PEDIATRICS Vol. 106 No. 2 August 2000, pp. 340-342

EXPERIENCE AND REASON:
Neonatal Alloimmune Neutropenia in Premature Monozygous Twins

	ABSTRACT

Top Abstract Introduction Discussion References

Alloimmune neonatal neutropenia (ANN) is an uncommon but potentially life-threatening disorder of the neonate and young infant. Hematologically, the mother's peripheral neutrophil count is normal. However, the passive transfer of maternal immunoglobulin G neutrophil-specific antibodies and the subsequent sensitization of fetal neutrophils can result in severe neutropenia in the neonate. Generally, ANN is a self-limiting condition, but with severe bacterial infection, mortality can be high. We present the clinical features of monozygous twins delivered at 33 weeks' postconception with this condition. This case report is unique in that it occurred in twins born prematurely and was attributable to antibodies against 2 neutrophil-specific antigens, NA1 and NB1. A brief review of the diagnosis, management, and treatment of ANN is presented.

Alloimmune neonatal neutropenia (ANN) is a syndrome with a pathogenesis analogous to that of hemolytic disease of the newborn and isoimmune thrombocytopenia.¹ Unlike hemolytic disease of the newborn, ANN can occur in the firstborn child.² Fetal neutrophil antigens, which are foreign to the pregnant mother but are inherited from the father, provoke the formation of maternal antibodies. These antibodies are of the immunoglobulin G (IgG) class and readily cross the placenta.³ We report monozygous twins delivered prematurely who presented with neutropenia in which the cause was an isoimmune process attributable to anti-NA1 and anti-NB1 antibodies.

	CASE REPORT

Monozygous male twins were born to a 32-year-old, gravida 2, para 1 mother, whose first child was a healthy term infant without complications. At 33 weeks of pregnancy, the twins, weighing 840 g and 1170 g at birth, respectively, were delivered by cesarean section secondary to discordant growth and absent end diastolic arterial blood flow in twin B. The infants appeared to be healthy and physical examination revealed no abnormalities. Routine laboratory examinations showed severe neutropenia: total white blood cell counts 4100/mm³ and 2400/mm³, and absolute neutrophil counts (ANC) 123/mm³ and 120/mm³, for twin A and twin B, respectively. Other hematologic and biochemical profiles were initially within normal limits. Blood cultures were obtained and ampicillin and gentamicin initiated for the treatment of potential sepsis in premature neonates with neutropenia.

The infants received intravenous (IV) IgG (750 mg/kg) on the second day of life with a slight improvement in ANC (714/mm³ and 336/mm³, respectively). Because the neutropenia was severe, recombinant human granulocyte colony-stimulating factor therapy (rhG-CSF, 10 µg/kg/d, SQ) was initiated on the third day of life. After 3 days of therapy, the ANCs increased to 9089/mm³ and 4095/mm³, respectively. Blood cultures were negative and further evaluation of the neutropenia was undertaken including obtaining of samples for maternal antineutrophil antibody studies. The total leukocyte counts of the parents were normal.

Granulocyte typing, granulocyte cross-matching, and assays of serum to detect neutrophil-specific antibodies and immune complexes were performed at the Neutrophil Serology Reference Laboratory of the American Red Cross, St Paul, Minnesota. The granulocyte agglutination assay and the granulocyte immunofluorescence assay were both positive using maternal serum.

Human leukocyte antigen (HLA) antibodies can react in these assays, so a monoclonal antibody immobilization of granulocyte antigens assay (MAIGA)⁴ was performed to differentiate between HLA and neutrophil-specific antibodies. Specifically, patient serum was incubated with cells that are: 1) homozygous for NA1 and NA2, and 2) positive for NB1. The cells were then washed and incubated with monoclonal antibodies specific for the NB1 antigen, and CD 16 and CD 18 antigens. Results were considered positive if the absorbance was 3 times the negative control. In our patients, the MAIGA detected specificity for the NA1 and NB1 antigens.

At 3 weeks of age, the twins were again neutropenic with ANCs of 288/mm³ and 160/mm³, respectively, and a second 3-day course of rhG-CSF therapy was administered with the ANCs rising to 2376/mm³ and 4472/mm³, respectively. The hospital courses of the twins were otherwise unremarkable, and they were discharged home at 24 and 31 days of age.

