Patient characteristics

The median age of all AA patients was 43 years (range, 18–97), and there were 109 male patients and 72 female patients. There were 107 patients (59.12%) in the high-MCV group and 74 patients (40.88%) in the normal-MCV group, including 125 patients (69.06%) in the NSAA group and 56 patients (30.94%) in the SAA group. Among the participants, 165 (91.16%) were initially treated with cyclosporine A plus androgen, and fewer were treated with cyclosporine A alone. The differences in clinical characteristics between the two groups were compared and analyzed. There were statistically significant differences in neutrophil count, red blood cell count, mean corpuscular volume, platelet count, reticulocyte count, and disease severity between the two groups (P < 0.05) (Table 1).

Table 1 The characteristics of the 181AA patients

Meanwhile, the results showed that the average MCV levels of NSAA patients and SAA patients were 104.63 fl and 98.13 fl, respectively, and the difference was statistically significant (P < 0.001). Moreover, the MCV level of NSAA patients was significantly higher than that of SAA patients (Fig. 1A).

Fig. 1

Mean corpuscular volume (MCV) level according to disease severity at diagnosis (non-severe and severe AA) (A). The degree of bone marrow cellularity in all AA patients. (II: significantly active; III: active; IV: reduced; V: extremely reduced) (B)

Comparison of bone marrow cell morphology at initial diagnosis

Of the 181 overall patients, 1.66% had significantly active bone marrow cellularity (II), 23.20% had active bone marrow cellularity (III), 69.61% had reduced bone marrow cellularity (IV), and 5.52% had extremely reduced bone marrow cellularity (V) (Fig. 1B). Between the two groups of patients, the proportion of bone marrow lymphocytes in the high-MCV group was lower than that in the normal-MCV group, and the difference was statistically significant (P = 0.016, Fig. 2A). At the same time, the average proportion of bone marrow granulocytes in the high-MCV group was significantly higher than that in the normal-MCV group (P = 0.027, Fig. 2B), and there was no significant difference in other indicators between the two groups (P > 0.05, Fig. 2C, D). In addition, the results showed that the average proportion of lymphocytes in NSAA patients was lower than that in SAA patients, while the average proportion of granulocytes was higher than that in SAA patients (P < 0.05, Fig. 2E, F). There was no significant difference in the level of the other two lines between the disease types (P > 0.05 Fig. 2G, H).

Fig. 2

Baseline bone marrow cell morphology comparison of lymphocytes, granulocytes, erythrocytes, and megakaryocytes in MCV subgroups (A–D), in disease severity at diagnosis (non-severe and severe AA, E–H)

MCV and ORR or DOR

By treatment with cyclosporine A plus androgen or cyclosporine A alone, there were 160(88.40%) surviving patients and 21(11.60%) deaths out of 181 patients. Remission occurred in 74 high-MCV patients, and the ORR was 69.10%, including 19 patients (17.76%) with a CR, and 55 patients (51.40%) with a PR. Remission occurred in 44 normal-MCV patients, with an ORR of 59.46%, including 19 patients with a CR (25.68%), and 25 patients with a PR (33.78%). There were significant differences in the ORR between the two groups (P = 0.049)(Fig. 3A). Furthermore, in the high-MCV group, the median DOR was 48 months, and in the normal-MCV group, the median DOR was 34.5 months. There were no significant differences in the DOR between the high-MCV and normal-MCV groups (P = 0.482)(Fig. 3B).

Fig. 3

The rates of best response in MCV subgroups (A). The DOR in MCV subgroups (B)

MCV and OS or PFS

The overall median follow-up time was 50 months (95%CI 22–82.5). The median OS and PFS in the high-MCV group have not yet been obtained. In the normal-MCV group, the median OS was not yet obtained, and the median PFS was 63 months (95%CI: 23.91–102.09). The 5-year OS of AA patients in the high-MCV group was higher than that in the normal-MCV group (96.99%, 76.23%, P < 0.001) (Fig. 4A), and the 5-year PFS of the former was also better than that of the latter (77.39%, 50.68%, P < 0.001) (Fig. 4B). At the same time, according to the results of univariate analysis and clinical experience, age, disease severity, white blood cell count, neutrophil count, platelet count, and reticulocyte count were included in Cox regression analysis, and normal MCV and SAA were independent risk factors for poor survival (Table 2).

