Butyrate Esterase Research Articles

Hematology, leukocyte cytochemical analysis, plasma biochemistry, and plasma electrophoresis of wild‐caught and captive‐bred Gila monsters (Heloderma suspectum)

The Gila monster (Heloderma suspectum), one of several venomous lizard species in the world, is found within the United States and Mexico and is recognized as an iconic symbol of the American Southwest. Thus, Gila monsters are of growing interest in the captive reptile trade and within zoological and educational institutions. The aims of this study were to determine results for CBCs, describe cytochemical reactions in WBCs, and obtain plasma biochemical and protein electrophoresis results from wild-caught and captive-bred H. suspectum. Ventral tail vein blood samples were obtained from 16 captive (14 wild-caught and 2 captive-bred) Gila monsters. CBCs, RBC morphometric analysis, plasma biochemical analysis, and protein electrophoresis were performed. Leukocytes were stained for peroxidase, Sudan black B (SBB), chloroacetate esterase, napthyl butyrate esterase, and leukocyte alkaline phosphatase, and blood smears were examined for the presence of hemoparasites. The median (range) PCV was 37% (22-50%) and WBC count was 4.6 × 10(3) /μL (3.3-6.4 × 10(3) /μL) with approximately 50% heterophils and fewer lymphocytes, basophils, azurophils, and monocytes in decreasing order. Cytochemical reactions were unique among reptiles with strong staining for peroxidase and SBB in monocytes/azurophils. Biochemical results were similar to those of earlier reports with slight increases in uric acid and urea concentrations. Plasma electrophoretic results indicated that albumin was approximately equal to the combined globulin fractions. Results of blood analysis in healthy wild-caught and captive-bred H. suspectum may be useful for monitoring health status in this species.

A highly positive nonspecific esterase reaction in a t(15;17)(q22;q12) acute promyelocytic leukemia

A 32-year-old man was admitted to the hematological department of our facility in November 2010 with fever, pharyngodynia, leucopenia, and thrombocytopenia. The patient had been in his usual state of health until approximately 2 weeks prior to admission, when he developed intermittent fevers, with temperatures of up to 39.0 C, epistaxis, and pharyngodynia. He was seen by his primary care physician 7 days prior to admission. Laboratory workup revealed leukopenia and thrombocytopenia, and he was referred to our hospital. Patient examination at the time of admission revealed no palpable swelling of the superficial lymph nodes. The liver and spleen were not palpable by abdominal exam. The complete blood count showed a reduced white blood cell (WBC) count of 2.5 9 10/L, a hemoglobin level of 13.1 g/dL and a platelet count of 92 9 10/L. The WBC differential revealed 38% neutrophils, 45% lymphocytes, and 8% blasts. Further laboratory tests revealed a coagulopathy consistent with disseminated intravascular coagulation (DIC); the D-dimer was positive (12.50 lg/mL) and the fibrin-fibrinogen deregulation products were elevated (44.7 lg/mL), while other tests of coagulation and serum and urine lysozyme levels were within normal limits. A May-Grunwald-Giemsa-stained bone marrow aspirate smear showed numerous hypergranular blasts with intense azurophilic granulation and Auer rods (faggot cells) (Fig. 1a). On cytochemical staining, the blasts were strongly positive on myeloperoxidase (MPO) staining (Fig. 1b), and strongly dual-positive on double-staining by naphthyl butyrate (nonspecific) esterase (NSE) and naphthol-ASD-chloroacetate esterase (CAE) (Fig. 1c). Sodium fluoride (NaF) inhibited NSE staining (Fig. 1d). Flow cytometry was performed on a specimen of bone marrow. The results showed predominant populations of CD33, CD13, CD34, CD14, HLA-DR, CD11b, CD117, CD64, CD15, CD65, CD71 and CD2. Cytogenetic analysis showed an abnormal karyotype, 46, XY, t(15;17)(q22;q12), in each of the 20 metaphase cells analyzed. The PML–RARA fusion transcript of the t(15;17) was confirmed by reverse-transcription polymerase chain reaction. With these findings, the patient fulfilled the diagnostic requirements for acute promyelocytic leukemia (APL) with t(15;17)(q22;q12). The patient received induction chemotherapy of alltrans-retinoic acid (ATRA), followed by anthracyclinebased chemotherapy, and achieved a hematologic remission. PML–RARA rearrangement transcript was negative by fluorescent in situ hybridization at the end of the induction treatment. The patient then began consolidation chemotherapy with idarubicin and cytarabine. APL is a subtype of acute myeloid leukemia (AML) with distinctive biologic and clinical features, characterized by a t(15;17) translocation fusing the PML and RARA genes. The introduction of ATRA therapy has dramatically improved the clinical outcome in APL over the last decade. However, prompt recognition and treatment of APL is vital for patient care, as life-threatening complications, such as bleeding, can present prior to diagnosis. Review of the peripheral blood smear with the bone marrow aspirate is necessary for the presumptive diagnosis of APL. Blasts are characterized by irregular and variable nuclear size and shape, bundles of Auer rods (faggot cells) and large, abundant, coalescent primary azurophilic granules. All the leukemic promyelocytes show a strong positive reaction to MPO. The leukemic promyelocytes are typically NSEnegative; however, approximately 25% of the cases are T. Aoki (&) T. Nishiyama N. Imahashi K. Kitamura Division of Hematology, Ichinomiya Municipal Hospital, 2-2-22 Bunkyo, Ichinomiya, Aichi 491-8558, Japan e-mail: taoki-cib@umin.net

