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Megakaryocyte cytoplasmic fragments and ragocytes in the peripheral blood of a domestic cat infected with Anaplasma

Megakaryocyte cytoplasmic fragments and ragocytes in the peripheral blood of a domestic cat... In this case report, the author reports three strong and highly unusual changes in the peripheral blood of a domestic cat infected with Anaplasma. A freshly made blood smear from the cat Anaplasma had a shower of about 100 variably large to huge cytoplasmic fragments of megakaryocyte cytoplasm along the feathered edge. These had budding of platelets at their margins. Neutrophils with red cytoplasmic inclusions resembling ragocytes and neutrophils with ingested round inclusions of nuclear material were seen in a 1-day-old EDTA blood sample but not the freshly made blood smear. The cells with nuclear material resembled LE cells or alternatively Tart cells. Keywords Domestic cat · Peripheral blood · Megakaryocyte cytoplasmic fragments · Ragocytes · LE cells · Anaplasma Introduction rheumatoid arthritis but even systemic lupus, Waldenström’s macroglobulinemia, multiple myeloma, Hodgin’s disease, Anaplasma infection in cats often does not cause a reduced and trypanosomiasis. Phagocytosis was positively associated platelet count. A review of Anaplasma in cats indicated that with rheumatoid factor but occurred with sera and synovial thrombocytopenia was identified in only 20/34 cats (Schäfer fluid without rheumatoid factor from patients with the other and Kohn 2020). A platelet count within reference intervals diseases (Cats et al. 1975). does not eliminate the possibility that there was increased LE cells are phagocytes that ingested a LE body, which destruction of platelets caused by the Anaplasma, but that is a homogenous, round clump of magenta colored mate- there is a great enough increase in platelet production to rial. This nuclear material is from dead cells and has been offset destruction of platelets. acted upon by antinuclear antibodies. LE cells were reported Ragocytes in synovial fluid of human patients have been once before in peripheral blood of a human lupus patient associated with rheumatoid arthritis (Cats et  al. 1975). (Hepburn 2001). The LE phenomenon can be recreated by Ragocytes and LE cells have been reported in synovial incubation of buffy coat leukocytes in vitro with plasma fluid of dogs and a cat with immune-mediated polyarthri- from patients with lupus. This is the basis of the LE test. tis (Anonymous 2022; Bolliger et al. 1994; Camus et al. Various methods have been used for the LE test, but one 2010). Ragocytes seen in joint fluid are neutrophils which method is to pass clotted venous blood through a wire mesh. have variably sized and variably colored, round cytoplasmic This damages leukocytes and releases nuclear material to inclusions that contain antibodies (IgM, IgG). Phagocytosis be exposed to autoantibodies against nuclear histones pos- of antibodies has been stimulated experimentally in vitro sibly in the serum. This mixture is centrifuged to obtain a by incubation of leukocytes together with synovial fluid buffy coat, which is then smeared on glass slides to allow or sera from human rheumatoid patients (Cats et al. 1975). a microscopic search for LE cells. It is important for the Granulocytes from clinically well people phagocytized IgM, operator to differentiate LE cells from Tart cells (Hepburn IgG, and C3 after incubation with sera from people with 2001). LE cells must be differentiated from Tart cells which are only phagocytes which had eaten a nucleus from a dead cell (nuclear phagocytosis). Formation of LE cells supports * H. Tvedten the diagnosis of lupus. Tart cell formation does not support University Animal Hospital Swedish University a diagnosis of lupus. Tart cells retain a distinct chromatin of Agricultural Sciences, Box 7038, 750 07 Uppsala, pattern in the round structure in the phagocyte. Tart was the Sweden Vol.:(0123456789) 1 3 Comparative Clinical Pathology name of a human patient who had many Tart cells in the The referring veterinarian asked us to evaluate what they bone marrow sample (Steensma 1999). called large platelets. The freshly made blood smear had about 104 large cytoplasmic fragments along the feathered edge which were interpreted to be fragments of megakar- Case description and discussion yocyte cytoplasm (Figs. 1 and 2). About 46 of