Auer bodies were first described by light microscopy as stick-like and spiculate bodies in the cytoplasm of leukemic cells by John Auer in 1906.1 Since then, numerous ultrastructural observations have demonstrated a fibrillar or crystallized substructure to this inclusion in acute leukemias and granulocytic sarcomas.2–4 Many investigations have revealed that Auer bodies are mostly found in acute myelogenous leukemia (AML), especially in acute promyelocytic leukemia (APL) with large numbers in the cytoplasm of leukemic cells.5,6 Recently, clinical investigations have indicated that raised numbers of Auer bodies and primary granules in neoplastic cells were associated with severe hemorrhage and disseminated intravascular coagulation in APL patients.7
Histochemical studies have demonstrated that Auer bodies contain oxidase, myeloperoxidase, phosphatase and stain with periodic acid-Schiff (PAS), and Sudan black. They are negative for lipase, glycogen and deoxyribonucleic acid.8 Combined morphological transmission electron microscopy (TEM) and ultrastructural cytochemistry have confirmed Auer bodies as containing acid phosphatase and myeloperoxidase.9 Given that the characteristics of Auer bodies are the same as those of primary granules and azurophilic granules in normal granulocytes, some researchers have presumed that Auer rods in APL might originate from the fusion of azurophilic granules.10
Here, we demonstrate the presence of large cytoplasmic bodies, which we refer to as giant inclusions, and which coexist with typical Auer bodies in the leukemic blasts in APL. The morphologic characteristics and distinctive substructure of these giant inclusions suggest an alternative origin for Auer bodies in the cells of APL.
2. MATERIALS AND METHODS
2.1. Clinical data and laboratory examination
Ten previously untreated APL patients were referred to the Blood Diseases Hospital, Tianjin, between 2012 and 2022. They included 8 males and 2 females, aged between 10 and 50 years old. All patients were subjected to light microscope morphology, flow cytometry, cytogenetic analysis and molecular biological characterization, as well as TEM and ultrastructural cytochemistry. All patients were characterized by hypercellularity of myeloid blasts, the cytogenetic abnormality t (15; 17) (q24; q21) and positivity of PML/RAR. Diagnoses were made on the basis of the World Health Organization classification of myeloid neoplasms and acute leukemia.11 For comparison, mononuclear cells from bone-marrow aspirates of anemic patients were observed as control cells for the leukemic cells of APL.
2.2. Wright-Giemsa and cytochemical stains
Bone-marrow aspirate smears were stained with Wright-Giemsa and cytochemical stains, which included Sudan black, myeloperoxidase, PAS staining, alpha-naphthyl butyrate esterase (α-NBE), and naphthol AS-D acetate esterase (AS-D NAE).
2.3. Transmission electron microscopy
A portion of the mononuclear cells from the bone-marrow aspirates was conventionally fixed and embedded in resin. Briefly, the samples were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, washed in phosphate-buffered saline, dehydrated in graded alcohols and embedded in Epon 812. Ultrathin sections at 60 nm were cut and stained with uranyl acetate and lead citrate. For detection of myeloperoxidase activity, mononuclear cells were incubated for 1 hour in Graham and Karnovsky medium, and then processed for electron microscopy as described above, and unstained sections observed by TEM.12,13
3.1. Wright-Giemsa stain and cytochemistry for myeloperoxidase
The Wright-Giemsa stain exhibited hyper-proliferation of promyelocytes in bone-marrow smears in all cases. All promyelocytes contained numerous round azurophilic granules, but a small proportion of cells included rod-like Auer bodies and irregularly shaped inclusions (Fig. 1A and B). The Auer bodies, inclusions and granules showed strong reactivity for myeloperoxidase (Fig. 1C).
3.2. General features of promyelocytes
Normally, neutrophils included slender profiles of rough endoplasmic reticulum (rER), primary granules in promyelocytes, and specific granules in myelocytes to segmented granulocytes; few of the above organelles are found in myeloblasts. There were no Auer bodies, irregularly shaped inclusions or expanded rER cisternae by morphological TEM or ultrastructural myeloperoxidase staining (Fig. 2).