Hydrolytic enzyme activities, carbon dioxide production and the growth of litter degrading fungi in different soil layers in a coniferous forest in Northern Finland

To establish the importance of the layered soil structure to microbial activities, the growth of litter degrading fungi, and the chemical status of forest soil in a cold climate, we studied carbon dioxide production and five hydrolytic enzyme activities in the humus, eluvial and illuvial layers at three sites in an acidic Pinus sylvestris forest soil at Inari, northern Finland. The enzyme activities were low (<30%) in extracts of soil compared to those in the corresponding soil layers, indicating that the enzymes were largely particle-bound. Carbon dioxide production, and β-glucosidase, β-xylosidase, α-glucosidase and butyrate esterase activities were highest in the humus layer in the soil column. The litter degrading fungi Agaricus bisporus, Agrocybe praecox, Gymnopilus luteofolius, Gymnopilus sapineus, Mycena galericulata and Stropharia rugosoannulata produced extracellular hydrolytic enzymes in the humus and eluvial soil layer plates, but not in the illuvial soil layer plates. Sulphatase activity was higher in the layers below the humus layer in the soil column. Carbon dioxide production, and β-glucosidase, β-xylosidase, α-glucosidase and butyrate esterase activities were significant below humus layer in the eluvial and illuvial layers in the soil column, indicating the importance of these deeper layers in microbial metabolic functioning and depolymerization of biopolymers in the soil column in cold climate in Northern Finland.

Cytochemistry of leukocytes from the family Macropodidae

The cytochemical staining characteristics of leukocytes from four species of Macropodidae and one species of Potoroidae were studied to investigate the cellular composition of leukocytes within the Macropodidae family and determine markers that may be useful for identifying cell lineage. Blood smears from the tammar wallaby (Macropus eugenii), the western grey kangaroo (Macropus fuliginosis), the red kangaroo (Macropus rufus), quokka (Setonix brachyurus) and a potoroid species, the woylie (Bettongia pencillata) were examined following reaction for Sudan Black B (SBB), peroxidase (PER), chloracetate esterase (CAE), α-naphthyl butyrate esterase (NBE) and alkaline phosphatase (ALP). Similar to domestic animal species, the neutrophils and eosinophils of macropodid species stained for both SBB and PER while monocytes and lymphocytes showed little or no reaction. CAE and NBE, however, were not useful as markers for macropodid neutrophils and monocytes, respectively. Significant variation between species was seen in ALP content. Tammar wallabies and quokkas demonstrated strong ALP activity within their neutrophils and eosinophils. In contrast, western grey kangaroos and red kangaroos contained no ALP activity in any of their leukocytes and staining in woylies was limited to weak reaction within some eosinophils.

