Anna Merino

Automatic Recognition of Atypical Lymphoid Cells From Peripheral Blood by Digital Image Analysis

OBJECTIVES The objective was the development of a method for the automatic recognition of different types of atypical lymphoid cells. METHODS In the method development, a training set (TS) of 1,500 lymphoid cell images from peripheral blood was used. To segment the images, we used clustering of color components and watershed transformation. In total, 113 features were extracted for lymphocyte recognition by linear discriminant analysis (LDA) with a 10-fold cross-validation over the TS. Then, a new validation set (VS) of 150 images was used, performing two steps: (1) tuning the LDA classifier using the TS and (2) classifying the VS in the different lymphoid cell types. RESULTS The segmentation algorithm was very effective in separating the cytoplasm, nucleus, and peripheral zone around the cell. From them, descriptive features were extracted and used to recognize the different lymphoid cells. The accuracy for the classification in the TS was 98.07%. The precision, sensitivity, and specificity values were above 99.7%, 97.5%, and 98.6%, respectively. The accuracy of the classification in the VS was 85.33%. CONCLUSIONS The method reaches a high precision in the recognition of five different types of lymphoid cells and could allow for the design of a diagnosis support tool in the future.

Massive intravascular haemolysis during Clostridium perfrigens sepsis of hepatic origin

An 83-yr-old woman was admitted to our hospital because of a 3-h history of fever and abdominal pain. She had a past history of cholangitis. Biochemical screening showed total bilirrubin 19.6 mg ⁄dL, lactate dehydrogenase (LDH) 2288 UI ⁄L and increased liver enzymes. An automatic blood cell count showed high leucocyte count (26.5 · 10 ⁄L), haemoglobin (Hb) 12.2 g ⁄dL, haematocrit (Hct) 0.36 L ⁄L, mean cell volume MCV 89 fl (Fig. 1A) and platelets count 156 · 10 ⁄L. After a short period in the hospital the patient showed haemodynamic failure. At this moment, Hb level was 6.0 g ⁄dL, Hct 0.08 L ⁄L and mean cell volume MCV 49 fl with a red blood cell (RBC) distribution size curve showing a marked left shift (Fig. 1B). Peripheral blood (PB) film showed severe anaemia with spherocytes and microspherocytes red blood cells (Fig. 2) and polymorphonuclear leucocytes markedly vacuoled, some of them with prominent Döhle bodies (Fig. 3). Coagulation tests suggested a disseminated intravascular coagulation. The visual examination of the blood samples revealed severe haemolysis. Abdominal computed tomography scan demonstrated a liver abscess.

Chronic nonspherocytic hemolytic anemia (CNSHA) and glucose 6 phosphate dehydrogenase (G6PD) deficiency in a patient with familial amyloidotic polyneuropathy (FAP)

SummaryA new glucose-6-phosphate dehydrogenase (G6PD) variant with severe erythrocytic G6PD deficiency and a unique pH optimum is described in a young patient with chronic nonspherocytic hemolytic anemia (CNSHA) and familial amyloidotic polyneuropathy (FAP). Chronic hemolysis was present in the absence of infections, oxidant drugs or ingestion of faba beans. Residual enzyme activity was about 2.6% and 63% of normal activity in erythrocytes and leucocytes, respectively. A molecular study using standard methods showed G6PD in the patient to have normal electrophoretic mobility (at pH 7.0, 8.0 and 8.8), normal apparent affinity for substrates (Km, G6P and NADP) and a slightly abnormal utilization of substrate analogues (decreased deamino-NADP and increased 2-deoxyglucose-6-phosphate utilization). Heat stability was found to be markedly decreased (8% of residual activity after 20 min of incubation at 46°C) and a particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH. Although molecular characteristics of this variant closely resemble those of G6PD Bangkok and G6PD Duarte, it can be distinguished from these and all other previously reported variants by virtue of its unusual pH curve. Therefore the present variant has been designated G6PD Clinic to distinguish it from other G6PD variants previously described.

Characterization and automatic screening of reactive and abnormal neoplastic B lymphoid cells from peripheral blood.

The objective was to advance in the automatic, image‐based, characterization and recognition of a heterogeneous set of lymphoid cells from peripheral blood, including normal, reactive, and five groups of abnormal lymphocytes: hairy cells, mantle cells, follicular lymphoma, chronic lymphocytic leukemia, and prolymphocytes.

Automatic classification of atypical lymphoid B cells using digital blood image processing

There are automated systems for digital peripheral blood (PB) cell analysis, but they operate most effectively in nonpathological blood samples. The objective of this work was to design a methodology to improve the automatic classification of abnormal lymphoid cells.