In contrast, in most of the promyeloblasts in APL, rER cisternae were noticeably expanded and filled with a homogeneous matrix, so that some of them looked like irregular lakes in the cytoplasm. Primary granules varied in size and electron density, whereas specific granules were seldom found in promyeloblasts. Sometimes, structures resembling a simple vesicle, with a clear unstructured content, were observed and it was difficult to know whether these structures were elements of rER or a variant of primary granules (Fig. 3A–D).
The ultrastructural myeloperoxidase stain exhibited distinct reactivity of the amorphous matrix within expanded rER cisternae, the numerous primary granules, as well as lake-like rER elements and giant inclusions in APL promyeloblasts (Fig. 3E–H).
3.3. Giant inclusions and lake-like rER elements
In the promyeloblasts of APL on TEM (Fig. 4A and B), all the giant inclusions containing a dense homogeneous matrix were surrounded by loops of membrane, giving the bodies a lace-like appearance. These loops of membrane were of uniform thickness—10 nm—and were dense as a result of enclosing the same material as in the central mass of the body. Auer bodies with typical features often coexisted with the giant dense inclusions in these promyeloblasts (Fig. 4C). All of the giant dense inclusions, Auer bodies, rER cisternae, and primary granules had strong reactivity to myeloperoxidase stain (Fig. 4D).
3.4. Pro-Auer bodies and primary granules
Some giant inclusions had features in common with Auer bodies in having a rod-like main body like Auer bodies, but retaining the lace-like dense membrane profiles like those around the dense giant inclusions (Fig. 5A–D). We refer to this kind of inclusion as a “pro-Auer body.” The pro-Auer bodies often had attached smaller loops of dense membrane than those around giant inclusions, but no fine lamellar texture or crystal in the center like those in typical Auer bodies (Fig. 5A–C).
There were prominent primary granules with myeloperoxidase reactivity in the cytoplasm of promyeloblasts; some of them were budding out from pro-Auer bodies and giant inclusions (Fig. 5D), and some of them were located within perinuclear spaces and expanded rER cisternae (Figs. 3H and 6A).
3.5. Substructure of Auer bodies
Auer bodies were often needle or rod-shaped, and had a smooth surface. Some of them contained homogeneous content, while others showed a fine lamellar or crystal-like inclusion (Fig. 6B–D). Crystalline inclusions, which may be precursors of the crystalline elements in Auer bodies, are present within some rER cisternae (Fig. 7).
Following the description in acute leukemia by Auer1 of the bodies named after him as Auer bodies or Auer rods, having a splinter-like appearance and tubular substructure on Wright’s stain by light microscopy, various pink-staining inclusions and granules occurring in leukemic blasts and nonleukemic cells have been reported.14 Some inclusions had a tubular substructure similar to Auer rods,15 but some inclusions were more voluminous and irregularly shaped, and were called “giant inclusions” or “megagranules.”16
Auer bodies were predominantly found in APL and were characterized by activity for Sudan black B and the PAS reaction cytochemically. It was thought that Auer bodies resulted from the fusion of primary granules in promyeloblasts based on their shared cytochemical reactions.17 Ultrastructural investigation demonstrated that Auer bodies were large membrane-bound organelles with a crystalline core, although some of them exhibited a more lamellar texture.9,18 The above descriptions of Auer bodies were consistent with findings in the present study based on cytochemistry and ultrastructure.
