Introduction: Neonatal neutropenia is often secondary to sepsis, low birth weight, pregnancy-induced maternal hypertension, and other conditions. Case Report: We report a case of asymptomatic isoimmune neutropenia in a pair of preterm twins. Genotyping confirmed that the mother was negative for HNA-1a, 1b, and 1c, consistent with an FcγRIIIb deficiency. The father was 1(a+b+c–) and the neonates were 1(a–b+c–). A strongly positive result was observed in the granulocyte immunofluorescence test against paternal neutrophils (IgG antibodies). IgG anti-CD16b isoantibodies were detected in the mother’s breast milk. Neutropenia resolved after 28 days without requiring any specific treatments. Discussion: Even though neonatal alloimmune neutropenia (NAN) is usually benign and self-limiting, some patients pre-sent with delayed separation of the umbilical cord, mild skin infections, omphalitis, or severe infections like pneumonia, sepsis, and meningitis. Thus, it is important to rule out NAN in case of neonatal neutropenia.

Neutropenia is defined as an absolute neutrophil count (ANC) <1.5 × 109/L [1], although the reference range varies depending on gestational age, birth weight, gender, and race [2]. It is severe if ANC is <0.5 × 109/L [3]. Neonatal neutropenia is common, usually transitory, asymptomatic, and secondary to sepsis, prematurity, neonatal alloimmune neutropenia (NAN), low birth weight, pregnancy-induced hypertension, or severe hemolytic disease of the fetus and newborn (HDFN) [1, 4, 5]. Sometimes, it is difficult to distinguish if sepsis preceded neutropenia or vice versa. NAN usually presents with monocytosis compensating the neutropenia, but hemoglobin and platelets remain normal [4, 5]. Neutropenia frequently exists at birth and may decrease during the first week of life [4]. Clinicians should consider further evaluation of neutropenia if no clear cause is present, or if ANC does not increase within 3–5 days or persists for >2 days [1, 2, 4].

We report 2 cases of NAN due to anti-FcγRIIIb isoimmunization in a pair of dichorionic female twins born at 354/7 weeks of gestation from a healthy, nonconsanguineous, 30-year-old mother with 3 previous healthy children. They weighed 1,980 and 1,748 g (i.e., the 36 and 18th percentile according to Spanish growth curves for twins). Routine blood tests 12 h after birth due to a risk of infection for group B Streptococcus showed leukopenia and severe neutropenia: a total white blood cell (WBC) count of 6.740 and 4.820 × 109/L, and ANC 0.067 and 0.048 × 109/L, respectively. Other hematological and biochemical profiles were normal. Neutropenia was thought to be secondary to early-onset neonatal sepsis. They received antibiotics despite being asymptomatic until blood and CSF cultures were negative. The presence of neutrophil antibodies in the maternal serum was tested with a granulocyte immunofluorescence test (GIFT), granulocyte agglutination test (GAT), and MAIGA (monoclonal antibody-specific immobilization of granulocyte antigen) assay, with fully concordant results. For the GIFT, GAT, and MAIGA studies, freshly isolated HNA-typed donor cells were used. Cross-matching with the granulocytes of the father could only be performed by the GIFT. For the GIFT assays, an FITC-conjugated F(ab’)2 fragment goat anti-human IgG anti-globulin (Jackson Immunoresearch, Laboratories Inc.) was used (Fig. 1). The IF results were assessed by flow cytometry (FACSCalibur platform. Becton Dickinson). A strong positive result was observed in the cross-match with the paternal granulocytes, as well as against all the panel cells used, except against granulocytes from a woman with an FcγRIIIb deficiency. The GAT was performed by standard methods against a panel of granulocytes, and the results were negative. The anti-FcγRIIIb antibody present in the maternal serum did not induce agglutination. Two monoclonal antibodies against the FcγRIIIb (CD16) were used in the MAIGA: DJ130c (Novus Biologicals) and LNK16 (Invitrogen). The results were in agreement with those observed with GIFT and the Luminex assay LABSCreen Multi. Maternal serum and breast milk were tested with the LABScreen multiassay, including HLA and human neutrophil antigen (HNA) antibody screening by Luminex technology: LABScreenTM Multi (One Lambda, Inc. CA, USA). Maternal neutrophils were negative for HNA-1a and 1b, while 50% expressed HNA-2. Maternal HNA genotyping performed with a multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigens confirmed that the mother was negative for HNA-1a, 1b, and 1c, consistent with an FcγRIIIb gene deficiency. The father was 1(a+b+c–), and the neonates were 1(a–b+c–). HLA antibodies were not detected. Anti-CD16b isoantibodies were detected by GIFT and MAIGA against freshly isolated HNA-typed donor cells and by Luminex. IgG anti-CD16b isoantibodies were detected in the mother’s breast milk. As expected, the fluorescence intensity obtained with the breast milk sample was lower than that observed with the serum sample, as the concentration of IgG in human milk is much lower. Nevertheless, as seen in Figure 2, the normalized background ratio (NBG) observed in the milk sample clearly showed a pattern of reaction against the beads carrying the FcγRIIIb glycoprotein.