Feature Analysis and Automatic Identification of Leukemic Lineage Blast Cells and Reactive Lymphoid Cells from Peripheral Blood Cell Images

Automated peripheral blood (PB) image analyzers usually underestimate the total number of blast cells, mixing them up with reactive or normal lymphocytes. Therefore, they are not able to discriminate between myeloid or lymphoid blast cell lineages. The objective of the proposed work is to achieve automatic discrimination of reactive lymphoid cells (RLC), lymphoid and myeloid blast cells and to obtain their morphologic patterns through feature analysis.

Chronic (B cell) lymphocytic leukaemia with unusual granulation

A 79-year-old patient with chronic lymphocytic leukaemia (CLL), in whom the neoplastic B lymphocytes from peripheral blood (PB) and bone marrow showed intracellular granules, is described. The diagnosis of CLL had been made in 1992 (clinical stage Rai 0, Binet A). The patient remained asymptomatic without any need for treatment until 5 years before this presentation, when a significant enlargement of lymph nodes in several areas was noted. Combination chemotherapy (CHOP: cyclophosphamide, doxorubicin, vincristine and prednisone) was started and subsequently replaced by a 3to 4-weekly dose of cyclophosphamide and intermediate dose prednisone. Clinical progression was evident over a period of several months, with increasing lymphocytosis and lymphadenopathy, unresponsive to chemotherapy. At this stage, an automatic blood cell count (Advia-120; Bayer, Leverkusen, Germany) showed a high leucocyte count (77.8 · 10/l) and anaemia (haemoglobin, Hb: 9.9 g/dl) with a normal platelet count. A PB film showed 96% lymphocytes with 90% of these containing variably sized, round, azurophilic cytoplasmic granules (left). Some cells also showed small vacuoles. Neoplastic lymphocytes did not show acid hydrolases on cytochemical staining. Immunophenotyping showed that 95% of lymphocytes expressed a characteristic CLL phenotype: B lineage (CD19) with co-expression of CD23 and CD5 and weak expression of CD20, CD22 and CD79b. The neoplastic population showed co-expression of immunoglobulin (Ig)D and IgM, a restricted lambda light chain pattern and high expression of CD38 (>30%) and ZAP-70 (>20%). Cytogenetic analysis showed t(1;4)(q31;q33) and trisomy 12. Transmission electron microscopy (TEM) showed that PB lymphocytes contained variably sized, irregular, dense, cytoplasmic granules (right, TEM · 10 000). The incidence of cytoplasmic inclusions in CLL lymphocytes is very low: membrane-bound vacuoles and crystal-like or filamentous inclusions have occasionally been reported. An immunoglobulin origin of the very unusual cytoplasmic granules in this patient is highly probable.

New quantitative features for the morphological differentiation of abnormal lymphoid cell images from peripheral blood

Aims This work aims to propose a set of quantitative features through digital image analysis for significant morphological qualitative features of different cells for an objective discrimination among reactive, abnormal and blast lymphoid cells. Methods Abnormal lymphoid cells circulating in peripheral blood in chronic lymphocytic leukaemia, B-prolymphocytic leukaemia, hairy cell leukaemia, splenic marginal zone lymphoma, mantle cell lymphoma, follicular lymphoma, T-prolymphocytic leukaemia, T large granular lymphocytic leukaemia and Sézary syndrome, normal, reactive and blast lymphoid cells were included. From 325 patients, 12 574 cell images were obtained and 2676 features (27 geometric and 2649 related to colour and texture) were extracted and analysed. Results We analysed the 20 most relevant features for the morphological differentiation of the 12 lymphoid cell groups under study. Most of them showed significant differences: 19 comparing follicular and mantle cells, 18 for blast and reactive cells, 17 for Sézary cells and T prolymphocytes and 16 for B and T prolymphocytes and 16 for villous lymphocytes. Moreover, a total of five quantitative features were significant for the discrimination among reactive and the set of abnormal lymphoid cells included. Conclusions Image analysis may assist in quantifying cell morphology turning qualitative data into quantitative values. New cytological variables were established based on geometric and colour/texture features to contribute to a more accurate and objective morphological assessment of lymphoid cells and their association with flow cytometry methods may be interesting to explore in the next future.

Massive erythrophagocytosis by peripheral monocytes and neutrophils in parvovirus-B19 autoimmune hemolytic anemia