At 5 weeks of life, the twins' ANCs again decreased to 696/mm³ and 650/mm³, and a third course of rhG-CSF therapy was initiated. At this time, they were managed on an outpatient basis receiving subcutaneous injections of rhG-CSF (total of 6 days) and weekly complete blood counts. The ANCs peaked at 9843/mm³ and 9720/mm³, with twin B's neutropenia resolving at this time (6 weeks of age). However, at 10 weeks of age, twin A's ANC again decreased to 616/mm³, for which he received rhG-CSF therapy every other day for a total of 3 doses. At this time twin A's neutropenia resolved, and both infants continued to have normal neutrophil counts without rhG-CSF therapy. Neither twin developed an infection during the period of neutropenia or rhG-CSF therapy. At 5 months of age, the twins were hospitalized for bronchiolitis, and both had normal neutrophil counts. They continue to be followed as routine pediatric patients.

	DISCUSSION

Top Abstract Introduction Discussion References

The neutrophil-specific antigensNA1, NA2, NB1, and NC1were first characterized by Lalezari et al⁵ in 1974. Other neutrophil-specific antigens include NB2, ND1, and NE1.^2,6,7 The N-series antigens are independent of other antigens that are present on the surface of the neutrophil, such as the HLAs⁸ and red cell antigens.⁹ Neutrophil-specific antigens have been identified on mature adult neutrophils, myeloid precursors,¹⁰ and cord blood neutrophils.¹¹ The specificity of ANN involves mainly the NA1 and NB1 antigens, and less commonly, the NA2 and NB2 antigens.¹²

Estimates of the frequency of ANN vary widely (0.2%-20%).^13-16 Underreporting of the incidence may occur because of the difficulty in detecting antineutrophil antibodies in the past and because only the most severely affected neonates are evaluated. It is likely that many infants who acquire these alloantibodies have mild neutropenia, or that the disease is undetected and/or undiagnosed.

The neutrophil count of the mother of infants with ANN is within normal limits, excluding the likelihood that she has an autoimmune neutropenia. The total white blood cell count in the infant may be normal, increased or decreased, but invariably, a severe neutropenia is present. Infants with ANN lack mature neutrophils in their peripheral blood and myeloid precursors can be present. A relative or absolute monocytosis may represent a degree of compensation for the neutropenia, particularly if the infant has a concurrent bacterial infection. Bone marrow biopsy of the infant reveals a hypercellular or hypocellular marrow with a left shift in the myeloid series.¹

In utero the fetus with ANN is protected from bacterial infection. However, during the first few days of life, localized or generalized infection can develop. As many as 61% of infants with neutropenia and documented overwhelming early-onset sepsis die.¹⁷ Therefore, any neonate with unexplained neutropenia should be evaluated for ANN.

Since 1987, 17 cases of ANN have been reported in neonates^12,13,18-21 and 7 of these were born prematurely.^13,17,20,21 There has been 1 previous report of ANN in monozygous twins.²⁰ In all cases, a single neutrophil antigen was responsible for the transient neutropenia, and the median length of ANN was 7 weeks¹⁸ (range: 12-90 days).

Intravenous immune globulin (IVIG) has been used successfully in the treatment of various IgG antibody-mediated hematologic disorders including neonates with passive autoimmune or isoimmune neutropenia, immune thrombocytopenia, immune neutropenia, autoimmune hemolytic anemia, pure red cell aplasia, and pure white cell aplasia. The mechanism of action of IVIG has been postulated as competitive inhibition of antibody binding or reticuloendothelial blockage.¹⁸ In ANN, this reverses the neutropenia by decreasing the rate of neutrophil clearance. Rarely, ANN can develop after the administration of IVIG.²¹

More recently, rhG-CSF has been used to treat various neonatal neutropenias.^22-29 Five of the 17 neonates described since 1987 received rhG-CSF. Four of the infants had resolution of the neutropenia after a course of rhG-CSF ranging from 3 to 6 days' duration.^13,17 The single patient who did not respond to rhG-CSF did not have antibodies to NA1 or NA2 antigens, suggesting that rhG-CSF therapy may not be effective in cases of ANN attributable to other neutrophil antigens.¹⁹ Therefore, testing of maternal serum to determine antibody specificity might be important.

