Finding the morphologic clues to neutrophilia etiology Karen Lusky February 2021—Granulocyte morphology may contain clues to neutrophilia etiology, and that was the focus of a CAP20 virtual presentation by Olga Pozdnyakova, MD, PhD, associate professor of pathology at Harvard Medical School and medical director of the hematology laboratory at Brigham and Women’s Hospital. Reactive changes can mimic myeloproliferative neoplasm, but myeloproliferative neoplasm can have reactive morphology, she said. Pathologists can piece together clinical and morphological clues, “especially in concert with the clinical team, that may help them decide whether the changes are more reactive or more neoplastic in nature,” she told CAP TODAY in a follow-up interview. Neutrophilia is defined as greater than 7.7 × 109/L or two standard deviations above the mean, and it is important to note whether it is present in the context of the left shift. Dr. Pozdnyakova Also important to note is whether neutrophilia is accompanied by other cytoses or cytopenias and what the neutrophil morphologic changes are, Dr. Pozdnyakova said. “And as always, it is essential to review the peripheral blood smear in the context of the clinical picture. Is neutrophilia symptomatic or incidental? How long has it been present? Is the patient taking medications?” The primary causes of neutrophilia are both constitutional and acquired. Leukocyte adhesion deficiency, familial myeloproliferative neoplasm, and Down syndrome are examples of constitutional etiologies. Acquired primary neutrophilias are usually associated with myeloproliferative neoplasms, CML being one of the most common, followed by chronic neutrophilic leukemia or other Philadelphia-negative myeloproliferative neoplasms, atypical chronic myeloid leukemia, or other myelodysplastic/myeloproliferative neoplasm (MDS/MPN) overlap syndromes. Secondary neutrophilia signifies reactive changes to various conditions, she said, such as smoking, infection and inflammation, medications (growth factors and corticosteroids are most common), and stress. Neutrophilia can be part of the paraneoplastic syndrome or be seen in patients with a nonfunctioning spleen. Secondary neutrophilias are more common than primary and generally accompanied by count changes and various morphological changes, which Dr. Pozdnyakova reviewed in her presentation. She described a 36-year-old female who presented with an absolute neutrophil count of 14,000 in the setting of ongoing infection and accompanied by the left shift (Fig. 1). Metamyelocytes, promyelocytes, and myelocytes were reported on the patient’s white blood cell differential. “The presence of anemia and low platelets are likely related to ongoing chemotherapy for primary CNS lymphoma. Review of the smear shows characteristic reactive changes of toxic granulation, cytoplasmic vacuolization, and the presence of Dohle bodies in segmented neutrophils and in bands.” In another case, a 59-year-old male presented with extensive metastatic adrenocortical carcinoma (Fig. 2). “The patient had significant leukocytosis of greater than 20,000,” and bandemia with neutrophilia, Dr. Pozdnyakova said. “And on review of the peripheral blood smear we noticed that some of the neutrophils contained blue-green inclusions indicative of liver failure.” Chemistry results were consistent with multisystem organ failure, with elevated ALT, AST, BUN, and creatinine. These inclusions have been “unfortunately termed ‘death crystals,’” she said, because their emergence is usually associated with imminent death. The patient died soon after presenting. Dr. Pozdnyakova reported the case of a 64-year-old male admitted with respiratory failure and hypertension after three weeks of low-grade fever, cough, headache, and periodic sweating (Fig. 3). He was a hunter who resided in New England and his wife reported several tick bites. His WBC count of almost 20,000, with 84 percent neutrophils, was associated with cytoplasmic inclusions consistent with Anaplasma morulae, a rickettsial bacterium. “The morphologic impression, once we reviewed the smear, was confirmed by the PCR test,” Dr. Pozdnyakova said. “Presentation of leukocytosis in this particular case was unusual because most of the patients present with leukopenia and thrombocytopenia. And presence of leukocytosis may indicate coinfection with babesiosis, since the same tick transmits all three pathogens. (Fig. 3, lower left: Babesia within red blood cells with a characteristic Maltese cross.) She reported that their patient hadn’t contracted other pathogens. Howell-Jolly body-like inclusions (Fig. 4) can imitate intracytoplasmic organisms. These are nuclear fragments that could be seen in connection with antiviral medications, immunosuppression, and sometimes