Novel Anti-Hemoglobin F Antibody in Bone Marrow Can Predict a Response to Antithymocyte Globulin and Cyclosporine in Patients with Aplastic Anemia. Retrospective Study of 28 Cases.
Authors: Ichihashi Ryoichi , Ito Masafumi , Mori Naoyoshi , Kojima Seiji 
Published: October 2019

Abstract
The distinction between hypoplastic myelodysplastic syndrome (MDS) from AA by morphology alone is sometimes difficult. It is required to invent a new tool to differentiate two diseases. We developed a novel polyclonal antibody by immunizing a rabbit with synthetic peptide of human HbF, which can detect of HbF-containing erythroblasts in paraffin-embedded hematpoietic tissues. HbF is a useful marker to distinguish MDS from AA. Although 42 of 51 MDS patients with cytogenetic abnormalities were positive with HbF, only 2 of 12 patients with typical AA were positive in previous study. (Ichihashi R, et al: Blood102: 428a, 2003). On the basis of previous findings, we hypothesized that HbF expression on bone marrow erythroblast might be applied to predict to IST in AA patients.
Material and methods: We investigated bone marrow samples from 24 patients with acquired AA who received IST with antithymocyte globulin (ATG) and cyclospoline (CSA) between 1993 and 2004 in Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. The diagnosis of AA was established by bone marrow findings and peripheral blood counts. As for grading, the disease was considered severe if at least two of the following were noted: a neutrophil count of less than 0.5×109/L, a platelet count of less than 20×109/L, a reticulocyte count of less than 20×109/L with hypocellular bone marrow. Moderate disease was defined by at least 2 of the following hematologic values: a neutrophil count of less than 1.0×10×109/L, a platelet count of less than 50×109/L and reticulocyte count of less than 60×109/L. 23 Patients had severe AA and 5 had moderate AA. Patient’s age ranged from 2 to 80 years (median: 25.7 years), Complete response (CR) was defined as a neutrophil count > 1.5×109/L, a platelet count > 100×109/L, and a hemoglobin level of > 11.0g/dl. Partial response (PR) was defined as a neutrophil count > 1.0×109/L, a platelet count > 30×109/L, and a hemoglobin level of > 8.0g/dl in patients with sever AA. It was defined as a neutrophil count > 1.0×109/L, a platelet count >3.0×109/L in patients with moderate AA. Formalin fixed, paraffin embedded bone marrow tissues were immunostained. We defined that HbF was positive when more than 3 erythroblasts were stained in a cluster.
Results: Overall response rate was a 46.4percentage of patients with CR and PR at 6 months after IST. Higher probability of response to IST was observed in patients with HbF-negative AA. Although 5 of 7 patient (71.4%) with HbF-negative AA responed to ISF, only 8 of 21 patient (38.1%) with HbF-positive AA responded. Of 15 patients who did not respond to IST, HbF positive erythreblasts were detected in 13 patients (86.7%).
Conclusion: These results suggest that staining of HbF positive erythroblast in pretreatment bone marrow samples may be helpful to predict a good response to IST especially in adults. HbF-positive AA may have common clinical and biological basis with MDS. We are planning to use this novel tool to distinguish AA from MDS prospectively.
HbF and Response to ATG, Adults HbF+ HbF− *All 5 cases: Not examined CR/PR 3 (3cases:DR2+) 3 (3cases:DR2+) NR/BMT 5(DR2+*) 0 HbF and Response to ATG, Children HbF+ HbF− CR/PR 5 (3cases :DR2+) 2 NR/BMT 8 (3case: DR2+) 2 (1case:DR2+)