Images in haematology: Faggot cells in an HIV‐positive patient with inv(16)/therapy‐related acute myeloid leukaemia

A 32-year-old human immunodeficiency virus (HIV)-positive male presented with fever and some haemorrhagic vesicles on the soft palate. Three years previously, he had been diagnosed with stage IVB, nodular sclerosis Hodgkin lymphoma, treated with six cycles of doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD), and achieved complete remission. On admission, a full blood count showed: white cell count 67AE4 · 10/l, haemoglobin concentration 85 g/l and platelet count 10 · 10/l. Peripheral blood films showed 10% blasts (some with granules), 25% promonocytes and 6% eosinophils. A variable number of Auer rods were observed in some neutrophils and band forms, occasionally being numerous (top). A bone marrow aspirate showed blast cells, promonocytes, monocytes, eosinophils and eosinophil precursors. In addition there were faggot cells – maturing cells of neutrophil lineage containing bundles of Auer rods (bottom). Cytochemical studies showed a proportion of cells positive for myeloperoxidase, chloroacetate esterase and butyrate esterase. Periodic acid–Schiff (PAS) positivity was observed in some atypical eosinophils. Immunophenotypic analysis showed two abnormal populations: the first of these accounted for 15% of the events and showed a CD34+, CD117+, CD33+, CD13++, CD15±, HLA)DR+, CD45+, cMPO+ and lysozyme–phenotype; the second accounted for 60% and showed a CD34), CD117±, CD13+, CD15+, HLA-DR+, CD11b+, CD14+, CD64+, CD36+, CD45++, cMPO± and lysozyme+ phenotype. Interphase fluorescence in situ hybridization analysis revealed inv(16) and excluded t(15;17). Cytogenetic analysis showed 47,XY,+8,inv(16)(p13q22)[20]. Molecular analysis confirmed inv(16) (MYH11-CBFB/ABL1 ratio:12AE47). The final diagnosis was therapy related-acute myeloid leukaemia (AML) with +8 and inv(16). He received two cycles of induction chemotherapy without attaining complete remission. Faggot cells are very characteristic of acute promyelocytic leukaemia but, as illustrated by this patient, they cannot be regarded as specific. They are a very uncommon feature of AML associated with inv(16).

A novel unbalanced whole-arm translocation der(3;10)(q10;q10) in acute monocytic leukemia

We describe here a novel unbalanced whole-arm translocation der(3;10)(q10;q10) in a 58-year-old man with acute monocytic leukemia. Bone marrow was massively infiltrated with 22.2% monoblasts, 55.4% promonocytes, and 5.6% monocytes. These monocytic cells were positive for myeloperoxidase and α-naphthyl butyrate esterase staining. Surface marker analysis revealed that they were positive for CD4, CD13, CD33, CD56, and HLA-DR but negative for CD14 and CD34. Chromosome analysis of the bone marrow cells showed 46,XY,+3,der(3;10)(q10;q10)[18]/46,XY[2]. Spectral karyotyping confirmed der(3;10)(q10;q10) as a sole structural abnormality. By acquisition of a normal chromosome 3 but not a chromosome 10, the der(3;10)(q10;q10) resulted in trisomy 3q and monosomy 10p. The +3,der(3;10)(q10;q10) is thought to play a crucial role in the pathogenesis of acute monocytic leukemia because of the gain of 3q or the loss of 10p.

Exposure to heptachlor: Evaluation of the effects on the larval and adult epidermis of Rana kl. esculenta

Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400 ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; α-naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmental toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria-rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed.

Characterization of a Mediterranean litter by13C CPMAS NMR: relationships between litter depth, enzyme activities and temperature