Fig. 4

Overall survival and progression-free survival in MCV subgroups (A and B)

Table 2 Logistic regression analysis of influencing factors of overall survival in AA patientsMCV of diverse population and OS or PFS

We further compared the results in subgroups of MCV across different populations (disease severity, sex, age). The results showed that the OS of the high-MCV group was better than that of the normal-MCV group in SAA patients, male patients, and patients younger than 35 years or aged 35–55 years (P < 0.05). In NSAA, SAA, male, female, and 35–55-year-old patients, the high-MCV group had better PFS than the normal-MCV group (P < 0.05). There was no significant difference in OS and PFS between MCV subgroups in other populations (P > 0.05)(Fig. 5A–N). Subgroup analysis of MCV according to disease severity showed that OS in male patients with high MCV was better than that in the normal-MCV group (P < 0.05), and PFS in male patients or patients aged 35–55 years was better than that in the normal-MCV group (P < 0.05). There was no significant difference in OS and PFS between the other groups (P > 0.05) (Supplementary information—Fig. S1).

Fig. 5

Long-term outcomes between MCV groups in diverse populations. A–D The OS and PFS between MCV subgroups in NSAA and SAA. E–H The OS and PFS between MCV subgroups in male and female. I–N The OS and PFS between MCV subgroups in the populations of age of < 35, age of 35–55, and age of ≥ 55 (year)

MCV and hemogram restoration

We compared the blood cell counts between the two MCV groups after treatment. In our study, The results showed that white blood cell (WBC) count, neutrophil (NE) count, hemoglobin (Hb) level, and platelet (PLT) count were significantly higher than before treatment, and the high-MCV group was higher than the normal-MCV group, among which Hb increased most significantly. The difference was statistically significant (P < 0.05) (Fig. 6).

Fig. 6

Hemogram restoration, comparison with the recovery of blood routine pre-treatment and post-treatment (A–D), the recovery of blood routine between two groups of patients (E–H)

MCV and reticulocyte parameters

In the results of the study comparing the levels of reticulocytes and their related parameters between the two groups, the relative and absolute reticulocyte counts, high-fluorescence reticulocyte (HFR), medium-fluorescence reticulocyte (MFR), and immature reticulocyte fraction (IRF) in the high-MCV group were significantly higher than those in the normal-MCV group (P < 0.001). At the same time, the low-fluorescence reticulocyte (LFR) was significantly lower in patients with high MCV than in those with normal MCV (P < 0.001) (Fig. 7).

Fig. 7

Reticulocyte parameters were stratified by MCV. The reticulocyte, reticulocyte percentage, HFR, MFR, LFR, IRF in the High-MCV subgroup compared with those in the Normal-MCV subgroup (A–F). (HFR: high-fluorescence reticulocyte; MFR: medium-fluorescence reticulocyte; LFR: low-fluorescence reticulocyte; IRF: immature reticulocyte fraction; MCV mean corpuscular volume)

To explore the association between reticulocyte parameters and MCV, this study further analyzed the correlation between reticulocyte and its parameters and MCV. The results showed that there was a positive correlation between MCV level and absolute reticulocyte count, relative reticulocyte count, high-fluorescence reticulocyte (HFR), medium-fluorescence reticulocyte (MFR), and immature reticulocyte fraction (IRF) (r = 0.384, r = 0.300, r = 0.430, r = 0.447, r = 0.507, respectively). At the same time, MCV level was negatively correlated with low-fluorescence reticulocyte (LFR) (r = − 0.514). All correlation analyses reached statistical significance (P < 0.001) (Fig. 8).

Fig. 8

Correlation between reticulocyte, reticulocyte percentage, HFR, MFR, LFR, IRF, and MCV (A–F). (HFR: high-fluorescence reticulocyte; MFR: medium-fluorescence reticulocyte; LFR: low-fluorescence reticulocyte; IRF: immature reticulocyte fraction; MCV mean corpuscular volume)