Diagnosis of a T-lineage acute lymphoblastic leukemia through digitalized cell analysis of the pleural effusion
A 31-year-old man was admitted to the emergency room in August 2012 due to chest pain, dyspnea, cough, and fever. The patient did not have a medical history of night sweats, weight loss, or bleeding. The patient’s initial blood count was normal (white blood cell count: 8.55 × 109/L; red blood cell count: 4.71 × 1012/L; hemoglobin: 14.3 g/dL; and platelet count: 194 × 1 0 12/L). No lymph node enlargement was detectable upon physical examination. The electrocardiogram was normal; the echocardiogram showed pericardial effusion and a chest X-ray revealed bilateral pleural effusion (). Due to the fact that the patient’s general status was worsening, a thoracentesis was required. The fluid from the pleural effusion was sent to the laboratory for routine investigation.\nThe Sysmex XE-5000™ (Sysmex Corporation, Kobe, Japan) hematology analyzer, equipped with the body fluid module, was used as previously described to obtain the patient’s cell count, and to achieve white blood cell differentiation into mononuclear (MN) and polymorphonuclear cells; we obtained a cell count of 14,000 cells/μL (90% MN cells, and 10% polymorphonuclear cells). Cell cluster spreading from the MN area into the high-fluorescence area was observed (); this cell cluster had higher forward and side scatter than normal cells (), meaning that those cells were bigger and more complex. Cytospin preparation (CytoFuge2; IRIS International, Chatsworth, CA, USA) was performed with the pleural fluid using 250–300 μL of the cell suspension (20 cells/μL) to obtain a cell pellet of approximately 5,000 cells. The slides were air-dried and stained with the modified Wright-Giemsa method. Slides were analyzed using the CellaVision® DM96 (CellaVision AB, Lund, Sweden). Morphological examination () of the pleural fluid revealed a massive infiltration of immature cells, with large and clefted nuclei; the cytoplasm was basophilic without granules, and was scant in volume, large in size, presenting with fine chromatin and evident nucleoli. To clarify the nature of the immature cells observed in the pleural fluid, flow cytometry analysis was performed. A panel of antibodies including surface CD3 (CD3), cytoplasmic CD3 (CD3Cy), CD7, CD4, CD8, CD2, CD5, cytoplasmic TdT (TdTCy), CD45, and CD1a was used. The analysis was performed using a FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA). Cytograms () showed that the cells (in red; P1) were CD45+/−, CD3−, CD7++, CD5+, CD2−, CD34+/−, CD1a−, CD3Cy+, TdTCy+, which were immature T-lineage cells. Therefore, a bone marrow aspirate was performed (), which showed an infiltration of lymphoblast cells. Flow cytometry analysis on peripheral blood and bone marrow () were also performed; according to the European Group for the Immunological Classification of Leukemia (EGIL) criteria, the malignancy was classified as Pro T-lineage acute lymphoblastic leukemia (T-ALL) (EGIL T1). Cytogenetic and fluorescence in situ hybridization analyses showed a normal karyotype.\nThe patient received induction therapy according to the Northern Italy Leukemia Group’s protocol. He achieved complete remission on November 2012. Two months later, he received cord blood allogeneic stem cell transplantation and died during aplasia due to septic shock.