A 66-year-old male attended Zhejiang Provincial Hospital of Chinese Medicine (Hangzhou, China) in September 2022 with ‘recurrent episodes of weakness and dizziness for 2 years’. The patient felt dizzy and weak, along with leukopenia, severe anemia and thrombocytopenia (Table I). Bone marrow aspirations performed at the First Hospital of Chun'an County (Chun'an Branch of Zhejiang Provincial People's Hospital; Zhejiang, China) in June 2020 showed strong hematopoietic function, with impaired maturation of leukocytes and megakaryocytes, and active erythropoiesis. The patient received oral folic acid (5 mg, 3 times a day) and methylcobalamin (0.5 mg, 3 times a day) to replenish hematopoietic raw materials and relieve dizziness. The patient was transfused with B-type platelets when severely lethargic. In September 2022, the patient came to Zhejiang Provincial Hospital of Chinese Medicine with worsening fatigue and acute pruritus of both legs. The general condition and the results of conventional investigations are detailed in Fig. 1, Fig. 2 and the following descriptive record.

At 1 day after admission, the bone marrow smear revealed pathological hematopoiesis and 3.5% primordial immature granulocytes. Proliferating leukocytes accounted for 72.5% of all cells, with a marked increase in eosinophils, accounting for 26% of leukocytes. The erythrocyte ratio was 16% and erythrocytes showed pathological hematopoiesis. Lymphocytes accounted for 7.5% of the total cell count and presented normal morphology. A total of 2 megakaryocytes could be found throughout the smear, the megakaryocyte lineage was inactive and small nucleated megakaryocytes were easily seen. Lobulated nuclei and multinucleated megakaryocytes were reduced (Fig. 3A and B). Bone marrow smear and cytochemical staining analysis were performed by Department of Hematology of Zhejiang Provincial Hospital of Chinese Medicine(Hangzhou, China). Wright-Giemsa stain was applied for 10 sec at room temperature, then the same amount (0.5–0.8 ml) of phosphoric acid buffer was added and mixed with the dye solution at room temperature for 25 min. Subsequently, the smear was washed with distilled water and the slides were air-dried. The mounted slides were then examined and photographed using a light microscope (magnification, ×1,000).

Flow cytometry analysis of the bone marrow revealed 1.85% medullary primitive cells with abnormal immunophenotype. Elevated proportions of eosinophils were detected, with the overall proportion of nucleated cells at 20.7%. The differentiation of granulocytes was abnormal and the expression of nucleated red blood cell CD36/CD71 expression was diminished.

Bone marrow biopsy showed the fat percentage at ~65%, with 35% active nucleated cell proliferation, significantly active granulocyte lineage proliferation and increased eosinophil levels. A less active red lineage proliferation was shown with 2–10 megaloblasts/high power field (HPF). Multinucleated megakaryocytes and small nucleated megakaryocytes with reduced fractionated nuclei were easily seen. Few mature lymphocytes and plasma cells were seen. Pathological biopsies of the bone marrow showed immunophenotypic abnormalities, exhibiting CD34 (individual +), CD117 (individual +), MPO, CD15, CD235a, E-cadherin (partial +), CD61 (megakaryocyte +), CD20 (individual +); CD3 (few +), CD138, κ and λ (few +). Histopathology was performed by the Department of Pathology of Zhejiang Provincial Hospital of Chinese Medicine. Bone marrow tissue was fixed in 10% neutral formalin for 30–50 min at room temperature and rinsed under running water. The tissues were dehydrated sequentially by ethanol gradient. An equal mixture of pure alcohol and xylene was added for 15 min and xylene I and II solutions were each infiltrated for 15 min. A mixture of equal amounts of xylene and paraffin was infiltrated for 15 min, and then put into paraffin I and paraffin II permeable wax for 50–60 min each. Samples were slice to a thickness of 5 µm. Hematoxylin and eosin (HE) staining was performed according to routine protocols. Briefly, after deparaffinization and rehydration, 5-µm-thick sections were stained with hematoxylin solution for 5 min at room temperature, followed by five washes in 1% acid ethanol and then rinsed in distilled water. Subsequently, the sections were stained with eosin solution for 3 min at room temperature, followed by dehydration in ethanol twice for 20 min each and clearing in xylene. The mounted slides were then examined and photographed using a light microscope (optical microscope; Leica; magnification, ×50).

Myelofibrosis grade 0 (reticular fiber). Perls (−) (Perls' Prussian blue Stain). Reticulated fiber staining was performed by the Department of Pathology of Zhejiang Provincial Hospital of Chinese Medicine. 5-µm-thick sections were deparaffinized and fixed, oxidized with potassium permanganate solution for 3 min and rinsed in running water. The sections were bleached with oxalic acid solution for 1–2 min and rinsed in running water. Sections were stained with ferric ammonium sulfate solution for 3 min and rinsed with deionized water for 10 sec. Staining with silver ammonia solution for 5 min, rinsing with deionized water for 5–10 sec. Formaldehyde solution reduction for 30 sec, rinse with running water. Gold chloride solution toning 1 min and rinse. Staining with sodium thiosulfate solution for 2 min, rinse with running water. Slices were dehydrated until transparent and sealed. Iron staining was performed by the Department of Pathology of Zhejiang Provincial Hospital of Chinese Medicine. Dried bone marrow smears were placed in acidic potassium ferricyanide solution and stained for 30 min at room temperature. Following rinsing with distilled water, nuclear solid red stain was restained for 10–15 min and rinsed with running water. Sections were dried and sealed. Finally, the mounted slides were examined and photographed using a light microscope (optical microscope; Leica; magnification, ×50).

