Abstract：
OBJECTIVE: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays.
METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity.
RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/μL vs. 4.5 × 103/μL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times).
CONCLUSIONS: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.
METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity.
RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/μL vs. 4.5 × 103/μL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times).
CONCLUSIONS: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.
摘要：
目的：分离中性粒细胞并随后检测抗人中性粒细胞抗原（HNA）抗体在临床医学中对自身免疫性中性粒细胞减少症的诊断至关重要。新生儿同种免疫性中性粒细胞减少症（NAIN）和输血相关急性肺损伤（TRALI）。本研究报告了两例无NAIN症状的母体抗Fc-γ-受体-IIIb（FcγRIIIb）免疫，并比较了免疫磁性阴性选择（IMNS）与传统葡聚糖/Ficoll在HNA血清学测定中分离中性粒细胞的效率。
方法：调查2例母体抗FcγRIIIb免疫，使用IMNS和葡聚糖/Ficoll从8mL全血中分离来自三个供体的嗜中性粒细胞。血清学检测包括粒细胞凝集和免疫荧光检测,粒细胞抗原的单克隆抗体固定和LABScreenMulti（一个Lambda）。IMNS和葡聚糖/Ficoll在细胞产量方面进行了比较，生存能力，时间,成本和纯度。
结果：在两种情况下均检测到具有FCGR3B基因缺失的母体抗FcγRIIIb抗体。新生儿和父亲表现出特定的基因组合：FCGR3B*02/FCGR3B*02（案例1）和FCGR3B*02/FCGR3B*03（案例2）。IMNS的表现优于葡聚糖/Ficoll，产生四倍以上的中性粒细胞（平均中性粒细胞计数：18.5×103/μLvs.4.5×103/μL),有效去除非中性粒细胞细胞并减少处理时间（30-40分钟vs.70-90分钟),虽然它产生了更高的成本(2.7倍)。
结论：2例母体抗FcγRIIIb抗体，与Nain无关,已确定。虽然在这些病例中没有发现中性粒细胞减少症,我们强调识别可能导致严重中性粒细胞减少的无症状病例的重要性.此外,IMNS是作为一种快速的，高产,高纯度中性粒细胞分离技术，有利于血清学检测抗HNA抗体。
方法：调查2例母体抗FcγRIIIb免疫，使用IMNS和葡聚糖/Ficoll从8mL全血中分离来自三个供体的嗜中性粒细胞。血清学检测包括粒细胞凝集和免疫荧光检测,粒细胞抗原的单克隆抗体固定和LABScreenMulti（一个Lambda）。IMNS和葡聚糖/Ficoll在细胞产量方面进行了比较，生存能力，时间,成本和纯度。
结果：在两种情况下均检测到具有FCGR3B基因缺失的母体抗FcγRIIIb抗体。新生儿和父亲表现出特定的基因组合：FCGR3B*02/FCGR3B*02（案例1）和FCGR3B*02/FCGR3B*03（案例2）。IMNS的表现优于葡聚糖/Ficoll，产生四倍以上的中性粒细胞（平均中性粒细胞计数：18.5×103/μLvs.4.5×103/μL),有效去除非中性粒细胞细胞并减少处理时间（30-40分钟vs.70-90分钟),虽然它产生了更高的成本(2.7倍)。
结论：2例母体抗FcγRIIIb抗体，与Nain无关,已确定。虽然在这些病例中没有发现中性粒细胞减少症,我们强调识别可能导致严重中性粒细胞减少的无症状病例的重要性.此外,IMNS是作为一种快速的，高产,高纯度中性粒细胞分离技术，有利于血清学检测抗HNA抗体。