For those neonates with prolonged neutropenia who respond to rhG-CSF therapy, subcutaneous administration of the drug might by an alternative to IV administration. As with the twins presented here, this would allow the infants to be discharged from the hospital earlier and would encourage normal parent-infant interactions. Of course, this alternative might not be appropriate for every patient. Close physician monitoring of the ANC and parental education on signs of infection are necessary. Although the neutropenia in most neonates with ANN is brief, the use of rhG-CSF for the treatment of ANN requires more comprehensive studies, as the potential effects of rhG-CSF on nonhematopoietic tissues in the developing neonate remain to be determined.³⁰

Jacob K. Felix, MD
Pediatrix Medical Group, Inc
Arnot Ogden Medical Center
Elmira, NY 14905

Darlene A. Calhoun, DO
Department of Pediatrics
Division of Neonatology
University of Florida College of Medicine
Gainesville, FL 32610-0296

	FOOTNOTES

Received for publication Oct 20, 1999; accepted Dec 10, 1999.

Reprint requests to (D.A.C.) University of Florida, Box 100296, Department of Pediatrics, Division of Neonatology, Gainesville, FL 32610-0296. E-mail: calhoda{at}peds.ufl.edu

	ABBREVIATIONS

ANN, alloimmune neonatal neutropenia; IgG, immunoglobulin G; ANC, absolute neutrophil count; IV, intravenous; rhG-CSF, recombinant human granulocyte colony-stimulating factor; HLA, human leukocyte antigen; MAIGA, monoclonal antibody immobilization of granulocyte antigens assay; IVIG, intravenous immune globulin.

