Practical challenges in peripheral blood smear evaluation

The recommended lymphocyte count at diagnosis is greater than 2 × 10⁹/L, but T-LGL can be diagnosed at a lower percentage in the presence of clinical features or proof of a clonal process. Lymphocytes should also account for greater than 15 percent of the white blood cell count (normal is about five percent).

Patients commonly have neutropenia and may have anemia, but thrombocytopenia is rare. Bone marrow red blood cell hypoplasia is common, and patients often have mild to moderate splenomegaly.

“This is very difficult to diagnose,” Dr. Chabot-Richards said. “Our first ancillary test that helps us out is the flow immunophenotype. It’s a mature, post-thymic, T-cell phenotype” with CD3, CD57, CD16, and CD2. The vast majority of cases are CD8 positive and T-cell receptor α/β positive. They can also show loss of normal T-cell antigens, most commonly CD5 and CD7.

A STAT3 mutation is seen in 70 percent of cases of T-LGL (“not specific; it’s seen in many other disorders”), while another subset of cases has a STAT5B mutation, which is more commonly seen in CD4-positive cases. “This is an interesting finding because they are also coming out with STAT3 inhibitors, so this is a potential target for treatment,” Dr. Chabot-Richards said.

The majority of T-LGL cases have positive T-cell gene rearrangement, and testing is required to prove clonality. The normal distribution of the lengths of the T-cell receptor gene should have a nice bell curve. In clonal processes, there is usually a single dominant peak. “In 30 percent of cases, the length of this sequence will actually change over the course of the disease. So you can’t compare in the same patient and necessarily prove it’s the same process.”

Patients who have multiple waxing and waning clones are similar to patients who have a stable clone, so it does not predict their disease.

“T-LGL cases do have a heterogeneous course,” Dr. Chabot-Richards said. They are typically indolent, and about half the time patients are symptomatic at diagnosis while the other half are found incidentally on the CBC.

Some cases spontaneously regress, “so the thinking is, the process is driven by chronic antigen stimulation and chronic inflammation. If you can control what is causing that, you can help treat the leukemia,” she said.

Other cases progress and require treatment, and normal progression is that the patients would have increasing cytopenias and become symptomatic. “Patients can have recurrent infections due to immune dysregulation” and other immune system problems, she said.

The traditional chemotherapy for these patients, if needed, is methotrexate or cyclophosphamide. Clinical trials are underway for STAT3 inhibitors.

The differential diagnosis would be B-cell disorders, which Dr. Wilson discussed, and other T-cell disorders: Sezary syndrome (CD4 positive in majority of cases, versus CD8), T-cell prolymphocytic leukemia (rapidly rising, very high WBC count and CD4 positive or, in a subset, CD4/CD8 double positive), and adult T-cell leukemia (usually CD4 and CD25 positive, CD57 negative).

“The NK cell disorders can be a little difficult to pick apart sometimes,” Dr. Chabot-Richards said. They are CD2, CD16, and CD56 positive and should be surface CD3 negative. T-cell clonality studies should be negative. “Instead, you can look at KIR receptors to see if that has clonal restriction,” which is a send-out test and done in only a few laboratories.

Chronic lymphoproliferative disorders of NK cells have morphology that is indistinguishable from large granular lymphocytic leukemia (LGL). Immunophenotype is key to distinguishing these disorders from LGL. Patients with aggressive NK-cell leukemia, on the other hand, are easier to distinguish because they have acute symptoms and morphologic abnormalities.

“We also have to distinguish T-LGL from reactive granular lymphocyte expansions,” she said. “This can be very difficult because there is a lot of overlap.” Viral infections—most commonly Epstein-Barr virus and cytomegalovirus—will be associated with a T-cell lymphocytosis.

“The big question is, when do we run flow cytometry on our patients with a cytosis?” (Fig. 2). If there is anything that looks monomorphic on the peripheral blood smear, and certainly if there is anything with a CLL-like phenotype, it is important to consider flow cytometry. Age greater than 65 is a factor. And studies have shown that a good lymphocyte count to consider reflex flow cytometry is greater than 6.7 × 10⁹/L, for a 95 percent sensitivity and 76 percent specificity for finding a neoplastic process.

Case No. 3 is that of a 47-year-old man with a history of medical problems including the following: a 1996 cadaveric renal transplant due to diabetes mellitus; lymph node diagnosed with an early post-transplant lymphoproliferative disorder in 2002; classic Hodgkin lymphoma diagnosed in 2003 and treated with five cycles of chemotherapy; and a second renal transplant in 2014 for end-stage renal disease owing to diabetes.

The patient was on a number of medications, among them Bactrim, mycophenolate mofetil, tacrolimus, and prednisone.

“I received a peripheral blood smear for review from the technologists who had questions about it,” Dr. Wilson said. The smear showed a mild, normochromic normocytic anemia with other lineages intact, and an increased number of abnormally segmented neutrophils. Abnormal neutrophil segmentation can be hyposegmented or hypersegmented. If it is hyposegmented, as in the smear reviewed, the two main considerations are a congenital or an acquired Pelger-Huët anomaly.