Fig. 1.

GIFT results observed in the cross-match of the maternal serum against paternal granulocytes, with an FITC-conjugated anti-human IgG anti-globulin. FITC is measured over the population of viable granulocytes, not stained with 7-amino-actinomycin D (Gate P3). MFI, median fluorescence intensity.

Fig. 1.

GIFT results observed in the cross-match of the maternal serum against paternal granulocytes, with an FITC-conjugated anti-human IgG anti-globulin. FITC is measured over the population of viable granulocytes, not stained with 7-amino-actinomycin D (Gate P3). MFI, median fluorescence intensity.

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Fig. 2.

Results observed in the LABScreen Multi assay for IgG HNA antibody investigation. The red line indicates the standard cut-off value of >5 used for serum samples. NBG, normalized background ratio.

Fig. 2.

Results observed in the LABScreen Multi assay for IgG HNA antibody investigation. The red line indicates the standard cut-off value of >5 used for serum samples. NBG, normalized background ratio.

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ANC gradually increased without requiring any specific treatments. The neonates were discharged on day 10 with a WBC count 10.25 and 5.04 × 109/L, and ANC 0.67 and 0.62 × 109/L, respectively. Neutropenia persisted for 28 days (Table 1). Clinical follow-up was done until the age of 7 months.

Table 1.

Trend of peripheral blood leukocytes and neutrophils (×109/L)

Trend of peripheral blood leukocytes and neutrophils (×109/L)
Trend of peripheral blood leukocytes and neutrophils (×109/L)

There are 3 types of immune-mediated neutropenia: alloimmune, isoimmune, and autoimmune [1, 4]. NAN is caused by the maternal sensitization against an antigen of fetal neutrophils inherited from the father [2, 6, 7]. The most frequently involved granulocyte antibodies are those from the HNA-1 family (HNA1a, 1b, 1c, and 1d) that represent polymorphisms of the FcγRIIIb or CD16b, and HNA2, 3a, 4a, 4b, and 5a [1-4]. NAN can also be caused by antibodies against the FcγRIIIb in mothers with a FcγRIIIb deficiency, resulting in the HNA-1 null phenotype, or by HLA class I antibodies against paternal antigens [2, 4, 5, 8]. An immunization against an entire glycoprotein that the patient lacks is defined as an isoimmunization (i.e., RhD isoimmunization in RhD-negative women, who lack all the RhD glycoprotein). An alloimmunization is caused by an antibody against a specific antigen, e.g., an anti-HNA-1a or an HNA-1b. It is often used indiscriminately (commonly for platelet antagonism, not isoimmune neonatal neutropenia). In some cases, NAN is caused by isoantibodies, if a mother lacks a complete HNA system-carrying structure (i.e., anti-FcγRIIIb isoantibodies or HNA-2 antibodies). However, there may be some confusion about which name can be best used for this condition [4]. Diagnosis requires identifying the antibody in the maternal serum or in the patient’s neutrophils [6, 7]. The study should be done as soon as possible because antibody titers decrease as time goes by [3]. It is also important to consider that all women with antibodies against HNA antigens may not be used as blood donors due to the potential risk of transfusion-related acute lung injury (TRALI).