SummaryOrganic matter mineralization of forest litter is catalysed by the action of different extracellular enzymes produced by microorganisms. Coupling enzyme activities with data on the general macromolecular structure of organic matter, provided by cross‐polarization magic angle spinning13C nuclear magnetic resonance (13C CPMAS NMR), allows researchers new insights into organic matter degradation processes. In this paper, the effect of the temperature of incubation on the degradation processes was evaluated in three distinct layers (OhLn, OhLv and OhLf) of an evergreen oak litter (Quercus ilexL.), located in the Mediterranean area of south‐eastern France. We studied degradation phenomena by a combination of13C CPMAS NMR and microbiological analysis. In order to determine the microbial activity of litter layers, three enzyme activities (laccase, cellulase and butyrate esterase) were measured in a 6‐month mesocosm study. Results showed an increase in the alkyl C toO‐alkyl‐C ratio and an increase of the phenolic C and carboxyl C regions, indicating a preferential degradation of polysaccharides. The aromaticity also increased with litter depth and degradation, and humification processes were more elevated at 30°C.anovashowed significant effects (P&lt; 0.001) of increased temperature, depth and time of degradation on microbiological variables. Further information is needed about the variations in temperature and temperature‐litter response and soil functions to link fundamental understanding of carbon stabilization, climate change and global C cycling.

A Peculiar Case of Acute Myeloid Leukemia Mimicking Plasmacytoid Dendritic Precursor Cell Leukemia

Differential diagnosis between plasmacytoid dendritic precursor cell leukemia (pDC leukemia) and acute myeloid leukemia (AML) with monocytic differentiation is difficult due to shared clinicopathological features ; however, such diagnosis is critical because the two leukemias are treated differently. Here we report a peculiar case of AML mimicking pDC leukemia. A 22-year-old man presented with leukocytopenia and bone marrow involvement of atypical plasmacytoid cells with a prominent nucleolus. In spite of positive cytochemical staining for NaF-sensitive naphthyl butyrate esterase, this case was diagnosed as pDC leukemia because the abnormal cells were positive for CD4, CD56, and CD123, and negative for myeloperoxidase and lysozyme. The patient achieved complete remission after 4 courses of combination chemotherapy, but relapsed four months later with leukemic manifestation and skin involvement. The morphology of the leukemia cells became myelomonoblastic, and some were immunohistochemically positive for lysozyme, suggesting AML. Although the patient received allogenic stem cell transplantation twice, he died of progressive disease. This case demonstrates the importance of cytochemical staining for naphthyl butyrate esterase in differential diagnosis between AML and pDC leukemia coexpressing CD4, CD56, and CD123.

Basophilic Blast Phase (B-BP) of Chronic Myelogenous Leukemia (CML).

Approximately 65% of patients with CMP BP exhibit a myeloid phenotype, 30% a lymphoid, and 5% of cases have and undifferentiated or mixed phenotype. Although basophilia is a common feature in both the peripheral blood and the bone marrow during the chronic phase of the disease, transformation to B-BP is a rare occurrence with only anecdotal cases reported. To evaluate the incidence and outcome of B-BP, we reviewed 410 patients with CML who underwent transformation to BP at M.D. Anderson Cancer Center between January 1999 and April 2007. Of them, only 2 (0.5%) cases were diagnosed as having B-BP.CASE 1: a 19-year-old woman with CML in accelerated phase (AP) failed allogeneic stem cell transplantation (allo-SCT), donor lymphocyte infusion (DLI), autologous SCT, IFN-α, splenectomy, and gemtuzumab. In 10/00 started imatinib therapy at 400 mg/d, achieving a complete hematologic response (CHR) that lasted until 04/03 when she transformed to BP. Gentuzumab was again administered and rendered a CHR, after which she immediately received SCT from her haploidentical mother, resulting in a complete molecular response (CMR) that lasted until 6/05. Therapy with imatinib 800 mg/d was unsuccessful. In 7/05 her white cell count (WBC) was 44x109/L with 25% blasts and 28% basophils. A bone marrow (BM) biopsy showed the presence of 36% blasts with coarse basophilic granules (negative for myeloperoxidase [MPO] and butyrate esterase) and 38% basophils. Flow cytometry demonstrated 65% blastic cells with positive markers of myeloid lineage (CD11c, CD13, CD33) without immaturity markers (CD34, HLA-DR, TDT) B or T lymphoid markers (CD10, CD19, CD20, CD22, CD2, CD3, CD5) or other myeloid markers (CD14, CD15, CD65, MPO). Cytogenetics revealed an isochromosome 17, +8, and t(9;22) in 19/20 metaphases. A b3a2 BCR-ABL1 (p210) transcript with no evidence of mutations within the kinase domain was detected. Given her critical condition the patient was received only supportive care and died shortly thereafter.CASE 2: a 39-year-old woman was diagnosed with CML in chronic phase (CP) in 12/04. She failed therapy with IFN-α, allo-SCT, DLI, imatinib 800 mg/d, PR1 vaccine, matched unrelated donor SCT, imatinib 800 mg/d in combination with tipifarnib, and nilotinib 600 mg twice daily, after which she progressed to BP. A CBC revealed a WBC of 21x109/L with 29% blasts and 66% basophils. A hypercellular BM biopsy revealed 44% blasts (MPO negative) with large cytoplasmic granules and 45% basophils. Blast exhibited an immunophenotype characterized by positivity of myeloid markers, negativity of MPO, B and T cell markers, and partial positivity of CD117. Karyotypic analysis showed that 16/19 metaphases carried t(9;22), −16, add(17)(p13). Molecular studies detected a e1a2 p190 BCR-ABL1 transcript. Therapy with dasatinib 70 mg twice daily for 16 weeks was ineffective and the patient was referred to hospice care. In conclusion, B-BP is a rare subtype of BP CML that exhibits high resistance to standard chemotherapy, allo-SCT, and targeted BCR-ABL kinase inhibitors.