these frag- ments of megakaryocyte cytoplasm were extraordinarily A tube of EDTA blood and one air-dried blood smear from a huge (Fig. 2). Some of the largest fragments looked like a 16-year-old cat with depression and vomiting were submitted complex of 2–4 smaller units. There was budding along the for laboratory analysis. The referring veterinarian’s serum margins of the structures suggesting the active formation amyloid A concentration was reported to be > 100 mg/L with of platelets. a reference value of < 7 mg/L and was thus very elevated. The appearance of neutrophils in a blood smear made The referring veterinarian asked us to confirm there were from the 1-day-old EDTA blood was different than that Anaplasma phagocytophilum inclusions in the neutrophils in neutrophils in the freshly made blood smear. About 24% of the blood smear. Hematology results were within reference the neutrophils in a smear made from the 1-day-old EDTA intervals, including the platelet count (210 × 10 /L) with a blood had variably sized, reddish cytoplasmic inclusions as reference interval of 170–490 × 10 /L. Anaplasma was iden- stained with a May Grunewald Giemsa stain (Fig. 3). No tified in 14–18% of neutrophils on the blood smears. reddish inclusions were seen in neutrophils in the freshly Fig. 1 Large fragments of megakaryocyte cytoplasm at the feathered neutrophil. The largest structure was a complex of three fragments. edge of a blood smear. Use a neutrophil (about 15  µm) or erythro- One neutrophil contains Anaplasma. May Grunewald Giemsa stain, cytes (about 6  µm) to indicate size of the larger structures. The 60 × objective diameters of these fragments were about 2 times the diameter of a 1 3 Comparative Clinical Pathology Fig. 2 Huge fragments of megakaryocyte cytoplasm of the feathered 9 times the diameter of a neutrophil. The right fragment resembles edge of a blood smear. The diameter of the left fragment was about 4 a complex of four smaller fragments. May Grunewald Giemsa stain, times the diameter of a neutrophil, and the right fragment was about 40 × objective made blood smear. Some monocytes also had the reddish disease of cats and dogs in certain regilons. The fragments inclusions. These cells resemble ragocytes reported previ- did not include a nucleus and were not megakaryocytes. ously in joint fluids of cats and dogs with immune-mediated Because it is a new observation, it is difficult to suggest a polyarthritis (Anonymous 2022; Bolliger et al. 1994; Camus unique name for the fragments which are intermediate in et al. 2010). Fewer neutrophils contained a round, dark blue size between megakaryocytes and platelets. structure resembling an altered nucleus (Fig. 3). Ragocytes Megakaryocytes produce platelets by extending very and nuclear phagocytosis were only seen in the smear made thin, almost thread-like extensions of cytoplasm with thicker from the day-old EDTA blood sample, which indicated they buds. These are termed proplatelets (Thon et al. 2010). Pro- formed in vitro. Similar numbers of neutrophils in both the platelets from megakaryocytes in the bone marrow and lung fresh and the 1-day-old blood smears had Anaplasma (14 extend out into the bloodstream. These then break into indi- and 18%). Similar numbers of neutrophils had one or more vidual platelets. Proplatelets have been studied in mouse small blue inclusions resembling Döhle bodies in both fetal liver megakaryocyte cultures. Proplatelets could be smears (18–22%). subdivided into two groups by their perimeter (Thon et al. The cat had three remarkable changes not previously 2010). The perimeter of large proplatelets was > 50 µm and described in peripheral blood of a cat with Anaplasma infec- about 30–50 µm for small proplatelets. Another interme- tion. The very large number of very large and variably large diate stage is termed a preplatelet (Thon et al. 2010). The to huge cytoplasmic fragments of megakaryocytes has not preplatelet is round-to-discoid form and about 2–10 µm in been described before even though Anaplasma is a common diameter. The size previously reported for proplatelets or 1 3 Comparative Clinical Pathology Fig. 3 Blood smear from day-old EDTA blood. One neu- trophil (upper center) contains a round dark body resembling an altered nucleus. Several neutro- phils contain various sized red inclusions and resembled rago- cytes. May Grunewald Giemsa stain, 60 × objective Funding Open access