NB1, the tissue antigen 5b and HLA-A and B remained within normal limits. antigens. Granulocyte immunofluorescence (2) The ANC remained below 0 5 x 109/l for was used to determine NA1, NA2, NB1, and 5b the first 12 months. phenotype and lymphocytotoxicity was used to (3) The level of anti-granulocyte antibody http://jcp.bmj.com/ determine HLA-A and B phenotypes. activity slowly decreased during the course of the disease. (4) Temporary increases in peripheral Results neutrophil counts were observed with the During the study period, within the Lothian development of Campylobacter enteritis and an region of Scotland which has a childhood urticarial illness. population of just over 96 000 aged from 0-10 (5) The incidence of infections (five upper on October 2, 2021 by guest. Protected copyright. years, five children presented with serologically respiratory tract infections, two fevers, one confirmed ANI (table 1). Three were female episode of mouth ulcers and one episode of and the median age at diagnosis was 8 months diarrhoea, in addition to the two illnesses (range 7-13 months). They all presented with described above) during the 24 month period of minor illnesses, and in three there was skin neutropenia could not be considered excessive sepsis. All had absolute neutropenia with ANC for a child of this age. ranging from 0 00-0-87 x 109/1, case 4 also had iron deficiency anaemia. Bone marrow examin- Discussion ation of these patients confirmed an increased The five patients described in this report have M:E ratio with "left shift" ofthe myeloid series clinical and laboratory features (table 1) but no specific arrest patterns. In cases 1-3 entirely consistent with the previous reports of there appeared to be an initial elevated T4:T8 ANI.2-7 These five cases, from a defined geo- lymphocyte subset ratio which normalised with graphical area, suggest that the annual time. Serum immunoglobulin concentrations incidence ofANI is in the order of 1/100 000 of were normal. the childhood population, although, because of The anti-granulocyte antibodies detected the relatively benign course ofthe disorder this were of the IgG class but weakly reactive IgM figure is likely to be an underestimate. antibodies were also detected in one patient. In The differential diagnosis of neutropenia in three patients the anti-granulocyte antibodies infancy was recently reviewed and an algorithm were specific for the NAI antigen. Anti-HLA for assessment of such infants described.'3 The antibodies were not detected. many causes of infant neutropenia can be In cases 1 and 5 the neutropenia has resolved divided into congenital and acquired (table 2). after 14 and 24 months respectively; in the The congenital syndromes may be associated other three cases neutropenia persists at with phenotypic abnormalities, immuno- Autoimmune neutropenia ofinfancy 433

1gG 3+ 2+ 1+ W+ Granulocyte normal limits. Monocytosis or eosinophilia 01 100- 4.5 167 2-7 serology may occur but do not seem to affect the rate of infection. Anaemia has been reported either -a---_ Total white cell count x 109/l due to coincidental iron deficiency or as a result

-- Absolute neutrophil of recurrent infections. In a study of 121 J Clin Pathol: first published as 10.1136/jcp.45.5.431 on 1 May 1992. Downloaded from 4- c count x 109/l children,2 the median age of diagnosis was 8 n D + V Diarrhoeal illness 0 URTIC Urticarial illness months (range 3-30) and the female to male 0) IgG Anti-granulocyte ratio was 6:4. All children had ANCs of less a) IgG than 1-5 x 109/l but in many cases neutrophils l_ 01 Opsonic index were undetectable. None of the children had any other serious illnesses; there was no evidence of other autoimmune diseases; no previous blood transfusions; and no drug ingestion that might have given rise to antibody Months formation. Most ofthe children presented with Typical course of autoimmune neutropenia ofinfancy (case 1). minor infections such as , gin- givitis, respiratory tract infections, gastroen- teritis, or skin sepsis. Diagnosis was often made only after the child had suffered several