Monosomy 7 as the sole abnormality of an acute basophilic leukemia

We report the case of a 72-year-old man who had the very rare disease acute basophilic leukemia with the sole chromosomal finding of a monosomy 7. Most nuclear cells in the peripheral blood and bone marrow samples were either basophils or blasts. The blasts showed negative reaction with myeloperoxidase, periodic acid Schiff, chloroacetate esterase, α-naphthyl butyrate esterase, acid phosphatase, and Sudan black B. Metachromatic features of the blasts, however, were observed with toluidine blue stain. Electron microscopic evaluation showed the typical ultrastructure, with basophil and immature mast cell granules. Cytogenetic study revealed monosomy 7 in all metaphase cells, and this finding was confirmed by fluorescence in situ hybridization. The Philadelphia chromosome was absent. Review of the literature revealed abnormalities in cases of ABL. To our knowledge, the case reported here is the first to have basophilic leukemia with monosomy 7 as the only chromosome abnormality.

Clinico-Biological Characteristics and Prognosis of Non-M3 Acute Myeloid Leukemia with High Percentage of Myeloperoxisase Positive Blasts. Single Center Experience.

Introduction: It has been suggested that acute myeloid leukemia (AML) showing mature phenotype is associated with favourable outcome. In a study recently published by JALSG, myeloperoxidase (MPO) positivity in over 50% of blasts had favourable prognostic impact, independent from karyotype, on achieving complete remission (CR), overall survival (OS) and disease free survival (DFS). No other studies have established the independent prognostic value of MPO expression.Objectives: Analyze the clinico-biological characteristics of AML with high percentage of MPO+ blasts and its impact on CR, OS and DFS.Material and methods: Between 1986 and 2005, 418 adult patients (median 53 years, range 15–80) were diagnosed with de novo non-APL AML and evaluated for percentage of MPO+ blasts. All patients received intensive chemotherapy. Diagnosis was made by optic microscopy of bone marrow (BM) aspirates stained with May-Grumwald giemsa, MPO, butyrate esterase and or non specific esterase. Cytogenetic and immunophenotype analysis was evaluated in 66% and in 76% of the cases respectively.Results: 118 patients (28%) showed a percentage of MPO+ blasts >75%. AML with MPO+ blasts >75% was associated with M1-M2-M4 subtypes, leucocytes >50×109/L, blasts in BM >70% and HLA-DR negativity (p<0.01). It was also significantly associated with favourable karyotype (11% vs 3% favourable, 52% vs 48% intermediate and 3% vs 15% unfavourable). Patients with AML and MPO+ blasts >75% obtained higher CR rate (71% vs 55%), due to less resistant disease (9% vs 22%, p<0.01). In multivariate analysis favourable karyotype, leukocytes <50×109/L and age <60 were favourable prognostic factors for CR. Median OS and DFS was higher in patients with AML and MPO+ blasts >75% (15 vs 7 months, p<0.001, y 41 vs 12 months, p<0.001, respectively). ). In multivariate analysis, favourable karyotype, leukocytes <50×109/L, age <60 years and MPO+ >50% were favourable prognostic factors for OS; and age <60 and MPO+ >75% were the only independent factors for DFS. Median DFS was higher in patients with AML and MPO+ blasts >75% in the intermediate cytogenetic risk group (59 vs 13 months, p=0.015), age <60 (109 vs 15 months, p=0.003), age >60 (13 vs 7 months, p=0.03), autologous stem cell transplantation (100 vs 9 months, p=0.04) and chemotherapy alone (16 vs 8 months, p=0.003).Conclusion: In our series, patients with AML and MPO+ blasts >75% show less chemoresistant disease and a longer remission duration, the latter independently from the karyotype. This biological characteristic could be useful in designing therapeutic strategies in patients that lack other prognostic markers.

Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles ( Caretta caretta)

A morphologic classification based on the cytochemical characteristics of blood cells of 35 juvenile loggerhead sea turtles ( Caretta caretta) is described. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and toluidine blue. The morphologic characteristics of erythrocytes were similar to those reported in green turtles. Six types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes, monocytes and thrombocytes. Except for the basophils, the rest of the white blood cells from loggerhead turtles had different cytochemical characteristics compared to blood cells from other sea turtle species. The leukocyte differential count was different from that reported for other sea turtle species. Heterophils were the most numerous leukocytes from these loggerhead turtles, followed by lymphocytes, eosinophils, monocytes and basophils. This paper provides a morphologic classification of blood cells of loggerhead sea turtles that is useful for veterinary surgeons involved in sea turtle conservation.

Unusual type of TLS/FUS-ERG chimeric transcript in a pediatric acute myelocytic leukemia with 47,XX,+10,t(16;21)(p11;q22)

We report on a case of pediatric acute myelocytic leukemia showing 47,XX,+10,t(16;21)(p11;q22) that resulted in an unusual TLS/FUS-ERG chimeric transcript. The leukemic cells showed erythrophagocytosis, positive reactions for myeloperoxidase and Sudan black B stains, and negative reactions for periodic acid–Schiff and α-naphtyl butyrate esterase stains as well as expression of myeloid antigens. We also confirmed a very rare type of TLS/FUS-ERG chimeric transcript by fusion of the 5′ part of the TLS/FUS gene in chromosome 16p11 and the 3′ part of the ERG gene in chromosome 21q22 using reverse-transcriptase polymerase chain reaction and direct sequencing. After achieving a complete remission with two cycles of induction chemotherapy, the patient received an umbilical cord blood transplantation.

Peculiar Morphological, Cytochemical, Biochemical and Immunophenotyping Features in One Case of Acute Myeloid Leukaemia at the Onset.

On July 28th 2005, a 53-year old woman was admitted to our Hematology Dpt. with fever (37.5 C), fatigue, marked gingiva hypertrophy, mild hepatomegaly and absence of lymphoadenopaty and splenomegaly. Laboratory tests showed high leukocyte count of 182 x109/L, moderately increased platelet count (438 x109/L), normocytic anemia (10,9 g/dl, MCV 84 fl). Biochemical parameters, including lysozyme, were in the normal range except for high level of lactate dehydrogenase (2524 UI/l). Peripheral blood (PB) smear showed the presence of 94% of blasts, without evident granules. At the cytochemical myeloperoxidase staining blast cells revealed the presence of single or multiple Auer rods, while cytoplasmic granules were positive to the α-naphtyl butyrate esterase. In the bone marrow (BM) aspirate blasts were 80% showing same morphological and cytochemical features as in peripheral blood, while the granulocytic maturating series was 14%. No evidence of morphologically identifiable monocytic series in PB neither in BM. Megakariocytes were slightly increased and there was trilineage dysplasia. Blast immunophenotyping in PB and in BM was as follow: CD45, CD33, CD13, CD7 and HLA-Dr positive, 36% MPO positive and 48% co-express CD11c an CD117. Moreover blasts were CD14, CD19 and CD56 negative. FAB diagnosis was AML-M4. Interphase Fluorescence in situ Hybridization (FISH) for detection of AML1/ETO and bcr/abl gene rearrangements was negative. After treatment with 2 g hydroxyurea and leucapheresis, the WBC count was still high (140 x109/L) while after 1g of aracytin, WBC count decreased to 31000/mm3. Patient is presently enrolled into the GIMEMA protocol AML-12.This case shows at the onset following abnormalities:- an increased platelet count is a rare evenience in an AML at the onset;- negativity for lysozyme;- absence of CD14;- presence of Auer rods in quite all the blast population, which is a very rare evenience associated with monocytic subtypes of acute myeloblastic leukemia (AML);- atypical homogenous cytochemical pattern of blasts, showing Auer rods MPO positive and granules ANBE positive;- absence of any morphological differentiation of monocytic series. S4479

