Krzysztof Lewandowski

A case of mast cell leukaemia with exon 9 KIT mutation and good response to imatinib

Background:  Mastocytosis is a myeloproliferative neoplasm characterized by the excessive proliferation of mast cells. Mast cell leukaemia (MCL), the aggressive form of this disease, requires cytoreductive therapy, such as cladribine, interferon‐alpha‐2b and, most recently, tyrosine kinase inhibitors – dasatinib or imatinib.

Microscopic examination of bone marrow aspirate in healthy adults - comparison of two techniques of slide preparation.

Introduction:  According to the International Council for Standardization in Hematology (ICSH) guidelines for the standardization of bone marrow specimens and reports, smears from bone marrow aspirates for microscopic examination should be prepared using two techniques simultaneously: the wedge‐spread and the crush technique. However, the outcomes of these techniques have never been compared.

Fludarabine Therapy in Waldenström's Macroglobulinemia Patients Treated Previously with 2-chlorodeoxyadenosine

Six patients with Waldenströms macroglobulinemia (WM) resistant to previous 2-chlorodeoxyadenosine (2-CdA) therapy were treated with fludarabine. Both initial and subsequent therapy was administered according to the most widely used protocols. The median number of 2-CdA cycles given to the patients was 3.7 (range 2-5), while fludarabine was 1.7 (range 1-2). As a result partial remission was achieved in two patients and stabilization of the disease in the remaining four in spite of the inadequate response or progressive disease after 2-CdA therapy. Median monoclonal IgM concentration fell from 29.4 (range 11.6-37.1) to 16.4 g/l (range 3.8-29.1). At the same time hemoglobin concentration increased from median 114 (range 89-129) to 122 g/l (range 97-133). Side effects consisted of neutropenia (three patients) and thrombocytopenia (two patients). Two patients are alive with progression of the disease noted after 12 and 21 months of observation. One patient died as the result of cardiac disease, two others died of infection 5 and 16 months after cessation of therapy in the course of disease progression. The remaining patient was lost to follow-up after 10 months of observation with continuous stabilization. Our observations suggest that in WM patients resistant to 2-CdA, stabilization of the disease or even remission can be obtained with fludarabine.

Erythrophagocytosis by neutrophils – a rare morphological phenomenon resulting in acquired haemolytic anaemia?

Erythrophagocytosis by neutrophils is a rare morphological phenomenon described in patients with clonal malignancies of haematopoiesis with myelodysplasia and in some haemolytic conditions including paroxysmal cold haemoglobinuria, haemolysis caused by snake‐bite, sickle cell anaemia and other defects of red cells. We describe a female patient who presented with acquired haemolytic anaemia. Erythrophagocytosis was found in around 35% of neutrophils of the peripheral blood. A similar picture was seen in the bone marrow, but with additional erythrophagocytosis by macrophages. These two processes were considered as the main causes of anaemia, but the first one seemed to be predominant. Malignancies, autoimmunisation disorders and infections were excluded. Immunosuppressive therapy with corticosteroids was implemented, but had to be stopped because of side effects. Long‐term normalization of peripheral blood morphology was achieved after splenectomy. Splenectomy may be considered a therapeutic option for patients with diagnosed neutrophil erythrophagocytic hyperactivity. Therapy with corticosteroids is also possible, but the long‐term effects remain unknown.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Cabot rings as a result of severe dyserythropoiesis in a dog

An 11-year-old female Dachshund was presented with depression, diarrhea, weight loss, and radiographic evidence of masses involving the liver, spleen, and cranial lobe of the right lung. Results of a CBC included severe nonregenerative anemia (HCT 14.2%, hemoglobin, 4.3 g/dL, reticulocytes 66,000/microL) with marked metarubricytosis (nucleated RBCs 6.39 x 10(3)/microL). Examination of the peripheral blood smear revealed marked erythroid dysplasia, including marked anisocytosis with a prevalence of macrocytes, Howell-Jolly bodies, diffuse basophilic stippling, and multinucleated and atypical nucleated RBCs. Neutrophil hypersegmentation and giant forms were also noted. Numerous erythrocytes, particularly polychromatophilic cells, contained inclusions consistent with Cabot rings, which appeared as delicate red-purple ellipsoid or figure 8 structures. Rarely, Cabot rings were observed extracellularly. The dog was treated symptomatically with blood transfusions, prednisone, erythropoietin, and vitamin supplementation, but the anemia progressively worsened. The dog was euthanized 2 months after presentation. Bone marrow aspirate and core biopsy specimens obtained at the time of euthanasia revealed marked dysplastic changes in all cell lines, especially dyserythropoiesis, along with infiltrating carcinoma cells. A necropsy was performed, and histologic examination revealed poorly differentiated adenocarcinoma of the lung with multiple metastases to the marrow, spleen, and liver. The final diagnosis was marked myelodysplasia secondary to metastatic adenocarcinoma. Cabot rings are found rarely in humans with myelodysplasia, but have not been described previously in dogs. Based on the findings in this case, Cabot rings may occur rarely in dogs with severe dyserythropoiesis.