	REFERENCES

Top Abstract Introduction Discussion References

1. Minchinton R, McGrath K Alloimmune neonatal neutropeniaa neglected diagnosis? Med J Aust. 1987; 147:139-141 [Medline]
2. Lalezari P, Murphy G, Allan F NB1, a new neutrophil specific antigen involved in the pathogenesis of neonatal neutropenia. J Clin Invest. 1971; 50:1108-1115
3. Contractor S, Eaton B, Stannard P Uptake and fate of exogenous immunoglobulin G in the perfused human placenta. J Reprod Immunol. 1983; 5:265-273 [CrossRef][Medline]
4. Bux J, Kober B, Kiefel V, Mueller-Eckhardt C Analysis of granulocyte-reactive antibodies using an immunoassay based on monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA). Transfus Med 1993; 3:157-162 [Medline]
5. Lalezari P, Nussbaum M, Gelman S, Spaet T Neonatal neutropenia due to maternal isoimmunization. Blood. 1960; 15:236-243 [Abstract/Free Full Text]
6. Verheugt F, von dem Borne A, von Noord-Bokhorst J, Nijenhuis L, Engelfriet C ND1, a new neutrophil granulocyte antigen. Vox Sang. 1978; 35:13-17 [Medline]
7. Claas F, Langerak J, Sabbe L, van Rood J NE1: a new neutrophil specific antigen. Tissue Antigens. 1979; 13:129-134 [Medline]
8. Thorsby E HLA antigens on human granulocytes studies with cytotoxic iso-antisera obtained by skin grafting. Scan J Haematol. 1969; 6:119-127
9. Dunstan R Status of major red cell blood group antigens on neutrophils, lymphocytes and monocytes. Br J Haematol. 1986; 62:301-309 [Medline]
10. Verheugt F, von dem Borne A, van Noord-Bokhorst J, van Elven E, Engelfriet C Serologic, immunochemical and immunocytological properties of granulocyte antibodies. Vox Sang. 1978; 35:294-303 [Medline]
11. Madyastha P, Blassman A, Levine D Incidence of neutrophil antigens on human cord neutrophils. Am J Reprod Immunol. 1984; 6:124-127
12. Rios E, Heresi G, Arevalo M Familial alloimmune neutropenia of NA2 specificity. Am J Pediatr Hematol Oncol. 1991; 13:296-299 [Medline]
13. Gilmore M, Stroncek D, Korones D Treatment of alloimmune neonatal neutropenia with granulocyte colony-stimulating factor. J Pediatr. 1994; 125:948-951 [CrossRef][Medline]
14. Levine D, Madyastha P Isoimmune neonatal neutropenia. Am J Perinatol. 1986; 3:231-233 [Medline]
15. Levine D, Madyastha P, Wade T, Levkoff A Neonatal isoimmune neutropenia. Pediatr Res. 1982; 16:296A
16. Verheugt F, voan Noord-Bookhorst J, von dem Borne A, Engelfriet, CP A family with allo-immune neonatal neutropenia: group-specific pathogenicity of maternal antibodies. Vox Sang. 1979; 36:1-8 [Medline]
17. Rodwell R, Gray P, Taylor K, Minchinton R Granulocyte colony stimulating factor treatment for alloimmune neonatal neutropenia. Arch Dis Child. 1996; 75:F57-F58
18. Hanada T, Shin R, Hosoi M, Saito K, Takita H Intravenous gammaglobulin in treatment of isoimmune neonatal neutropenia. Eur J Pediatr. 1988; 148:218-219 [CrossRef][Medline]
19. Bedu A, Rohrlich P, Fenneteau O, Cartron J Failure of granulocyte colony-stimulating factor in alloimmune neonatal neutropenia. J Pediatr. 1995; 127:508-509 [Medline]
20. Yoshida N, Shikata T, Sudo S, Alloimmune neonatal neutropenia in monozygous twins. Acta Paediatr Scand. 1991; 80:62-65 [Medline]
21. Lassiter H, Bibb K, Bertolone S, Patel C, Stroncek D Neonatal immune neutropenia following the administration of intravenous immune globulin. Am J Pediatr Hematol Oncol. 1993; 15:120-123 [Medline]
22. Kocherlakota P, La Gamma E Recombinant human granulocyte colony-stimulating factor (rhG-CSF) increased the absolute neutrophil count (ANC) and decreased the incidence of infections in persistent preeclampsia-associated-neutropenia in VLBW neonates. Pediatr Res. 1997; 41:937
23. Kocherlakota P, La Gamma EF. Human granulocyte colony-stimulating factor may improve outcome attributable to neonatal sepsis complicated by neutropenia. Pediatrics. 1997;100(1). URL: http://www. pediatrics.org/cgi/content/full/100/1/e6
24. Kocherlakota P, La Gamma EF Preliminary report: rhG-CSF may reduce the incidence of neonatal sepsis in prolonged pre-eclampsia associated neutropenia. Pediatrics. 1998; 102:1107-1114 [Abstract/Free Full Text]
25. Murray J, McClain K, Wearden M Using granulocyte colony-stimulating factor for neutropenia during neonatal sepsis. Arch Pediatr Adolesc Med. 1994; 148:764-766 [Medline]
26. Nankervis C, Seguin J Granulocyte colony-stimulating factor for neutropenia in neonatal sepsis. Arch Pediatr Adolesc Med 1995; 149:218-219 [Medline]
27. Calhoun DA, Lunøe M, Du Y, Hutson AD, Veerman M, Christensen RD Granulocyte colony-stimulating factor serum and urine concentrations in neutropenic neonates before and after the intravenous administration of recombinant granulocyte colony-stimulating factor. Pediatrics. 2000; 105:392-397 [Abstract/Free Full Text]
28. Calhoun DA, Christensen RD Kostmann syndrome in preterm neonates. Pediatrics. 1997; 99:259-261 [Free Full Text]
29. Juul S, Calhoun DA, Christensen RD Evaluation of "idiopathic neutropenia" in very-low-birth-weight infants. Acta Paediatr Scand. 1998; 87:963-968
30. Calhoun DA, Donnelly WH, Du Y, Dame JB, Li Y, Christensen RD The distribution of granulocyte colony-stimulating factor receptor (G-CSF-R) and its messenger RNA expression in the human fetus. Pediatr Res. 1999; 43:333-338