Morphologic characterization of specific granules in Greyhound eosinophils

"Vacuolated" eosinophils (ie, eosinophils with empty, nonstaining granules) have been described previously in normal Greyhounds. However, to our knowledge, detailed studies of granules in vacuolated and normal eosinophils in this breed have not been performed. The objective of this prospective study was to characterize some of the morphologic, ultrastructural, and cytochemical staining features of specific (primary) granules in both normal and vacuolated eosinophils in Greyhound blood. Morphologic features of eosinophils in Wright's- and Diff-Quik-stained peripheral blood smears from 49 Greyhounds were compared with 200 blood smears from non-Greyhound dogs. Transmission electron microscopy was done on blood from 3 Greyhounds with vacuolated eosinophils and 3 with normal eosinophil granules. Blood smears from 4 of these dogs also were stained cytochemically with alkaline phosphatase (AP), chloracetate esterase (CAE), and alpha naphthyl butyrate esterase (ANBE). The morphologic features and tinctorial properties of vacuolated and normal eosinophils were compared. Twenty-six Greyhounds (53%) had vacuolated eosinophils and 23 (47%) had normal granulated eosinophils in smears stained with Wright's stain. Only 1% of eosinophils were vacuolated in non-Greyhound dogs. Twenty of the 23 (85%) Greyhounds with normal granulated eosinophils on Wright's-stained smears had vacuolated eosinophils in smears stained with Diff-Quik. Ultrastructurally, no morphologic differences were observed between granules of vacuolated and normal eosinophils. Both vacuolated and normal eosinophils in Greyhounds were positive for AP and negative for CAE and ANBE, as expected for normal dogs. Vacuolated eosinophils in Greyhounds likely reflect, at least in part, differential staining properties of the specific granules with different hematologic stains. Ultrastuctural and cytochemical features of eosinophil granules were similar in normal and vacuolated eosinophils from Greyhounds.

American paddlefish leukocytes demonstrate mammalian‐like cytochemical staining characteristics in lymphoid tissues

American paddlefish Polyodon spathula leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, α‐naphthyl butyrate esterase and β‐glucuronidase. American paddlefish monocytes, lymphocytes and granulocytes stained positive for acid phosphatase. Monocytes stained positive for α‐naphthyl butyrate esterase. Lymphocytes that stained positive for α‐naphthyl butyrate esterase were designated type A. Lymphocytes that stained positive with antibodies to the L chain of white sturgeon Acipenser transmontanus immunoglobulin (Ig) were designated type B. Type A and type B lymphocytes stained positive for β‐glucuronidase. All leukocytes observed were negative for Sudan Black B. Monocytes, lymphocytes and granulocytes were present in the renal haematopoietic tissue, spleen, thymus, pericardial myeloid tissue, lamina propria of the spiral valve, and in meningeal myeloid tissue located dorsal to the brain, at the base of the brain and around the notochord. Peyer's patches were present in the gut. Morphological characteristics of leukocytes stained with Wright's and haematoxylin and eosin and appeared very similar to those of other fish species.