infec- deficiency, or be pure neutrophil abnor- tions or an incidental blood count revealed malities. This group of patients tends to neutropenia. More seriously affected children present with serious infections in the very early have presented with pneumonia, central sepsis, months of life, with the exception of cyclical or (one study showed an increase in neutropenia which may present later with less vulval abscesses in girls, caused particularly by serious infections, often superficial, and often Pseudomonas aeruginosa.9). No cases of ANI so with a positive family history."4 Clues to the far described have died as a consequence of acquired causes may be elicited from the his- their disease. tory and consequent appropriate investiga- Longitudinal studies'> of infants with ANI tions. have demonstrated the clinical course of the The diagnosis of ANI depends on history disorder. The median duration of disease is and examination and upon positive serological around 30 months (range 6-60 months) but results, although these will not be available at 95°% of children recover by 4 years of age.2 the time of presentation. Autoimmune Thus far no children who have recovered from neutropenia has been reported in children and this condition have been described with any adults infected with the human immuno- secondary recurrence of neutropenia or other deficiency virus, so evidence for this infection autoimmune problems. Neutropenia in ANI should also be sought.516 can be profound but increased neutrophil The bone marrow findings in ANI are not production can occur during infections, diagnostic and any process causing increased especially as antibody levels decrease. Some http://jcp.bmj.com/ synthesis (for example, infection) or increased children show an increased level of minor destruction of neutrophils (for example, allo- infections during the course of the disease immune neutropenia) will give a similar pic- while others remain well. Generally, no treat- ture. Marrow examination excludes neutro- ment, apart from efficient personal hygiene and penia caused by proliferative disorders or occasional (usually oral) for minor aplasia. infections, is required. Children who are subject to more serious recurrent infections on October 2, 2021 by guest. Protected copyright. Clinical features of autoimmune may be treated with intravenous human neutropenia ofinfancy immunoglobulin (IVIG) to induce temporary All patients have severe neutropenia at presen- increases in the ANC. IVIG treatment has tation; the ANC is usually less than 0-5 x 10s/1 been used effectively for acute infections, sur- but the total white blood count is always within gical interventions, and for longer term protec- tion until there is spontaneous improvement m Table 2 Causes ofneutropenia in infancy the ANC.17-19 Bone marrow aspiration has been performed Congenital Acquired in almost all children with ANI so far reported, Neutrophil abnormality only Infection related primarily to exclude other causes of Kostmann's syndrome Viral neutropenia. In ANI, the bone marrow tends to (severe congenital neutropenia) Bacterial Cyclical neutropenia Rickettsial show an active picture with no abnormalities of Myelokathexis Fungal cell morphology. There is an increased myeloid Lazy leucocyte syndrome Parasitic to erythroid ratio (M:E'ratio) and some patients Associated with lymphocyte abnormalities Immune mediated Reticular dysgenesis Iso-immune have been reported to show arrest of matura- Agammaglobulinaemia (materno-fetal, multi-transfusion) tion ofthe myeloid series at various stages,5 but X-linked agammaglobulinaemia Auto-immune Dysgammaglobulinaemia (ANI, SLE, HIV) this does not correlate with any difference in Associated with other conditions Marrow infiltration clinical severity.4 Adrenaline and hydrocort- Cartilage hair hypoplasia Leukaemia Schwachmann syndrome Lymphoma isone stimulation ofthe bone marrow can cause Fanconi's syndrome Neuroblastoma increased release of neutrophils into the cir- Chediak-Higashi syndrome Amino acidopathies culation but the response is usually poor or Marrow aplasia negligible.25 These findings are not diagnostic Vitamin deficiency Vitamin B12 for ANI, which can only be confirmed by the Folate presence of anti-granulocyte antibodies. Copper Lalezari et al2 found that serum immuno- Drugs and toxins globulin levels were normal in patients with 434 Lyall, Lucas, Eden