funding provided by Swedish University of Agri- pre-platelets is much smaller than the structures seen in this cultural Sciences. cat. An erythrocyte should be about 6 µm in diameter. Nei- ther the previously described proplatelets or pre-platelets are Declarations as large or round as the structures in this cat’s blood, which had diameters of 2 to 10 times the diameter of a neutrophil Ethics approval This is not applicable to a case report. No procedures (Fig. 2). were performed except for routine patient care. Ragocytes and LE cells have been reported in joint fluid but not in peripheral blood. The numerous ragocytes in the Consent to participate This is not applicable to a case report. No pro- cedures were performed except for routine patient care. But in addition, blood smear can support a conclusion that the neutrophils the University Animal Hospital of the Swedish University of Agricul- containing an altered nucleus were LE cells because for- tural Sciences obtains written consent from all patient owners to use mation of both is stimulated by a similar immune process. information from routine patient care for publications. Morphology is used for differentiation of LE cells from Tart Consent for publication The author consents for Comparative Clini- cells (Hepburn 2001; Steensma 1999). LE cells have a LE cal Pathology to publish this manuscript and may take ownership of body, which is a homogenous, round clump of magenta- it after doing so. colored material. Identification of LE cells in the LE test was earlier one of the criteria used to diagnose lupus. Tart Conflict of interest The author declares no competing interests. cells are only phagocytes which had eaten a nucleus from a dead cell (nuclear phagocytosis) and are not diagnostic for Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- lupus. Tart cells retain a distinct chromatin pattern in the tion, distribution and reproduction in any medium or format, as long round structure in the phagocyte. Cells in this cat were not as you give appropriate credit to the original author(s) and the source, light magenta colored but were often quite darkly stained. provide a link to the Creative Commons licence, and indicate if changes Some had a granular texture suggesting a retained chromatin were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated pattern and which would indicate that they were more likely otherwise in a credit line to the material. If material is not included in Tart cells and not typical LE cells. the article's Creative Commons licence and your intended use is not This cat recovered well to antibiotic treatment for Ana- permitted by statutory regulation or exceeds the permitted use, you will plasma and appeared normal after a few days of treatment. need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . No follow-up testing was performed. Therefore, it was not determined if the cat had similar cytoplasmic fragments before or after this hematological evaluation. 1 3 Comparative Clinical Pathology Schäfer I, Kohn B (2020) Anaplasma phagocytophilum infection in cats References a literature review to raise clinical awareness. J Feline Medicine and Surgery 22:428–441 Anonymous (2022) Immune-mediated polyarthritis in a cat. eClinpath Steensma DP (1999) Fifty years of Tart cells. Mayo Clin Proc 74:936–938 Cornell University. Available at https:// eclin path. com/ cytol ogy/ Thon JN, Montalvo A, Patel-Hett S, Devine MT, Richardson J, Ehrlicher synov ial- fluid/ le- ragoc ytes/. Accessed 15 Jan 2022 A, Larson MK, Hoffmeister K, Hartwig JH, Italiano JE Jr (2010) Bolliger AP, DeCamp C, Tvedten H (1994) What is your diagnosis? Cytoskeletal mechanics of proplatelet maturation and platelet Vet Clin Pathol 23(4):109–110 release. J Cell Biology 191(4):861–874 Camus MS, Almy FS, Cienava EA (2010) A ragtime joint. Immune- mediated polyarthropathy in a dog. Vet Clin Pathol 39(1):4 Publisher's Note Springer Nature remains neutral with regard to Cats A, Lafeber JM, Klein F (1975) Immunoglobulin phagocytosis by jurisdictional claims in published maps and institutional affiliations. granulocytes from sera and synovial fluids from various rheuma- toid and non-rheumatoid diseases. Ann Rhem Dis 34:146–155 Hepburn AL (2001) The LE Cell Rhematology 40:826–827 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Comparative Clinical Pathology Springer Journals