Primary Cutaneous Natural Killer Cell Blastic Lymphoma

An 81 yo male presented with several asymptomatic firm 1–5 cm red purple plaques on the trunk and lower extremities associated with a mild pancytopenia. Histological examination revealed a diffuse, monotonous dermal infiltrate of atypical medium sized cells with fine chromatin and scanty cytoplasm. Immunoperoxidase staining demonstrated positivity for CD 45, CD43, CD4, Bcl‐2, and TdT; but was negative for cytokeratin, melan‐A, CD30 and hematopoietic lineage specific markers. A subsequent bone marrow aspirate demonstrated a dense population of cells that were morphologically consistent with blasts. Immunophenotyping by flow cytometry revealed lesional cells that expressed CD56, CD4, CD7, CD5, HLA‐DR and TdT. However, lineage specific markers for B‐cells (CD19, CD20, cCD79a, and CD10), T‐cells (sCD3 and cCD3), and myeloid cells (CD13, CD33, CD117, cCD13, CD14, CD41, CD61, myeloperoxidase, and alpha napthyl butyrate esterase) were not expressed. Molecular studies by PCR exhibited no evidence of T‐cell receptor or heavy chain gene rearrangements. Collectively, these findings are consistent with a primary cutaneous blastic natural killer cell lymphoma. Blastic natural killer cell lymphomas are characterized by a high incidence of cutaneous involvement and an aggressive clinical course. Our patient responded dramatically to one cycle of CHOP chemotherapy with resolution of his cutaneous tumors.

Acute Monocytic Leukaemia with Cutaneous Manifestation

A 50‐year‐old Caucasian male presented with generalised skin rash. Gingival hyperplasia and hepatosplenomegaly were also noted. His laboratory data showed hemoglobin of 10.3 g/dL, white blood cell count of 203.6 K/uL and platelet count of 72 K/uL. Peripheral blood smears revealed that 67% of the white blood cells was monoblasts, 25% was promonocytes and atypical monocytes. Bone marrow aspirate smears showed that 97% of the nucleated cells were composed of monoblasts and promonocytes. Approximately 80% of the immature cells showed intense non‐specific butyrate esterase activity. The morphologic and cytochemical findings were compatible with a diagnosis of acute monocytic leukaemia. Skin biopsy taken from the left medial thigh revealed a Grenz zone and a dense infiltrate of atypical cells throughout the dermis. The cells were positive for KP1 (CD68) immunohistochemical stain. The findings were consistent with cutaneous manifestation of leukaemia (leukaemia cutis). The patient’s condition deteriorated rapidly despite chemotherapy and expired 10 days after initial presentation. Leukaemia cutis is more common in acute monoblastic/monocytic leukaemia (AMoL) than in other subtypes of leukaemia. Leukaemia gingival hyperplasia is another characteristic feature of AMoL. Dissemination to the skin is generally associated with a poor prognosis.

Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure.

Hematologic data are used routinely in the health care of humans and domestic mammals. Similar data for fish are largely fragmentary or have not been collected. The primary purpose of this study was to determine hematologic reference intervals for koi, an ornamental strain of the common carp (Cyprinus carpio). Secondarily, the morphology, cytochemical reactions, and ultrastructure of koi blood cells were characterized. A CBC was performed manually on heparin-anticoagulated blood specimens using Natt and Herrick's diluent and a Neubauer-ruled hemacytometer. Leukocyte differential counts were done on Wright-Leishman- and Diff-Quik-stained blood smears. Cytochemical reactions of koi leukocytes were determined using commercial kits. Transmission electron microscopy was performed to characterize the ultrastructural features of koi blood cells. Hematologic reference intervals were established for healthy koi for PCV (30-34%), hemoglobin concentration (6.3-7.6 g/dL), RBC count (1.7-1.9 X 10(6)/ microL), WBC count (19.8-28.1 X 10(3)/ microL), RBC indices, and differential leukocyte counts. Lymphocytes were the predominant leukocyte (accounting for up to 80% of all leukocytes), whereas eosinophils were rare. Basophils were positive with PAS staining. Naphthol AS-D chloroacetate esterase activity was observed only in eosinophils. alpha-Naphthyl butyrate esterase and beta-glucuronidase activities were positive in monocytes. Some lymphocytes were reactive for alpha-naphthyl butyrate esterase and acid phosphatase activity. Ultrastructurally, leukocytes, erythrocytes, and thrombocytes were identified on the basis of cytoplasmic organelles and granule appearance. Hematologic reference intervals and knowledge of the cytochemical reactions and ultrastructural characteristics of koi leukocytes will help standardize hematologic studies in this species.