The chromosomes were normal. Hematological oncogene mutation detection showed U2AF1:NM_006758.2:exon2:c.C101T:p.(S34F) 42.46%, ASXL1:NM_015338.5:exon12:c.2422delC:p.(P808Lfs*10) 37.09%, ETV6:NM_015338.5:exon3:c.C313T:p.(R105X)(nonsense mutation) 33.97% and ETV6:NM_001987.4:exon6:c.G1106A:p.(R369Q)(missense mutation) 5.34%. Fusion genes ETV6-PDGFRA, FIP1L1-PDGFRA, TEL-PDGFRB, KIF5B-PDGFRA, STRN-PDGFRA, and PCM1-JAK2 were negative.

The patient had been leukopenic, anemic and thrombocytopenic for 2 years. The patient bone marrow smear shows morphologic dysplasia of erythrocytes and megakaryocytes. The 2016 WHO classification system was used to diagnose the patient with MDS with multilineage dysplasia (10). Three scoring methods were used to assess the MSD risk in accordance with the ‘2019 Update on Diagnosis, Risk-stratification, and Management’: The International Prognostic Scoring System (IPSS), the WHO classification-based Prognostic Scoring System (WPSS) and the Revised International Prognostic Scoring System (IPSS-R), where the patient scored, 0.5 (intermediate risk), 2 (intermediate risk) and 5 (high risk) respectively (11). However, the patient's eosinophil ratio (26.3% of leukocytes) and IgG4 (493 mg/dl) level were persistently abnormally elevated, which were inconsistent with the symptoms of MDS. The patient and his relatives requested for the MDS-related treatment to be withheld and only be provided with symptomatic treatment, including blood transfusion and granulocyte colony-stimulating factor leukostimulation.

At day 2 after admission, the ultrasound showed regular liver morphology, the left lobe of the liver was enlarged, with an upper and lower diameter of 11.1 cm and the anterior and posterior diameters were 6.8 cm, while the right liver volume was also normal, with a smooth surface. The echogenicity of the liver area was dense and evenly distributed with well-defined blood vessels. The spleen was full in shape, 4.6 cm thick, with uniform echogenicity and a hyperechoic nodule of ~0.6 cm by 0.4 cm in size, with a clear border and regular shape.

MRI results showed hepatic macrosomia, ferrous deposits and decreased T2W1 signal in the liver parenchyma and a large spleen. However, normal pancreatic morphology, no obvious abnormal kidney morphology and no clear posterior peritoneal structures were seen (Fig. 3C). From the imaging results, liver lesions were considered, and a liver tissue biopsy was completed 10 days post admission. Hepatic puncture pathology (Fig. 3D-F) revealed cell swelling, metachromatic deposition of iron-containing heme and moderate chronic inflammatory cell infiltration in the confluent area. Pathological biopsies of the liver showed immunophenotypic abnormalities, exhibiting Ki-67 (<3% +), CK8 (+), CD10 (+), CD34 (vascular + in the confluent area), HBsAg (−), HBcAg (−), Hep (+), IgG (partial +), IgG4 (+ >10/HPF), CD20 (−), CD3 (lymphocytes +) and CD138 (+). The immunohistochemical staining for IgG and IgG4 was performed by Department of Pathology of Zhejiang Provincial Hospital of Chinese Medicine. Sections 5-µm-thick were placed in 10% neutral formalin and then stained with 12.5% IgG (ZA-0448, Amresco) or IgG4 (ZA-0576, Amresco) fluorescent antibody stained for 60 min at room temperature. Excess fluorescent antibody was poured off, and the sections were washed twice in phosphate buffered saline(PBS) at pH 7.2–7.4 with agitation for 5 min each time, and then washed with distilled water for 1 min to remove salt crystals. It was buffered with 50% buffer (0.5 mol/L carbonate buffer pH 9.0–9.5) and blocked with glycerol. The mounts were then examined and photographed using a light microscope (magnification, ×100).

From these results IgG4-RD diagnosis was confirmed, and the patient was prescribed 20 mg prednisone twice daily for 3 weeks. Skin pruritus of both lower extremities improved dramatically, rashes were reduced, sizes of the liver and spleen were decreased, the lymph node sizes in the groin were reduced, blood cell counts were elevated compared with previous levels and IgG4 was reduced to 398 mg/dl (Fig. 2; Table I). After 52 days of hospitalization, the patient refused to continue treatment and requested to be discharged. In the first telephone follow-up on in March 2023 the patient reported a stable blood count, improved skin pruritus and no other significant discomfort. However, in the second follow up in September 2023 the patient reported low platelets and hemoglobin and was transfusion-dependent due to the lack of standardized treatment.