Dear Editor, In response to the interesting case of diffuse large B cell lymphoma (DLBCL) and hemophagocytosis in peripheral blood (PB) reported in [1] associated to a fatal outcome, we would like to point out the relevance to detect this rare phenomenon and the clinical utility for this morphologic finding. A 37-year-oldmale, with clinical history of severe combined immunodeficiency (SCID), Evans syndrome, splenectomy, and autoimmune hemolytic anemia, was admitted to the hospital because of rapidly progressive fatigue, jaundice, and dark urine. The SCID was diagnosed after repeated respiratory infections during infancy; it was associated to hypogammaglobulinemia and he received replacement therapy with intravenous immunoglobulin (IvIg). The first determination of Ig in our center in 1996 (18 years old) showed IgA<0.25 g/l, IgM<0.54 g/l, and IgG 5.9 g/l values. On physical examination, he showed pallor and no lymphadenopathy was palpable. His full blood count showed hemoglobin (Hb) concentration 63 g/l, leucocytes (WBC) 67 × 10/l, monocytes 4.7 × 10/l, neutrophils 39.5 × 10/l, platelets 440 × 10/l, and absolute reticulocyte count 100 × 10/l. During several years, he showed a markedly elevatedWBC and it was interpreted as a leukemoid reaction in a splenectomized patient in the context of acute infections. Differential count in the current episode was neutrophils 60%, lymphocytes 33%, and monocytes 7%. Inversion on the CD4/ CD8 ratio was foundwithout any signs of clonality (IGHVFR1 polyclonal, TCR polyclonal). Biochemical tests showed increased LDH 1562 IU/l (normal <450), total bilirubin 15.6 mg/dl (normal <1.2), being indirect bilirubin 13.5 mg/dl (normal <0.6), and haptoglobin value was below the detection limit. Direct Coombs test was positive for IgG and negative for C3b/C3d. A specific real-time polymerase chain reaction for Parvovirus B19 was performed obtaining a viral load of 10,310 copies/ml. Automated PB examination using the CellaVision DM96 (Lund, Sweden) revealed massive erythrophagocytosis by monocytes and neutrophils. Images acquired on an Olympus BX43 microscope at ×1000 magnification are shown in Fig. 1. A bone marrow aspirate showed normal cellularity with isolate islands of giant erythroblasts and numerous figures of erythrophagocytosis. On day 2, he presented despite high dose steroids and RBC transfusion a rapidly worsening of his cardiorespiratory condition (dyspnea and chest pain) associated to increased values of myocardial enzymes. Hb values dropped to 28 g/l and serum lactate increased. In this situation, the patient was transferred to the intensive care unit (ICU) for sedation, orotracheal intubation, and mechanical ventilation. High dose IvIg (1 g/ kg/d for 3 days), rituximab (375 mg/m, the second dose was given at day 11), and a bolus of intravenous cyclophosphamide (750 mg/m) were administered. Over the following days, Hb and reticulocyte values increased. Serum lactate levels turned to normal allowing extubation after 4 days. Erythrophagocytosis was no longer seen in PB after the first dose of IvIg and on day 18, he was discharged. The analytical signs of hemolysis improved under * Anna Merino amerino@clinic.cat

Blood film findings in severe babesiosis.

A 66-year-old woman with an intact spleen was admitted to our hospital with shock and multiorgan failure. She had been born in India, but was resident in the United States, in a rural area of New Jersey. She had been on vacation in Europe for 3 weeks and had suffered fever and chills for 1 week, and nausea, vomiting and abdominal pain for 2 d. She had been admitted to another hospital where blood analysis showed anaemia (haemoglobin concentration: 81 g/l), a low platelet count (40 9 10/l), low prothrombin index (56%), and high creatinine (265 2 lmol/l; normal range 26 5–114 9) and bilirubin (171 04 lmol/l; normal <20 5) levels. During the next few hours her clinical condition deteriorated and she was transferred to our hospital. On admission she showed respiratory, hepatic and renal failure, anaemia, lymphopenia, thrombocytopenia, high lactate dehydrogenase (3500 iu/l; normal: 250–450) and negative direct and indirect Coombs tests. The peripheral blood film revealed a large number of ring-shaped parasites within the erythrocytes, initially interpreted as malaria. Red cells contained two or more parasites with 1–3 chromatin dots (left). The parasites were pleomorphic and occasionally showed the pointed ends of four parasites in contact, giving a characteristic Maltese cross formation (left). More than 20% of red cells showed pyriform parasites. Ring forms were seen extracellularly and within neutrophils (right). All these findings suggested the diagnosis of babebiosis. The patient lived in an endemic region; she had no history of known tick contact but contact with deer was reported. Treatment with quinidine and clindamycin, both administered intravenously, was initiated 2 d after her initial presentation and she also received an exchange transfusion. Following the exchange, parasitaemia decreased markedly to 0 3%. Haemodialysis was necessary because of persistence of renal failure. Quinidine and clindamycin were stopped after 2 d because of neurological deterioration and oral atovaquone and intravenous azithromycin were started. In the next 3 d the parasitaemia decreased further and became negative. Hepatic, renal and respiratory function recovered. In the United States, most reported cases of babesiosis have been caused by Babesia microti and are acquired in the northeast of the country. The presence of extracellular ring trophozoites, parasites within neutrophils, pleomorphism and the smaller size of the ring forms together with the absence of haemozoin can be useful in differentiating malaria and babesia parasites.