Giant inclusions were often demonstrated in neutrophils from patients with the Chediak-Higashi syndrome (CHS), occasionally found in acute myelomonocytic leukemia. These giant granules contained heterogeneous deposits and filamentous materials, and were thought to result from the fusion of primary and secondary granules based on their activity of myeloperoxidase under pathologic conditions.19,20
In this study, the homogeneous dense matrix, the positivity for myeloperoxidase, and the surrounding lace-like dense membrane mark them as different from the giant granules in CHS. The dense lace-like membrane associated with giant inclusions in our study was continuous with rER profiles nearby, some of which looked like the double-membrane-limited lysosomes found in CHS monocytes by combined electron microscopy and ultrastructural cytochemistry.21
An ultrastructural study demonstrated that prominent dilated rER, multi-laminar rER and complex stellate arrangements of rER appeared to be morpho-genetically related in APL.22 In this study, all the giant inclusions, Auer bodies, rER, and primary granule were characterized by high electron density and activity of myeloperoxidase of APL. This suggested that the giant inclusions might originate from expanded rER cisternae with abundant synthesized matrix within the cisternae. The presumption was substantiated by common myeloperoxidase activity of the giant inclusions and rER in promyeloblasts.23
Additionally, some giant inclusions showed an intermediate state between giant inclusions and Auer bodies, including a rod-like main body, furcate terminals and fewer, small membrane loops. We interpret the membrane loops as membrane being removed as the content of the body condenses and the body, as a whole, evolves from a more rounded or oval giant inclusion (asterisked in Fig. 4, for example) to a more slender and less voluminous rod-like pro-Auer body (Fig. 5). We termed these structures “pro-Auer bodies” because of the suggestion of a transition from giant inclusions to mature Auer bodies. Interestingly, in promyeloblasts, the myeloperoxidase stain demonstrated some primary granules located in the perinuclear space and within pro-Auer bodies, with a few of them budding off pro-Auer bodies. It suggested the possibility that Auer bodies originated from expanded rER cisternae rather than from the fusion of primary granules (Fig. 8). However, this presumption is contradicted by the idea in the literature that Auer bodies result from the fusion of cytoplasmic granules in promyeloblasts,2,10 and the novel hypothesis requires further identified using advanced devices.
ER is a dynamic membrane and serves many roles, including calcium storage, protein synthesis, transport and folding, lipid and steroid synthesis, and carbohydrate metabolism.24 Performing above diverse functions requires such distinct domains of ER with different architectures as tubules, sheets, and nuclear envelope.25,26 These structures are consisted with variant morphologies of Auer bodies and giant inclusions in this study, although transformation processes of giant inclusions associated with ER and rER to Auer bodies need to be demonstrated by dynamic techniques such as super-resolution microscopy and 3D correlative fluorescence and electron microscopy that developed in recent years.27
It is also possible that there is a morphological heterogeneity to Auer bodies, reflecting abnormalities that are almost a hallmark of the neoplastic process. One aspect of their development relates to how the crystalline component arises. This remains to be addressed in future work, although our preliminary findings include crystalline inclusions found within some rER cisternae; in a process as not yet defined, these rER cisternae may enter into the developmental process of Auer rod formation as illustrated in our Figure 7.
This study suggests the novel idea that expanded rER cisternae transform into giant inclusions and then Auer bodies partly as a result of the intracisternal accumulation of compounds rich in peroxides, and the progressive condensation of internal material by the release of loop-like membranous elements; further, that primary granules were directly released from pro-Auer bodies in a process which bypasses the Golgi apparatus in promyeloblasts of APL.
. Auer J. Some hitherto undescribed structures found in the large lymphocytes of a case of acute leukaemia. Am J Med Sci 1906;131:1002–1014.
. Bainton DF, Friedlander LM, Shohet SB. Abnormalities in granule formation in acute myelogenous leukemia. Blood 1977;49(5):693–704.
. Fukushi K, Nakasato N, Narita N, Yoshida K. Electron microscopic study of the Auer body. Acta Pathol Jpn 1972;22(3):509–515. doi:10.1111/j.1440-1827.1972.tb01848.x.
. Fujinaga S. Fine structure and development of Auer bodies. Kyushu J Med Sci 1964;15:59–68.
. Castoldi GL, Liso V, Specchia G, Tomasi P. Acute promyelocytic leukemia: morphological aspects. Leukemia 1994;8(Suppl 2):S27–S32.
. Yue QF, Xiong B, Chen WX, Liu XY. Comparative study of the efficacy of Wright-Giemsa stain and Liu’s stain in the detection of Auer rods in acute promyelocytic leukemia. Acta Histochem 2014;116(6):1113–1116. doi:10.1016/j.acthis.2014.05.005.
. Ikezoe T. Advances in the diagnosis and treatment of disseminated intravascular coagulation in haematological malignancies. Int J Hematol 2021;113(1):34–44. doi:10.1007/s12185-020-02992-w.
. Ackerman GA. Microscopic and histochemical studies on the Auer bodies in leukemic cells. Blood 1950;5(9):847–863.