with myelodysplastic syndrome, Dr. Pozdnyakova reported, pointing out the “spherical nuclear fragments where they are usually peripherally placed” in the neutrophils. “In this case, with a patient with end-stage renal disease status post-kidney transplant, those Howell-Jolly body-like inclusions were likely associated with an ongoing infection because you can also see toxic granulation and some vacuolation in neutrophils.” In another case, a 62-year-old female with relapsed diffuse large B-cell lymphoma had an ongoing infection (Fig. 5). “The CBC shows neutrophilia with some left shift, six percent myelocytes, microcytic anemia, and very low platelets of 22,000,” Dr. Pozdnyakova reported. “Review of the neutrophils shows some of the changes we have already seen and associated with infection, such as toxic granulation, cytoplasmic vacuolization, Dohle bodies, and Howell-Jolly body-like inclusions.” Yet the cells also display prominent nuclear dysplastic changes, abnormal nuclear lobation in the form of hypolobated nuclei, she said. Dr. Pozdnyakova noted in Fig. 5 (two left images in top row and one image on bottom left) monolobated nuclei, usually called Stodtmeister cells. She pointed also to bilobed nuclei (lower middle and upper far right), referred to as pseudo Pelger-Huët cells. “Some cells also show cytoplasmic hypergranulation. These morphologic findings are concerning for neoplastic rather than reactive changes,” she said. A molecular analysis identified ASXL1 and DNMT3A mutations consistent with treatment-related MDS. “And in this case, it is possible that hypergranulation and vacuolization are a sign of dysplasia rather than infection.” Dysplastic changes in neutrophils can present in many forms, Dr. Pozdnyakova cautioned, presenting what she later described to CAP TODAY as the most recent difficult case (Fig. 6). The 72-year-old female patient had a known history of clonal hematopoiesis and presented with progressive leukocytosis of greater than 30,000. The WBC count showed neutrophilia and monocytosis. Ten percent of the cells were called basophils. “Upon review it was clear that the granules in those cells that were called basophils are much larger than in typical basophils and are reminiscent of encapsulated intracellular yeast, particularly Histoplasma, which corroborated the clinical impression of disseminated fungal infection,” Dr. Pozdnyakova said. “However, Histoplasma urine antigen came back negative.” Dr. Pozdnyakova then requested a myeloperoxidase stain, which accentuated forms that turned out to be dysplastic primary granules in the setting of progression of the MDS/MPN overlap syndrome. And once a peripheral blood smear was reviewed, it demonstrated other more typical dysplastic changes, she said, noting a pseudo-Pelger-Huët nucleus. A session attendee noted that few laboratories offer myeloperoxidase. Dr. Pozdnyakova acknowledged that the Dana-Farber Cancer Institute hematology laboratory is likely “the last man standing” in offering the myeloperoxidase cytochemical stain. They use nonspecific esterase, MPO, PAS, and specific esterase for all new cases of acute leukemia. “It’s a very rapid test, and it allows us to put a case into an acute myeloid leukemia with monocytic differentiation category, or just typical AML if it’s MPO positive, very fast,” she said—“or into acute lymphoblastic leukemia if it’s PAS positive.” Administration of growth factors is another important cause of neutrophilia, presenting with the morphologic finding of a significant degree of left shift that frequently includes blasts, Dr. Pozdnyakova noted. However, occasionally this clinical information isn’t readily accessible. In the case of a 56-year-old male patient with Burkitt lymphoma who was taking Granix for neutropenia, the blast count was two percent (Fig. 7). She said she has come across cases with blast counts reaching 10 percent. The additional morphologic features to look for in such cases are prominent toxic changes and nuclear- cytoplasmic dyssynchrony. “For example, you can see retention of primary granules with very mature nucleus,” she said. In looking at the promyelocytes, “sometimes you can see prominent perinuclear hoffs. Very often neutrophilia is accompanied by thrombocytosis,” but it wasn’t seen in this case. Having reviewed the possible causes of neutrophilia and their related morphologic changes, most of which were reactive, Dr. Pozdnyakova introduced a key case: a 69-year-old male who presented with chronic leukocytosis and neutrophilia (Fig. 8). “The CBC showed a WBC count close to 30,000, with an absolute neutrophil count of over 24,000. There was no significant left shift.” The myelocyte count was one percent. The CBC was notable for four percent basophilia, a concerning fact in this case,