**3. H&E-stained AML**

#### **3.1. Identifying erythrocytic lineages and AML by H&E-stained BM clots**

To determine the utility of H&E-stained BM clots for the diagnosis of megaloblastic leukemia, megaloblastoid cells in MDS, or erythroleukemia, the H&E staining properties of erythrocytic lineages were considered. Pro-erythroblasts are identifiable by their basophilic cytoplasm, transparent nucleus, and multiple deformed nucleoli. A basophilic erythroblast is smaller and has a relatively large nucleus, bearing thick or coarse chromatin (**Figure 3A**). The nucleus occupies a relatively small part of the cell compared to that in basophilic erythroblasts. Importantly, owing to two highly contrasting colors in H&E staining, two distinct types of erythroblasts can be observed: polychromatic and orthochromatic. The polychromatic erythroblast has a lacy nucleus and baso-eosinophilic cytoplasm, while the orthochromatic erythroblast has eosinophilic cytoplasm, in which the nucleus is small, pyknotic, homogeneous, and structure-less, and ultimately becoming blue-black (**Figure 3B**).

encountered; in these cases, myeloblastic leukemic cells were observed. They have similar cytological features as those of normal myeloblasts: (i) eosinophilic, rough cytoplasm and (ii) a prominent nucleolus. **Figure 4A** shows leukemic marrow consisting of myeloblastic leukemic cells. M4, acute myelomonocytic leukemia (**Figure 4C**), and M5, acute monocytic leukemia (**Figure 4D**), have common features of monoblastic cells and are relatively easily identifiable due to a U-shaped nucleus with prominent dual nucleoli. Chromatin condensation was observed in the immature phenotype of monocytic leukemic cells in M4 and M5. The latter is a mix of monoblasts and monocytes and has pyknotic and condensed chromatin. In M6, acute erythroleukemia, atypical erythroblast-like leukemic cells were observed. The rough margin of the nucleus and basophil is a feature of these leukemic cells. In M7, acute megakaryocytic leukemia, the cells are transparent and lobulated without a distinct nucleus, which is observed in MDS. Furthermore, abnormal, deformed, and intensely stained nuclei

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**Figure 4.** AML diagnosis using H&E staining. (A) Two M2 leukemic cells (AML with maturation) of various sizes mimic myeloblastic morphology and show irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (B) Two M3 leukemic cells mimic pro-myelocytic morphology but present irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (C) Two M4 acute myelomonocytic leukemic (AMMoL) cells, mimicking monocytic and myeloblastic morphology but presenting irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (D) M5 acute monocytic leukemic cells are a mixture of two types of cells: Monoblasts and pro-monocytes. They mimic monoblastic morphology but present an irregular transparent nucleus with prominent eosinophilic nucleoli. Monoblasts have roughly circular and delicate lacy chromatin nuclei with one or two prominent nucleoli. Pro-monocytes have more convoluted nuclei, and the nucleoli are not prominent (600×). (E) M6 leukemic cells mimic erythroblastoid morphology but present irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (F) M7 leukemic cells mimic myeloblastic

morphology but present irregular transparent multilobed nuclei with prominent eosinophilic nucleoli (600×).

are observed in MDS/MPN.

#### **3.2. Identifying myeloblasts and AML by H&E-stained BM clots**

In this study, the utility of H&E-stained BM clots in diagnosing classic AML subtypes was also examined. H&E-stained clots were prepared for M2, M3, M4, M5, M6, and M7 according to the classic French-American-British classification. Cases of M2, acute leukemia with maturation, as specified in WHO 2008 classification, were the most frequent AML cases

**Figure 3.** Erythroid lineages detected by H&E staining. (A) Two basophilic erythroblasts represent large nuclei (represented by arrows). Nucleoli are not prominent relative to pro-erythroblasts. The cytoplasm is basophilic, rough, and heterogeneously condensed (600×). (B) Three metachromatic erythroblasts represent lacy and oval nuclei (indicated by two arrowheads). The cytoplasm is baso-eosinophilic, rough, and heterogeneously condensed. Two orthochromatic erythroblasts represent condensed and oval nuclei (indicated by two arrowheads). The cytoplasm is eosinophilic and rough (600×).

encountered; in these cases, myeloblastic leukemic cells were observed. They have similar cytological features as those of normal myeloblasts: (i) eosinophilic, rough cytoplasm and (ii) a prominent nucleolus. **Figure 4A** shows leukemic marrow consisting of myeloblastic leukemic cells. M4, acute myelomonocytic leukemia (**Figure 4C**), and M5, acute monocytic leukemia (**Figure 4D**), have common features of monoblastic cells and are relatively easily identifiable due to a U-shaped nucleus with prominent dual nucleoli. Chromatin condensation was observed in the immature phenotype of monocytic leukemic cells in M4 and M5. The latter is a mix of monoblasts and monocytes and has pyknotic and condensed chromatin. In M6, acute erythroleukemia, atypical erythroblast-like leukemic cells were observed. The rough margin of the nucleus and basophil is a feature of these leukemic cells. In M7, acute megakaryocytic leukemia, the cells are transparent and lobulated without a distinct nucleus, which is observed in MDS. Furthermore, abnormal, deformed, and intensely stained nuclei are observed in MDS/MPN.