Megakaryocyte cytoplasmic fragments and ragocytes in the peripheral blood of a domestic cat infected with Anaplasma

Comparative Clinical Pathology , Volume OnlineFirst – Sep 3, 2022

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Abstract

In this case report, the author reports three strong and highly unusual changes in the peripheral blood of a domestic cat infected with Anaplasma. A freshly made blood smear from the cat Anaplasma had a shower of about 100 variably large to huge cytoplasmic fragments of megakaryocyte cytoplasm along the feathered edge. These had budding of platelets at their margins. Neutrophils with red cytoplasmic inclusions resembling ragocytes and neutrophils with ingested round inclusions of nuclear material were seen in a 1-day-old EDTA blood sample but not the freshly made blood smear. The cells with nuclear material resembled LE cells or alternatively Tart cells. Keywords Domestic cat · Peripheral blood · Megakaryocyte cytoplasmic fragments · Ragocytes · LE cells · Anaplasma Introduction rheumatoid arthritis but even systemic lupus, Waldenström’s macroglobulinemia, multiple myeloma, Hodgin’s disease, Anaplasma infection in cats often does not cause a reduced and trypanosomiasis. Phagocytosis was positively associated platelet count. A review of Anaplasma in cats indicated that with rheumatoid factor but occurred with sera and synovial thrombocytopenia was identified in only 20/34 cats (Schäfer fluid without rheumatoid factor from patients with the other and Kohn 2020). A platelet count within reference intervals diseases (Cats et al. 1975). does not eliminate the possibility that there was increased LE cells are phagocytes that ingested a LE body, which destruction of platelets caused by the Anaplasma, but that is a homogenous, round clump of magenta colored mate- there is a great enough increase in platelet production to rial. This nuclear material is from dead cells and has been offset destruction of platelets. acted upon by antinuclear antibodies. LE cells were reported Ragocytes in synovial fluid of human patients have been once before in peripheral blood of a human lupus patient associated with rheumatoid arthritis (Cats et  al. 1975). (Hepburn 2001). The LE phenomenon can be recreated by Ragocytes and LE cells have been reported in synovial incubation of buffy coat leukocytes in vitro with plasma fluid of dogs and a cat with immune-mediated polyarthri- from patients with lupus. This is the basis of the LE test. tis (Anonymous 2022; Bolliger et al. 1994; Camus et al. Various methods have been used for the LE test, but one 2010). Ragocytes seen in joint fluid are neutrophils which method is to pass clotted venous blood through a wire mesh. have variably sized and variably colored, round cytoplasmic This damages leukocytes and releases nuclear material to inclusions that contain antibodies (IgM, IgG). Phagocytosis be exposed to autoantibodies against nuclear histones pos- of antibodies has been stimulated experimentally in vitro sibly in the serum. This mixture is centrifuged to obtain a by incubation of leukocytes together with synovial fluid buffy coat, which is then smeared on glass slides to allow or sera from human rheumatoid patients (Cats et al. 1975). a microscopic search for LE cells. It is important for the Granulocytes from clinically well people phagocytized IgM, operator to differentiate LE cells from Tart cells (Hepburn IgG, and C3 after incubation with sera from people with 2001). LE cells must be differentiated from Tart cells which are only phagocytes which had eaten a nucleus from a dead cell (nuclear phagocytosis). Formation of LE cells supports * H. Tvedten the diagnosis of lupus. Tart cell formation does not support University Animal Hospital Swedish University a diagnosis of lupus. Tart cells retain a distinct chromatin of Agricultural Sciences, Box 7038, 750 07 Uppsala, pattern in the round structure in the phagocyte. Tart was the Sweden Vol.:(0123456789) 1 3 Comparative Clinical Pathology name of a human patient who had many Tart cells in the The referring veterinarian asked us to evaluate what they bone marrow sample (Steensma 1999). called large platelets. The freshly made blood smear had about 104 large cytoplasmic fragments along the feathered edge which were interpreted to be fragments of megakar- Case description and discussion yocyte cytoplasm (Figs. 1 and 2). About 46 of these frag- ments of megakaryocyte cytoplasm were extraordinarily