Forgotten value of the rapid, inexpensive, and lineage specific myeloperoxidase cytochemical stain in urgent clinical scenarios
Authors: Janne V. Rand, Prabhjot Kaur
Journal: American Journal of Hematology (2016)

Differentiating between Acute Promyelocytic Leukemia and Myelomonocytic/Monocytic Leukemias Acute promyelocytic leukemia (APL) is characterized by the t(15;17) PML-RARA translocation, and is treated with All trans retinoic acid. This leukemia can present with life-threatening DIC and must be diagnosed as soon as possible upon presentation. Morphologically in APL, promyelocytes typically have abundant cytoplasm and a characteristic bilobed nuclear morphology with numerous Auer rods. In the microgranular variant, promyelocytes can be difficult to differentiate from promonocytes in the peripheral blood. A 51-year-old female with a past medical history only notable for adult onset diabetes and hypercholesterolemia, presented with a 2-week history of progressive shortness of breath, fever, and epistaxis with gingival bleeding and easy bruising. A CBC showed a white blood cell (WBC) count of 2,310/l, hemoglobin of 5.4, and platelet count of 23,000. The patient was admitted and peripheral smear examination showed 34% blasts with irregular folded or bilobed nuclei, fine nuclear chromatin, and sparsely granular cytoplasm (Image 1A). Due to the hypogranular nature of the blasts, morphological features were indeterminate as to whether the blasts were monocytic or promyelocytic in origin. The MPO cytochemical stain was performed and showed complete positivity in the blasts. The patient was given a dose of ATRA based on provisional morphologic impression while awaiting definitive diagnosis. The bone marrow biopsy later showed a final diagnosis of microgranular variant of APL. FISH studies came back positive for t(15;17) with fusion of PML/RARA loci and the patient was appropriately followed-up with induction therapy. Distinguishing between Different Thresholds for Apheresis in New Acute Leukemias Hyperleukocytosis is a complication of acute leukemias and can result in life-threatening leukostasis. Apheresis is performed when symptoms of leukostasis and/or critical values are reached. Critical WBC counts are conventionally defined as higher than 100 × 109/l in AML and >300 × 109/l in acute lymphoblastic leukemia1. In cases where morphologic features of a newly presenting acute leukemia are too poorly differentiated to characterize, MPO cytochemistry can be performed and interpreted within 10 min. A 48-year-old female with a past medical history of invasive breast cancer treated with doxorubicin and cyclophosphamide 2 years earlier, presented with lymphadenopathy, fatigue, and blood work concerning for acute leukemia. Peripheral smear examination revealed 60% blasts with round to slightly irregular nuclear contours, fine chromatin, prominent nucleoli, and a moderate amount of basophilic cytoplasm. No Auer rods or granules were seen (Image 1B). Her WBC count was 131,980/l with a hemoglobin of 6.9 and platelet count of 103,000, and morphologically it was unclear whether the blasts were of lymphoid or myeloid origin. Her low hemoglobin prompted need for transfusion, but her high WBC count proved to be a contraindication and a quick determination as to whether or not she needed emergent apheresis needed to be made. The WBC level had reached the critical level for apheresis in the myeloid criteria but not yet the lymphoid criteria. A MPO stain was performed within 10 min and confirmed the blasts were of myeloid lineage (inset). Urgent leukopheresis was initiated and the patient's WBC count improved. She was then transfused for her low hemoglobin. The clinicians initiated induction treatment for AML. Flow cytometric analysis and subsequent bone marrow biopsy later confirmed myelomonocytic leukemia with a phenotype positive for CD45 (dim), HLADR, CD13, CD33, CD56(partial), CD64, and MPO(partial), and negativity for CD34, CD117, CD11b. Mixed Phenotype Acute Leukemias can be Missed by Flow Cytometry Accurate diagnosis of mixed phenotype acute leukemia (MPAL) is important for designation of myeloid lineage for therapeutic and prognostic implications. The 2008 WHO criteria for MPAL require MPO as a specific marker for myeloid lineage differentiation. The WHO considers CD13 and CD117 as myeloid associated antigens but not specific enough1-4. The WHO provides no numerical guidelines to allow for the designation of myeloid lineage. These cases may express a low level of MPO positivity that can be missed by flow analysis where the small signal may be interpreted as signal from more maturing granulocytes within the blast gate. Cytochemical interpretation based on visualization of stain within the blasts can be very specific. A 17-year-old female who was previously healthy, presented with fatigue, leukocytosis, anemia and thrombocytopenia. Her WBC count was 142,000/l with a hemoglobin of 3.5 and platelet count of 15,000. Her peripheral smear showed 91% blasts with fine nuclear chromatin and inconspicuous nucleoli. A subset displayed “hand mirror” morphology (L1 subtype by FAB classification) (Image 1C). Flow cytometric analysis revealed that greater than 90% of the blasts expressed cytoplasmic CD3 (supportive of a T cell lineage), TdT, and CD34 as well as surface co-expression of myeloid associated antigens CD13 and CD117. About 10% of the blast gated cells, also co-expressed cytoplasmic myeloperoxidase (cMPO), and cytochemical stains revealed MPO activity in 10% of blasts (Image 1D). The findings of cytoplasmic CD3 by flow analysis and cytoplasmic MPO by flow and cytochemical stain fulfilled the requirements for a MPAL, T/myeloid type, as per The WHO 2008 monogram 3. (A): Peripheral smear showing microgranular variant of APL. The blasts appear very similar to promonocytes and can be confused with those of acute myelomonocytic leukemia (FAB M4) and acute monocytic leukemia (FAB M5). In this case the blasts all stained with MPO (inset view). (Wright-Giemsa, original magnification ×1,000, inset ×1,000). (B): Peripheral smear showing poorly differentiated blasts in a newly diagnosed case of acute leukemia with a WBC of >100,000/l. A MPO stain confirmed myeloid origin. (Wright-Giemsa, original magnification ×500, inset ×1,000). (C, D): Peripheral smear of a MPAL with blast showing fine chromatin and hand mirror morphology, typically seen in lymphoblasts (Panel C). The blasts expressed cCD3 by flow cytometry and were positive for MPO per cytochemical staining (Panel D), confirming mixed lineage phenotype. (Wright-Giemsa, original magnification ×1,000). Both authors (Dr. Janne Rand and Dr. Prabhjot Kaur) contributed equally in regards to searching EMR for relevant cases, taking images and write up.