**Figure 4.** AML diagnosis using H&E staining. (A) Two M2 leukemic cells (AML with maturation) of various sizes mimic myeloblastic morphology and show irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (B) Two M3 leukemic cells mimic pro-myelocytic morphology but present irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (C) Two M4 acute myelomonocytic leukemic (AMMoL) cells, mimicking monocytic and myeloblastic morphology but presenting irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (D) M5 acute monocytic leukemic cells are a mixture of two types of cells: Monoblasts and pro-monocytes. They mimic monoblastic morphology but present an irregular transparent nucleus with prominent eosinophilic nucleoli. Monoblasts have roughly circular and delicate lacy chromatin nuclei with one or two prominent nucleoli. Pro-monocytes have more convoluted nuclei, and the nucleoli are not prominent (600×). (E) M6 leukemic cells mimic erythroblastoid morphology but present irregular transparent nuclei with prominent eosinophilic nucleoli (600×). (F) M7 leukemic cells mimic myeloblastic morphology but present irregular transparent multilobed nuclei with prominent eosinophilic nucleoli (600×).

**Figure 3.** Erythroid lineages detected by H&E staining. (A) Two basophilic erythroblasts represent large nuclei (represented by arrows). Nucleoli are not prominent relative to pro-erythroblasts. The cytoplasm is basophilic, rough, and heterogeneously condensed (600×). (B) Three metachromatic erythroblasts represent lacy and oval nuclei (indicated by two arrowheads). The cytoplasm is baso-eosinophilic, rough, and heterogeneously condensed. Two orthochromatic erythroblasts represent condensed and oval nuclei (indicated by two arrowheads). The cytoplasm is eosinophilic and

rough (600×).

**3. H&E-stained AML**

12 Myeloid Leukemia

**3.1. Identifying erythrocytic lineages and AML by H&E-stained BM clots**

and structure-less, and ultimately becoming blue-black (**Figure 3B**).

**3.2. Identifying myeloblasts and AML by H&E-stained BM clots**

To determine the utility of H&E-stained BM clots for the diagnosis of megaloblastic leukemia, megaloblastoid cells in MDS, or erythroleukemia, the H&E staining properties of erythrocytic lineages were considered. Pro-erythroblasts are identifiable by their basophilic cytoplasm, transparent nucleus, and multiple deformed nucleoli. A basophilic erythroblast is smaller and has a relatively large nucleus, bearing thick or coarse chromatin (**Figure 3A**). The nucleus occupies a relatively small part of the cell compared to that in basophilic erythroblasts. Importantly, owing to two highly contrasting colors in H&E staining, two distinct types of erythroblasts can be observed: polychromatic and orthochromatic. The polychromatic erythroblast has a lacy nucleus and baso-eosinophilic cytoplasm, while the orthochromatic erythroblast has eosinophilic cytoplasm, in which the nucleus is small, pyknotic, homogeneous,

In this study, the utility of H&E-stained BM clots in diagnosing classic AML subtypes was also examined. H&E-stained clots were prepared for M2, M3, M4, M5, M6, and M7 according to the classic French-American-British classification. Cases of M2, acute leukemia with maturation, as specified in WHO 2008 classification, were the most frequent AML cases

**Figure 5.** Dysplastic megakaryocytes detected by H&E staining. Megakaryocytes have round, dispersed nuclei (A, B, C). Two or three dispersed nuclei are observed (600×).

**3.3. Dysplastic megakaryocytes in refractory anemia with excess blasts and MDS/MPN**

nuclear form, and dispersed round nucleoli were observed.

which have blots in the cytoplasm (indicated by arrows).

MDS patients (**Figure 5A**–**C**).

In H&E-stained clot specimens of refractory anemia with excess blasts and MDS, abnormal megakaryocytes, which include micromegakaryocytes and megakaryocytes with large mono-

**Figure 7.** Dysplastic myelocytes observed by H&E staining. (A–D) dysplastic metamyelocytes maturing into neutrophils,

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Morphological changes and increase in the number of megakaryocytes are informative for diagnosis. Micromononuclear megakaryocytes are detected in MDS, while large, over-mature megakaryocytes have been observed to increase in number in myeloproliferative neoplasms [12]. In anaplastic anemia, the megakaryocytes decrease definitively in number without any evident morphological abnormality [13]. By two-color contrast, megakaryocytic morphological abnormality is more distinct than that by M-G staining. Nuclear-cytoplasmic dissociation was more identifiable in H&E-stained specimens. As shown in **Figure 5**, polynucleated cells and cells with lobulated nuclei were identified in pro-erythroblasts of BM clot samples of

**Figure 6.** Dysplastic erythrocytes observed by H&E staining. (A, B) nuclear-cytoplasmic dissociation. Arrows represent the dysplastic erythroblasts that have baso-eosinophilic cytoplasm. (C, D) abnormal pro-erythroblasts. Arrows represent the abnormally hyperlobated nuclei.