The reported incidence of NAN is 2 per 1,000 live births [3, 5-7, 9], although it is not exactly known [2, 4] because anti-HNA antibody screening and identification assays are laborious [4]. NAN frequently goes undiagnosed, since blood tests are not routinely performed in newborns [3, 5-7, 9] and it is often detected when WBC counts are performed for nonrelated reasons, as in this case report [4]. Sometimes, the neutropenia is attributed to sepsis, prematurity, intrapartum asphyxia, maternal hypertension or pre-eclampsia [1, 6]. The clinical course of NAN is usually benign and self-limiting. Symptoms vary from none to a delayed separation of the umbilical cord or mild skin infections, and omphalitis, or severe, even life-threatening infections like pneumonia, sepsis, and meningitis [1, 4] mostly caused by S. aureus, S. epidermidis, and E. coli [8].

The main treatment consists of preventing and treating acute infections [3, 4]. Prophylactic antibiotics or antifungals such as fluconazole can be considered in severe cases [3, 4]. The use of intravenous immunoglobulin and recombinant human G-CSF is controversial and may be useful in some cases [1-6]. G-CSF can be considered for severe infections, although it has not increased infection-free survival [1, 4]. Intravenous immunoglobulin can be used as a second-line treatment for life-threatening infections or very recurrent infections [1, 3, 4].

Alloantibodies are cleared spontaneously during the first 6 weeks, but neutropenia can last for up to 6 months [1, 2, 4, 5]. WBC counts should be performed until neutropenia disappears, which usually happens after a decrease in monocytes [5]. In this study, granulocyte-specific antibodies against HNA were detected in both twins and in the maternal sera and breast milk. IgA, IgG, IgM, IgD, and IgE are present in human breast milk, although at lower levels than in the serum, and the absorption of orally transferred immunoglobulins is inefficient [10]. Transmission of clinically significant amounts of maternal IgG anti-KEL via breast milk in nursing murine pups has been previously reported, although no cases of resulting anemia were observed despite intestinal absorption of IgG being more efficient in mice than in humans [10]. The predominant antibody in colostrum and breast milk is secretory IgA (not tested in our laboratory), although secretory IgM and IgG are present as well. There is a theoretical risk of maintained passage of alloantibodies to infants through breast milk, although, to the best of our knowledge, no cases have been reported to date. It has been described in breastfed infants with HDFN (causing prolonged hemolysis) and in the infants of mothers with immune thrombocytopenia (causing persistent thrombocytopenia) [9, 11, 12]. Discontinuation of breastfeeding is not recommended unless the neutropenia becomes unmanageable. Our patients’ mother decided to bottle-feed, and we only obtained breast milk to analyze. Thus, we do not know what effect breastfeeding would have had on the neutropenia. Breast milk contains IgG against FcγRIIIb. Maternal granulocyte antibodies might be the cause of the prolonged neutropenia observed in some cases of NAN.

It is important to diagnose NAN, to be able to choose the best prophylactic and/or therapeutic options, predict the clinical course, and diagnose NAN in future children. The risk of recurrence is very high, and WBC and ANC screening should be performed in future neonates [2, 4, 5]. Further research is warranted to evaluate if maternal granulocyte antibodies present in breast milk may contribute to a prolonged neutropenia in these patients.

The patients’ parents gave us written permission to publish this case report. The patients are not identifiable through this paper.

The authors have no conflicts of interest to declare.

The authors received no specific funding for this work.

B.V.D.B. and S.M.-E. drafted the initial manuscript, reviewed the literature, and reviewed and revised the manuscript. J.J.R.-S. wrote the immunohematologic part, critically reviewed the manuscript for important intellectual content, and reviewed and revised it. C.C.S. helped draft the immunohematologic part, and critically reviewed the manuscript for important intellectual content and revised it. A.B.L. reviewed the literature, critically reviewed the manuscript for important intellectual content, and revised it. M.A.L.-V. helped draft the initial manuscript, critically reviewed it for important intellectual content, and revised it.

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