ANI but, in a study of 14 patients, Bussel et a14 recovering a normal ANC by at least 5-6 years found that one had elevated total immuno- of age. Our study suggests that the disease has globulin levels and four had elevated IgM an annual incidence in the order of 1/100 000 of levels. In four patients total IgG levels were the childhood population. The diagnosis of normal but decreased levels of IgG2 and IgG3 ANI depends on serological confirmation of were found. The T4:T8 lymphocyte ratio was anti-granulocyte antibodies. Bone marrow J Clin Pathol: first published as 10.1136/jcp.45.5.431 on 1 May 1992. Downloaded from within normal limits in 10/121 cases tested.2 aspiration is mandatory to exclude other more Assessment of neutrophil function has not serious causes of neutropenia. shown any conspicuous abnormality, although Children symptomatically more severely only very small numbers of patients have been affected can be treated with IVIG to improve examined.4 Serum granulocyte colony- their neutrophil count; this can be given for an stimulating factor levels are elevated in chil- acute crisis or regularly to maintain a higher dren with ANI and fall as the ANC improves, ANC. The level of anti-granulocyte antibody implying an autonomous feed back response in in the serum often begins to wane prior to this condition.20 improvement in the ANC and this can give an indication ofwhen recovery will begin to occur. Granulocyte serology There are no known sequelae ofthis condition. Anti-granulocyte antibodies detected in cases 1 Lalezari P, Jiang AF, Yegen L, Santorineou M. Chronic of ANI are usually of the IgG class, but may autoimmune neutropenia due to anti-NA2 antibody. N EnglJ Med 1975;293:744-7. occur together with IgM antibodies.2 These 2 Lalezari P, Kharshidi M, Petrosova M. Autoimmune antibodies often have specificity for the NAI neutropenia ofinfancy. J Pediatr 1986;109:764-9. 3 Conway LT, Clay ME, Kline WE, RamsayNKC, Krivit W, antigen, although other specificities have been McCullough J. Natural history of primary autoimmune reported.2" In Lalezari's study,2 antibodies neutropenia in infancy. Pediatrics 1987;79:728-33. 4 Bussel JB, Abboud MR. Autoimmune neutropenia ofchild- specific for NAI (or which had NAl-related hood. CRC Crit Rev Oncol Hematol 1987;7:37-51. activity) were identified in 17/121 patients, 5 Onisawa S, Ichimura T, Saito K, Sekine I. Five cases of autoimmune neutropenia in infancy or early childhood. while in another study7 anti-NAl antibodies Acta Paediatr Jpn [Overseas Ed] 1989;3:587-94. were identified in 17/36 patients. 6 Ducos R, Madyastha PR, Warnier RP, Glassman AB Shirley LR. Neutrophil agglutinins in idiopathic chronic Many different techniques have been des- neutropenia of early childhood. Am J Dis Child 1986; cribed for the detection of granulocyte reactive 140:65-8. 7 McCullough J, Clay M, Press C, Kline W. In: Granulocyte antibodies,7 but in the first international serology: a clinical and laboratory guide. Chicago: granulocyte serology workshop,' the granu- American Society of Clinical Pathologists Press, 1988. 8 Madyastha PR, Fudenberg HH, Glassman AB, Madyastha locyte agglutination test and the granulocyte KR, Smith CL. Autoimmune neutropenia in early immunofluorescence test were the most widely infancy: a review. Ann Clin Lab Sci 1982;12:356-67. 9 Jonsson OG, Buchanan GR. Chronic neutropenia during used and reliable techniques. The most succes- childhood. A 13-year experience in a single institution. sful laboratories in this workshop used more Am JDis Child 1991;145:232-5. 10 Verheught FWA, von dem Borne AEGKr, van Noord- than one technique for the investigation of Bokhorst JC, Engelfriet CP. Autoimmune neutropenia: antisera and it is clear that no single technique The detection of granulocyte autoantibodies with can detect all types of granulocyte reactive immunofluorescence test. Br JHaematol 1978;39:339-50. 