A tube of EDTA blood and one air-dried blood smear from a huge (Fig. 2). Some of the largest fragments looked like a 16-year-old cat with depression and vomiting were submitted complex of 2–4 smaller units. There was budding along the for laboratory analysis. The referring veterinarian’s serum margins of the structures suggesting the active formation amyloid A concentration was reported to be > 100 mg/L with of platelets. a reference value of < 7 mg/L and was thus very elevated. The appearance of neutrophils in a blood smear made The referring veterinarian asked us to confirm there were from the 1-day-old EDTA blood was different than that Anaplasma phagocytophilum inclusions in the neutrophils in neutrophils in the freshly made blood smear. About 24% of the blood smear. Hematology results were within reference the neutrophils in a smear made from the 1-day-old EDTA intervals, including the platelet count (210 × 10 /L) with a blood had variably sized, reddish cytoplasmic inclusions as reference interval of 170–490 × 10 /L. Anaplasma was iden- stained with a May Grunewald Giemsa stain (Fig. 3). No tified in 14–18% of neutrophils on the blood smears. reddish inclusions were seen in neutrophils in the freshly Fig. 1 Large fragments of megakaryocyte cytoplasm at the feathered neutrophil. The largest structure was a complex of three fragments. edge of a blood smear. Use a neutrophil (about 15  µm) or erythro- One neutrophil contains Anaplasma. May Grunewald Giemsa stain, cytes (about 6  µm) to indicate size of the larger structures. The 60 × objective diameters of these fragments were about 2 times the diameter of a 1 3 Comparative Clinical Pathology Fig. 2 Huge fragments of megakaryocyte cytoplasm of the feathered 9 times the diameter of a neutrophil. The right fragment resembles edge of a blood smear. The diameter of the left fragment was about 4 a complex of four smaller fragments. May Grunewald Giemsa stain, times the diameter of a neutrophil, and the right fragment was about 40 × objective made blood smear. Some monocytes also had the reddish disease of cats and dogs in certain regilons. The fragments inclusions. These cells resemble ragocytes reported previ- did not include a nucleus and were not megakaryocytes. ously in joint fluids of cats and dogs with immune-mediated Because it is a new observation, it is difficult to suggest a polyarthritis (Anonymous 2022; Bolliger et al. 1994; Camus unique name for the fragments which are intermediate in et al. 2010). Fewer neutrophils contained a round, dark blue size between megakaryocytes and platelets. structure resembling an altered nucleus (Fig. 3). Ragocytes Megakaryocytes produce platelets by extending very and nuclear phagocytosis were only seen in the smear made thin, almost thread-like extensions of cytoplasm with thicker from the day-old EDTA blood sample, which indicated they buds. These are termed proplatelets (Thon et al. 2010). Pro- formed in vitro. Similar numbers of neutrophils in both the platelets from megakaryocytes in the bone marrow and lung fresh and the 1-day-old blood smears had Anaplasma (14 extend out into the bloodstream. These then break into indi- and 18%). Similar numbers of neutrophils had one or more vidual platelets. Proplatelets have been studied in mouse small blue inclusions resembling Döhle bodies in both fetal liver megakaryocyte cultures. Proplatelets could be smears (18–22%). subdivided into two groups by their perimeter (Thon et al. The cat had three remarkable changes not previously 2010). The perimeter of large proplatelets was > 50 µm and described in peripheral blood of a cat with Anaplasma infec- about 30–50 µm for small proplatelets. Another interme- tion. The very large number of very large and variably large diate stage is termed a preplatelet (Thon et al. 2010). The to huge cytoplasmic fragments of megakaryocytes has not preplatelet is round-to-discoid form and about 2–10 µm in been described before even though Anaplasma is a common diameter. The size previously reported for proplatelets or 1 3 Comparative Clinical Pathology Fig. 3 Blood smear from day-old EDTA blood. One neu- trophil (upper center) contains a round dark body resembling an altered nucleus. Several neutro- phils contain various sized red inclusions and resembled rago- cytes. May Grunewald Giemsa stain, 60 × objective Funding Open access funding provided by Swedish University of Agri- pre-platelets