Fig 1.
Distribution of control non-megaloblastic anemia participants & Analysis of bone marrow collected from non-megaloblastic anemia controls and megaloblastic anemia cases using Leishman’s staining.
(a) Analysis of non-megaloblastic anemia control subjects using bone marrow aspirate normocytic-normochromic anemia (62%, 31/50); normocytic-normochromic with microcytic hypochromic anemia (24%, 12/50) and microcytic hypochromic anemia (14%, 7/50) (b) Photomicrographs (at 100X magnification) of Leishman’s stained bone marrow samples collected from control non-megaloblastic anemia and cases with megaloblastic anemia. Bone marrow from control subjects showed normal cellularity with erythroid cells showing—normal maturation (Black arrow),—myeloid series cells and megakaryocytes along with mature neutrophils (Red arrow) and Band forms (Yellow arrow). Megaloblastic anemia cases showed erythroid hyperplastic cells (Pink arrow) and open chromatin (Red arrow). Cells with more cytoplasm (Yellow arrow), giant sized nuclei (metamyelocytes, Black arrow) and band forms (neutrophils, Green arrow) and nuclear hyper segmentation and abnormal maturation (megakaryocytes) were observed only in MBA cases but not in control subjects.
Table 1.
Demographics of Study Participants.
Table 2.
Complete Blood Cells Count (CBC) Characteristics of Study Participants.
Table 3.
Distribution of non-megaloblastic anemia control subjects and megaloblastic anemia cases based on VB9 and VB12 status.
Table 4.
Control non-megaloblastic anemia subjects with LOW VB9 (Normal range: 5.00ng/ml– 20ng/mL) but NORMAL VB12 (Normal range: 211.00–911.00pg/ml).
Table 5.
Megaloblastic anemia subjects with LOW VB9 and VB12 (Normal range: VB9–5.00–20.00ng/ml, VB12–211.00–911.00pg/ml).
Table 6.
Megaloblastic Anemia subjects with LOW VB9 But NORMAL VB12 (Normal range: VB9–5.00–20.00ng/ml, VitB12–211.00–911.00pg/ml).
Table 7.
Megaloblastic anemia subjects with NORMAL VB9 BUT LOW VB12 (Normal range: VB9–5.00–20.00ng/mL, VitB12–211.00–911.00pg/mL).
Fig 2.
Patients Suffering From Megaloblastic Anemia Contain Low Vitamin-B9 And Vitamin-B12 In The Plasma.
(a) Low vitamin-B9 (Folic Acid) levels were observed in 90% (45/50) cases: Compared to controls, 90% megaloblastic anemia cases contain low folate levels (<5.0ng/mL) in the plasma. However, only 26% (13/50) controls contain folate levels lower than normal (<5.0ng/mL). (b) Low vitamin-B12 levels were observed in 78% (39/50) cases: Compared to controls, 78% megaloblastic anemia cases contain low vitamin-B12 levels (<200pg/mL) in the plasma. All most all controls contain vitamin-B12 values higher than 200pg/mL. (c) Megaloblastic bone marrow cases contain at least 2 fold lower vitamin-B9 and vitamin-B12 levels compared to control subjects: Analysis of vitamin-B9 and vitamin-B12 in the plasma of control and megaloblastic anemia cases revealed at least a 2-fold decrease in the levels of these two important vitamins compared to control subjects.
Fig 3.
Patients Suffering From Megaloblastic Anemia Contain Very High Plasma Homocysteine (HCys) Compared to Non-Megaloblastic Anemia Subjects.
Among the 30 megaloblastic anemia subjects 24 subjects (80%) had very high homocysteine levels in the plasma. However only 20% non-megaloblastic control subjects showed high plasma homocysteine. Therefore, very low VB9 and VB12, present in the megaloblastic anemia subjects, is responsible for elevated HCys in these subjects.
Fig 4.
P53 Expression Is Elevated In The Megaloblastic Anemia.
(a): Analysis of p53 expression in the bone marrow biopsies using immunocytochemistry: Bone marrow biopsy from control non-megaloblastic anemia, and cases with megaloblastic anemia were fixed and processed for immunohistochemical staining as described in materials and methods. The stained sections were observed under microscope and photographed using 40X magnification. The data showed more number of intensely expressed p53 protein in the megaloblastic anemia patients compared to control subjects. The breast carcinoma tissue with wild type p53 was used as positive control for p53 staining while the tissue from colon with no p53 expression served as negative control. (b) Quantitative assessment of p53 expression in megaloblastic anemia: Expression of p53 was quantified by counting the number of low, moderate (medium) and heavily stained cells (expressed p53 protein) and the results represented in terms of percentage total cells. The data showed a statistically significant ~5 fold increase in the heavily stained cells compared to controls. A similar percentage decrease was observed in the number of unstained cells, with more unstained cells observed in controls. (c) Relative distribution of p53 expressing cells with different degrees of staining in megaloblastic anemia subjects. MBA subjects with low VB9 and low VB12, low VB9 but normal VB12, normal VB9 but low VB12 and normal VB9 and normal VB12 were analyzed for the expression of p53 using IHC as described in materials and methods. The data showed a very high percentage p53 stained (heavy and moderate staining) cells in subjects deficient in VB9 and VB12 compared to subjects with normal VB9 and VB12 (P < 0.05) or subjects with having low VB9 or low VB12 (P < 0.05). (d) Relative distribution of p53 expressing cells with different degrees of staining in non-megaloblastic anemia control subjects. The graph shows the expression of p53 in different non-megaloblastic anemia subject categories—Normocytic Normochromic Anemia (NNA)– 31 individuals; Normocytic Normochromic and Microcytic Hypochromic Anemia (NNMA)–marked with * - 12 individuals; Microcytic Hypochromic Anemia (MHA)–marked with decrease—7 individuals. Further analysis showed the differences in p53 expression in subjects with low VB9 and low VB12 (only one individual ie., Subject #77), low VB9 but normal VB12 (Table 4), normal VB9 but low VB12 (Table e in S1 File, only one individual ie., Subject #85) and normal VB9 and normal VB12 (Refer Tables D and E in S1 File) were analyzed for the expression of p53 using IHC as described in materials and methods. The data showed a very high percentage (>75%) unstained and moderately stained (for p53) cells in 48 out of 50 subjects having at least normal VB12.