11 Hadley A, Holbum AM. The detection of anti-granulocyte http://jcp.bmj.com/ antibodies. antibodies by chemiluminescence. Clin Lab Haematol 1984;6:351-61. The initiating process in the development of 12 Decary F, Vermeulen A, Engelfriet CP. A look at HL-A anti-granulocyte antibodies is still unknown. It antisera in the indirect immunofluorescence technique (IIFT). In: Amos DB, Van Rood JJ, eds. Histocompati- may be speculated that ANI results from an bility testing. Copenhagen, Munksgaard: 1975:380-90. unusual response to common infection, as has 13 Roskos RR, Boxer LA. Clinical disorders of neutropenia. Pediatr Rev 1991;12:208-12. been postulated in the pathogenesis of acute 14 Wright DG, Dale DC, Fauci AS, Wolff SM. Human cyclic idiopathic thrombocytopenic purpura ofchild- neutropenia: clinical review and long term follow up of patients. Medicine 1981;60:1-13. on October 2, 2021 by guest. Protected copyright. hood.23 The high incidence of antibodies 15 McCance-Katz EF, Hoecker JL, Vitale NB. Severe specific for NAI in this disorder may also neutropenia associated with anti-neutrophil antibody in a patient with acquired immunodeficiency syndrome provide a key to the understanding of this related complex. Ped Infect Dis J 1987;6:417-8. condition. Studies into the nature of the 16 Lobut JB, Reinert P, Kohout G, Lemerle S, Bernaudin F, Cohen R. Autoimmune neutropenia disclosing AIDS in a neutrophil antigens and the receptors on the child. Ann Med Intern (Paris) 1987;138:416-8. neutrophil surface2425 have shown that the 17 Bussel J, Lalezari P, Fikrig S. Intravenous treatment with gamma globulin of autoimmune neutropenia of infancy. neutrophil specific antigens NAl and NA2 are J Pediatr 1988;112:298-301. closely bound to the neutrophil Fc y receptor 18 Hilgartner MW, Bussel J. Use of intravenous gamma- globulin for the treatment of autoimmune neutropenia of 111 (CD16) for immunoglobulin G (FcR 111). childhood and autoimmune hemolytic anaemia. Am JMed FcR 111 is a low affinity receptor for IgG and 1987;83 (Suppl 4A):25-9. 19 Kurtzberg J, Friedman HS, Chaffee S, et al. Efficacy of binds IgGi and IgG3 dimers only. Expression intravenous gamma globulin in autoimmune mediated ofthis receptor by neutrophils can be increased pediatric blood dyscrasias. Am J Med 1987;83 (Suppl 4A):4-9. by cytokines released during the inflammatory 20 Mizuno Y, Hara T, Nagata M, et al. Serum granulocyte process. The neutrophil can also secrete FcR colony-stimulating factor levels in chronic neutropenia of infancy. Pediatr Hem Oncol 1990;7:377-81. 111 in soluble form when stimulated. This 21 McCullough J. Autoimmune granulocytopenia. In: Engel- receptor may not only have a role in the cellular friet CP, van Loghem JJ, von dem Borne AEG Kr, eds. Research monographs in immunology. Vol 5. Immuno- immune system inducing phagocytosis and haematology. Amsterdam: Elsevier, 1984:257-4. antibody dependent cellular cytotoxicity but, 22 Lucas GF, Carrington PA. Results of the first granulocyte when act as a serology workshop. Vox Sang 1990;59:251-6. released, may ligand and 23 Lusher JM, Iyer R. Idiopathic thrombocytopenic purpura modulate IgG production by lymphocytes.26 in children. Semin Thromb Hemost 1977;33:175-99. 24 Rothko K, Kickler TS, Clay ME, Johnson RJ, Stroncek DF. The possible interrelationship(s) between the Immunoblotting characterisation of neutrophil antigenic NA1/NA2 antigens, the FcR III receptor, and targets in autoimmune neutropenia. Blood 1989;74: the in 1698-703. anti-neutrophil antibodies ANI remains 25 Huizinga TWJ, Kleijer M, Tetteroo PAT, Roos D, von dem to be elucidated. Bome AEG Kr. Biallelic neutrophil NA-antigen is associated with a polymorphism on the phosphinositol- linked Fc gamma Receptor 111 (CD16). Blood 1990; Conclusion 75:213-7. is a a 26 Huizinga TWJ, Roos D, von dem Borne AEG Kr. Neutro- ANI benign condition which follows phil Fc gamma receptors: a two way bridge in the immune relatively chronic course with all children system. Blood 1990;75:1211-4.