Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, UK; International Blood Group Reference Laboratory, NHS Blood and Transplant, Bristol, UK.
Transfusion. 2014 May;54(5):1305-16. doi: 10.1111/trf.12484. Epub 2013 Nov 17.
Quantitation of fetomaternal hemorrhage (FMH) is performed to determine the dose of prophylactic anti-D (RhIG) required to prevent D immunization of D- women. Flow cytometry (FC) is the most accurate method. However, maternal white blood cells (WBCs) can give high background by binding anti-D nonspecifically, compromising accuracy.
Maternal blood samples (69) were sent for FC quantitation of FMH after positive Kleihauer-Betke test (KBT) analysis and RhIG administration. Reagents used were BRAD-3-fluorescein isothiocyanate (FITC; anti-D), AEVZ5.3-FITC (anti-varicella zoster [anti-VZ], negative control), anti-fetal hemoglobin (HbF)-FITC, blended two-color reagents, BRAD-3-FITC/anti-CD45-phycoerythrin (PE; anti-D/L), and BRAD-3-FITC/anti-CD66b-PE (anti-D/G). PE-positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors.
Elevated numbers of neutrophils were present in 80% of patients. Red blood cell (RBC) indices varied widely in maternal blood. D+ FMH values obtained with anti-D/L, anti-D/G, and anti-HbF-FITC were very similar (r = 0.99, p < 0.001). Correlation between KBT and anti-HbF-FITC FMH results was low (r = 0.716). Inaccurate FMH quantitation using the current method (anti-D minus anti-VZ) occurred with 71% samples having less than 15 mL of D+ FMH (RBCs) and insufficient RhIG calculated for 9%. Using two-color reagents and anti-HbF-FITC, approximately 30% patients had elevated F cells, 26% had no fetal cells, 6% had D- FMH, 26% had 4 to 15 mL of D+ FMH, and 12% patients had more than 15 mL of D+ FMH (RBCs) requiring more than 300 μg of RhIG.
Without accurate quantitation of D+ FMH by FC, some women would receive inappropriate or inadequate anti-D prophylaxis. The latter may be at risk of immunization leading to hemolytic disease of the newborn.
定量检测胎儿母体出血（FMH）是为了确定预防 D 免疫所需的预防用抗 D（RhIG）剂量，D 阴性妇女需要预防 D 免疫。流式细胞术（FC）是最准确的方法。然而，母体白细胞（WBC）可能会因非特异性结合抗 D 而产生高背景，从而影响准确性。
在 Kleihauer-Betke 试验（KBT）分析和 RhIG 给药后，将 69 份母体血液样本送往 FC 定量检测 FMH。使用的试剂包括 BRAD-3-异硫氰酸荧光素（FITC；抗 D）、AEVZ5.3-FITC（抗水痘带状疱疹[抗 VZ]，阴性对照）、抗胎儿血红蛋白（HbF）-FITC、双色混合试剂、BRAD-3-FITC/抗-CD45-藻红蛋白（PE；抗 D/L）和 BRAD-3-FITC/抗-CD66b-PE（抗 D/G）。通过门控消除分析中的 PE 阳性 WBC。对母体样本和女性供体进行全血细胞计数。
80%的患者存在中性粒细胞数量升高。母体血液中的红细胞（RBC）指数差异很大。使用抗 D/L、抗 D/G 和抗 HbF-FITC 获得的 D+FMH 值非常相似（r=0.99，p<0.001）。KBT 和抗 HbF-FITC FMH 结果之间的相关性较低（r=0.716）。使用当前方法（抗 D 减去抗 VZ）进行不准确的 FMH 定量检测时，71%的样本 D+FMH（RBC）小于 15mL，计算出的 RhIG 不足 9%。使用双色试剂和抗 HbF-FITC，大约 30%的患者存在升高的 F 细胞，26%的患者不存在胎儿细胞，6%的患者存在 D-FMH，26%的患者存在 4 至 15mL 的 D+FMH，12%的患者存在超过 15mL 的 D+FMH（RBC），需要超过 300μg 的 RhIG。
如果不通过 FC 准确定量检测 D+FMH，一些女性将接受不适当或不足的抗 D 预防。后者可能有免疫新生儿溶血病的风险。