The quantification of reticulocyte maturation and neocytolysis in normal and erythropoietin stimulated rats.

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis. Copyright

Microscopic examination of bone marrow aspirates in malignant disorders of haematopoiesis—a comparison of two slide preparation techniques

It is mandatory to perform microscopic examinations of bone marrow aspirates during the diagnosis of many neoplastic haematopoiesis disorders. In 2008, The International Committee for Standardization in Hematology recommended the use of two types of slides for the microscopic evaluation of bone marrow: wedge-spread film and crush film slides. Because these techniques have not yet been compared, we performed such a comparison. Routine bone marrow samples from 250 patients diagnosed due to different neoplastic haematological disorders were evaluated. The major differences between the two compared techniques were identified in 13 patients with non-Hodgkin’s lymphoma, seven patients with systemic mastocytosis and 11 patients with acute leukaemias or myelodysplastic syndromes or chronic myelomonocytic leukaemia. Differences were noted also in many patients with multiple myeloma, but the clinical significance of these discrepancies was rather modest. The major causes of the differences observed seemed to be the dilution of marrow with blood cells and the focal growth of many neoplastic cells. We believe that the crush technique is more advantageous compared to the wedge-spread films. Therefore, we recommend the use of crush films as the primary method for establishing a diagnosis or for making therapeutic decisions based on the microscopic examination of bone marrow.

Potential leukemic cells engraftment after hematopoietic stem cell transplantation from unrelated donors with undiagnosed chronic leukemia

BACKGROUND Donor-related neoplasms are a potential complication of treatment strategies involving stem cell transplantation. Although mechanisms for detection of short-term complications after these procedures are well developed, complications with delayed onset, notably transmission of chronic diseases such as chronic myeloid leukemia (CML), have been difficult to assess. Consequently, we studied the potential of human CML cells to engraft hematopoietic tissues after intravenous implantation in mice. METHODS Human peripheral blood cells, collected from CML patients presenting with moderately increased white blood cells count before treatment, were transplanted into sub-lethally irradiated, immunodeficient mice. Five weeks after transplantation the nuclear cells were isolated from the murine bone marrow, spleen, and peripheral blood and were used to quantitatively detect human CD45 antigen by flow cytometry; qRT-PCR was used to detect the BCR-ABL1 fusion gene, and the human or murine beta-glucuronidase housekeeping gene was used to examine human-murine chimerism. RESULTS We found that all evaluated animals had donor chimerism at the selected interval after transplant and the presence of a specific BCR-ABL1 fusion gene transcript was also detected. CONCLUSIONS Our results suggest that the risk of neoplasm transmission cannot be eliminated during hematopoietic stem cell transplantation from undiagnosed CML donors with borderline leukocytosis. The obtained data confirms the potential of leukemic cells to viably engraft the hematopoietic organs post-transplantation in an immunosuppressed recipient.

Transient improvement of skin symptoms in an adult patient with pediatric-onset cutaneous mastocytosis treated with interferon-α

Department of Dermatology, Venerology and Allergology, Medical University of Gda nsk, Gdansk, Poland, Department of Allergology, Medical University of Gda nsk, Gdansk, Poland, Department of Pathology, Medical University of Gda nsk, Gdansk, Poland, Department of Biology and Medical Genetics, Medical University of Gda nsk, Gdansk, Poland, and Department of Clinical Analytics, Medical University of Gda nsk, Gdansk, Poland