. White JG. Fine structural demonstration of acid phosphatase activity in Auer bodies. Blood 1967;29(4):667–682.
. Freeman JA. Origin of Auer bodies. Blood 1966;27(4):499–510.
. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016;127(20):2391–2405. doi:10.1182/blood-2016-03-643544.
. Roels F, Wisse E, Brest BDE, et al. Cytochemical discrimination between catalases and peroxidases using diaminobenzidine. Histochemistry 1975;41(4):281–312. doi:10.1007/BF00490073.
. Buccheri V, Shetty V, Yoshida N, Morilla R, Matutes E, Catovsky D. The role of an anti-myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry. Br J Haematol 1992;80(1):62–68. doi:10.1111/j.1365-2141.1992.tb06401.x.
. Yanagihara ET, Naeim F, Gale RP, Austin G, Waisman J. Acute lymphoblastic leukemia with giant intracytoplasmic inclusions. Am J Clin Pathol 1980;74(3):345–349. doi:10.1093/ajcp/74.3.345.
. Newburger PE, Novak TJ, McCaffrey RP. Eosinophilic cytoplsmic inclusions in fetal leukocytes: are Auer bodies a recapitulation of fetal morphology. Blood 1983;61(3):593–595.
. Lejeune F, Turpin F, Lortholary P. Unusual giant inclusions in blast cells during acute transformation of chronic myeloid leukemia. Cytochemical and ultrastructural study. Pathol Biol (Paris) 1978;26(1):7–11.
. Schmalzl F, Huhn D, Asamer H, Rindler R, Braunsteiner H. Cytochemistry and ultrastructure of pathologic granulation in myelogenous leukemia. Blut 1973;27(4):243–260. doi:10.1007/BF01637437.
. Gorius JB, Houssay D. Auer bodies in acute promyelocytic leukemia. Demonstration of their fine structure and peroxidase localization. Lab Invest 1973;28(2):135–141.
. Payne CM, Harrow EJ. A cytochemical and ultrastructural study of acute myelomonocytic leukemia exhibiting the pseudo-Chediak-Higashi anomaly of leukemia and “splinter-type” Auer rods. Am J Clin Pathol 1983;80(2):216–223. doi:10.1093/ajcp/80.2.216.
. White JG, Clawson CC. The Chédiak-Higashi syndrome; the nature of the giant neutrophil granules and their interactions with cytoplasm and foreign particulates. I. Progressive enlargement of the massive inclusions in mature neutrophils. II. Manifestations of cytoplasmic injury and sequestration. III. Interactions between giant organelles and foreign particulates. Am J Pathol 1980;98(1):151–196.
. White JG, Clawson CC. The Chédiak-Higashi syndrome: ring-shaped lysosomes in circulating monocytes. Am J Pathol 1979;96(3):781–798.
. Parkin JL, Brunning RD. Unusual configurations of endoplasmic reticulum in cells of acute promyelocytic leukemia. J Natl Cancer Inst 1978;61(2):341–348.
. Zassadowski F, Ades L, Schlageter MH, et al. Auer rods and differentiation in acute promyelocytic leukemia. Br J Haematol 2008;142(6):998–1000. doi:10.1111/j.1365-2141.2008.07282.x.
. Obara CJ, Moore AS, Lippincott-Schwartz J. Structural diversity within the endoplasmic reticulum-from the Microscale to the Nanoscale. Cold Spring Harb Perspect Biol 2022;19:a041259. doi:10.1101/cshperspect.a041259.
. Reid DW, Nicchitta CV. Diversity and selectivity in mRNA translation on the endoplasmic reticulum. Nat Rev Mol Cell Biol 2015;16(4):221–231. doi:10.1038/nrm3958.
. Westrate LM, Lee JE, Prinz WA, Voeltz GK. Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem 2015;84:791–811. doi:10.1146/annurev-biochem-072711-163501.
. Kounatidis I, Stanifer ML, Phillips MA, et al. 3D Correlative Cryo-structured illumination fluorescence and soft X-ray microscopy elucidates reovirus intracellular release pathway. Cell 2020;182(2):515–530.e17. doi:10.1016/j.cell.2020.05.051.