**Figure 7.** Dysplastic myelocytes observed by H&E staining. (A–D) dysplastic metamyelocytes maturing into neutrophils, which have blots in the cytoplasm (indicated by arrows).

#### **3.3. Dysplastic megakaryocytes in refractory anemia with excess blasts and MDS/MPN**

In H&E-stained clot specimens of refractory anemia with excess blasts and MDS, abnormal megakaryocytes, which include micromegakaryocytes and megakaryocytes with large mononuclear form, and dispersed round nucleoli were observed.

Morphological changes and increase in the number of megakaryocytes are informative for diagnosis. Micromononuclear megakaryocytes are detected in MDS, while large, over-mature megakaryocytes have been observed to increase in number in myeloproliferative neoplasms [12]. In anaplastic anemia, the megakaryocytes decrease definitively in number without any evident morphological abnormality [13]. By two-color contrast, megakaryocytic morphological abnormality is more distinct than that by M-G staining. Nuclear-cytoplasmic dissociation was more identifiable in H&E-stained specimens. As shown in **Figure 5**, polynucleated cells and cells with lobulated nuclei were identified in pro-erythroblasts of BM clot samples of MDS patients (**Figure 5A**–**C**).

**Figure 6.** Dysplastic erythrocytes observed by H&E staining. (A, B) nuclear-cytoplasmic dissociation. Arrows represent the dysplastic erythroblasts that have baso-eosinophilic cytoplasm. (C, D) abnormal pro-erythroblasts. Arrows represent

**Figure 5.** Dysplastic megakaryocytes detected by H&E staining. Megakaryocytes have round, dispersed nuclei (A, B, C).

the abnormally hyperlobated nuclei.

Two or three dispersed nuclei are observed (600×).

14 Myeloid Leukemia

In the erythroblast lineage, nuclear-cytoplasmic dissociation (**Figure 6A** and **B**) [2], evident as hemoglobin production in the cytoplasm without enucleation, is identifiable in H&E-stained specimens because of distinct two-color contrast.

**5. Discussion**

**hematological neoplasm**

with microvacuoles are atypical findings [14].

compose the core concepts of hematological neoplastic entities.

**5.1. Terminology: dysplastic, atypical, and blastic findings and interrelationship with** 

Dysplastic, atypical and abnormal, and blastic findings, together with hypercellularity, compose the core concepts of hematological neoplastic entities in BM pathology. It is possible to understand the interrelationship of the entities by organizing concepts on the basis of their morphological findings (**Figure 8**). Dysplasia refers to specified morphological features that are distinct from other abnormal or atypical morphologies. As an important example, dysplastic megakaryocytes have circular, dispersed nuclei, which differ from atypical and irregular megakaryocytes in MPN and MDS/MPN. Thus, the terms "abnormal" and "atypical" should be used to describe the morphological changes other than morphological changes in MDS. The cytological spectrum of atypical lymphocytes is extremely broad. For example, small- to large-sized cells with often regular or indented nuclei, moderately condensed chromatin, moderate-to-faint basophilic cytoplasm without azurophilic granules and sometimes

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On the other hand, the term "blast" refers to "immature" cells, which have a transparent nucleus with a prominent clear nucleus. For example, the "centroblast" is a morphological concept that refers to lymphocytes that have a specified morphological feature, such as prominent nucleoli representing an immunologically activated state. As another example, "B-lymphoblasts" refer to the activated B-lymphocytes that undergo hypersomatic mutations and the class switch of immunoglobulin genes. In other words, blastic lymphocytes correspond to the lymphocytes with gene recombination. Myeloblasts and erythroblasts, however, do not necessarily correspond to specific genetic alterations but correspond to the activated state of the first stage of differentiation. Distinguishing these morphological features from hyperplastic marrow will elucidate the mutual relationships among AML, AML arising from MDS, MDS, MDS/MPN, and MPN. MDS, AML, and MPN are specific entities on the basis of dysplastic, blastic (>20% of nucleated cell in BM), and hypercellular findings, respectively.

**Figure 8.** Interrelationships of hematopoietic neoplasms. Dysplastic, blastic findings, together with hypercellularity,

Multinuclear abnormal pro-erythroblasts are also the hallmark of dysplasia (**Figure 6C** and **D**) [2]. Furthermore, basophilic heterogeneity in the cytoplasm is observed. Neutrophils exhibited a "blot" or localized heterogeneous staining in the cytoplasm, which is frequently seen in H&E-stained specimens (**Figure 7A**–**C**) [2]. In addition, a ring-shaped nucleus, which is observed in murine neutrophils, was observed in MDS and MDS/MPN (**Figure 7D**) [2].