Congenital Pelger-Huët anomaly was first described in 1928 by Dr. Karl Pelger as a defect in neutrophil segmentation with neutrophils having short and compact nuclei containing very clumped chromatin. In 1931, Dr. G. J. Huët recognized this to be a benign, autosomal dominant, inherited disorder, which is now called Pelger-Huët anomaly. The cause was discovered in 2000 to be due to lamin B receptor (LBR) gene mutations.

The heterozygous form of Pelger-Huët anomaly affects nearly one in 5,000 people in the United States. The homozygous form is rarely seen because it is lethal in utero in most cases.

The LBR gene codes for LBR protein on the inner surface of the nuclear membrane. “This protein allows for trafficking of heterochromatin and nuclear lamins, which form the scaffolding proteins of the nuclear membrane,” Dr. Wilson said.

Mutations result in a decreased amount of this functional protein and decreased segmentation of nuclei.

In the congenital form of Pelger-Huët anomaly, the LBR protein amount determines the degree of segmentation (Fig. 3). Most cases (heterozygous) have bilobed nuclei that are symmetrical in appearance and are separated by thin chromatin strands, often described as resembling pince-nez glasses, or thicker strands shaped more like a dumbbell. Only a few cells, termed Stodtmeister cells, are monolobated with nuclei resembling peanuts or coffee beans. These cells are more frequently observed in acquired pseudo-Pelger-Huët anomaly. Chromatin is usually very clumped. The cytoplasm appears normal, so the cells function normally and patients usually do not have associated cytopenias.

Acquired pseudo-Pelger-Huët anomaly can be iatrogenic or secondary to a neoplastic disorder. Iatrogenic causes are most commonly associated with transplant medications, though a number of other drugs can be responsible, as well as infections and inflammatory disorders. About five percent of renal transplant patients receiving medications have transient pseudo-Pelger-Huët anomaly.

“Hypotheses for the pseudo-Pelger-Huët appearance to the neutrophils are that drugs, infection, or inflammation cause downregulation of LBR gene expression, or that they interact with the LBR proteins to downregulate protein function,” Dr. Wilson said.

A peripheral blood smear from a two-year-old with a severe bacterial infection showed a number of pseudo-Pelger-Huët-type nuclei with asymmetrical nuclear lobation and Stodtmeister cells (Fig. 4). “You could see the neutrophilic left shift with toxic granulation and reactive appearing monocytes in the background, so this was not a congenital Pelger-Huët anomaly.”

“When pseudo-Pelger-Huët anomaly is observed on a blood film, concern for a myelodysplastic syndrome [MDS] often arises,” she said. A study of patients with acquired pseudo-Pelger-Huët anomaly (Wang E, et al. Am J Clin Pathol. 2011;135[2]:291–303) found that on average, a greater proportion of neutrophils have pseudo-Pelger-Huët-type nuclei in the iatrogenic causes than with MDS. The neutrophil hyposegmentation also resolves with adjustments of medications or disease resolution.

To assist in diagnosing MDS (Fig. 5), “there are a number of findings we look for on peripheral blood smear review that we consider dysplastic,” Dr. Wilson said, although none is specific or pathognomonic. “You have to make sure that everything reactive is excluded that could cause similar changes.” Anemia is the most common cytopenia associated with MDS and is usually normochromic normocytic, or macrocytic, with some anisopoikilocytosis, and coarse basophilic stippling can be seen. “One of the easiest dysplastic changes to recognize in the peripheral blood is hypogranulation of the neutrophil cytoplasm,” for which she advises caution and a well-stained smear.

“In a patient with MDS (Fig. 6), neutrophils with normal appearing cytoplasmic granules should always be present on the same slide as hypogranular neutrophils to ensure adequacy of staining,” Dr. Wilson said. Pseudo-Pelger-Huët cells associated with MDS differ from congenital Pelger-Huët anomaly by their increased nuclear heterogeneity, increased N:C ratios, and less condensed chromatin.

The 2016 revision to the WHO classification of myeloid neoplasms revises the MDS categories. “The new classification uses a combination of the number of cytopenias, the number of cell lineages that have more than 10 percent dysplasia per lineage, and the percent blasts in peripheral blood and bone marrow to define seven major categories,” Dr. Wilson said. The WHO also recommends providing in reports the cytologic features described as being dysplastic. The requirement for MDS classification based on blasts has also changed. Previously, if there was one percent blasts in the peripheral blood, it was at least an MDS unclassifiable. “Now you have to repeat with a second blood smear at a different time to confirm the one percent blasts. Bone marrow testing is required additionally to assess for ring sideroblasts and to do cytogenetics, particularly to look for chromosome 5q deletions.”

Amy Carpenter Aquino is CAP TODAY senior editor.