is much smaller than the structures seen in this cultural Sciences. cat. An erythrocyte should be about 6 µm in diameter. Nei- ther the previously described proplatelets or pre-platelets are Declarations as large or round as the structures in this cat’s blood, which had diameters of 2 to 10 times the diameter of a neutrophil Ethics approval This is not applicable to a case report. No procedures (Fig. 2). were performed except for routine patient care. Ragocytes and LE cells have been reported in joint fluid but not in peripheral blood. The numerous ragocytes in the Consent to participate This is not applicable to a case report. No pro- cedures were performed except for routine patient care. But in addition, blood smear can support a conclusion that the neutrophils the University Animal Hospital of the Swedish University of Agricul- containing an altered nucleus were LE cells because for- tural Sciences obtains written consent from all patient owners to use mation of both is stimulated by a similar immune process. information from routine patient care for publications. Morphology is used for differentiation of LE cells from Tart Consent for publication The author consents for Comparative Clini- cells (Hepburn 2001; Steensma 1999). LE cells have a LE cal Pathology to publish this manuscript and may take ownership of body, which is a homogenous, round clump of magenta- it after doing so. colored material. Identification of LE cells in the LE test was earlier one of the criteria used to diagnose lupus. Tart Conflict of interest The author declares no competing interests. cells are only phagocytes which had eaten a nucleus from a dead cell (nuclear phagocytosis) and are not diagnostic for Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- lupus. Tart cells retain a distinct chromatin pattern in the tion, distribution and reproduction in any medium or format, as long round structure in the phagocyte. Cells in this cat were not as you give appropriate credit to the original author(s) and the source, light magenta colored but were often quite darkly stained. provide a link to the Creative Commons licence, and indicate if changes Some had a granular texture suggesting a retained chromatin were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated pattern and which would indicate that they were more likely otherwise in a credit line to the material. If material is not included in Tart cells and not typical LE cells. the article's Creative Commons licence and your intended use is not This cat recovered well to antibiotic treatment for Ana- permitted by statutory regulation or exceeds the permitted use, you will plasma and appeared normal after a few days of treatment. need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . No follow-up testing was performed. Therefore, it was not determined if the cat had similar cytoplasmic fragments before or after this hematological evaluation. 1 3 Comparative Clinical Pathology Schäfer I, Kohn B (2020) Anaplasma phagocytophilum infection in cats References a literature review to raise clinical awareness. J Feline Medicine and Surgery 22:428–441 Anonymous (2022) Immune-mediated polyarthritis in a cat. eClinpath Steensma DP (1999) Fifty years of Tart cells. Mayo Clin Proc 74:936–938 Cornell University. Available at https:// eclin path. com/ cytol ogy/ Thon JN, Montalvo A, Patel-Hett S, Devine MT, Richardson J, Ehrlicher synov ial- fluid/ le- ragoc ytes/. Accessed 15 Jan 2022 A, Larson MK, Hoffmeister K, Hartwig JH, Italiano JE Jr (2010) Bolliger AP, DeCamp C, Tvedten H (1994) What is your diagnosis? Cytoskeletal mechanics of proplatelet maturation and platelet Vet Clin Pathol 23(4):109–110 release. J Cell Biology 191(4):861–874 Camus MS, Almy FS, Cienava EA (2010) A ragtime joint. Immune- mediated polyarthropathy in a dog. Vet Clin Pathol 39(1):4 Publisher's Note Springer Nature remains neutral with regard to Cats A, Lafeber JM, Klein F (1975) Immunoglobulin phagocytosis by jurisdictional claims in published maps and institutional affiliations. granulocytes from sera and synovial fluids from various rheuma- toid and non-rheumatoid diseases. Ann Rhem Dis 34:146–155 Hepburn AL (2001) The LE Cell Rhematology 40:826–827 1 3

Journal

Comparative Clinical PathologySpringer Journals

Published: Sep 3, 2022

Keywords: Domestic cat; Peripheral blood; Megakaryocyte cytoplasmic fragments; Ragocytes; LE cells; Anaplasma

References