A question that I think can be complicated for a MIR examiner. We are told of a leukocytosis at the expense of lymphocytes with a B-CLL phenotype. It seems that there are no other alarming data. No clinical data are given (so we assume that the patient is asymptomatic). No cytopenias... Conclusion: FOLLOW UP. The main hesitation with the answer should be with option 1. Why do I consider it incorrect? Because the TP53 mutation, although it establishes prognosis, is not indicated at diagnosis, but when treatment is going to be initiated (first line and prior to successive lines in case they are needed). It would be possible to go too far in this question in which the percentage given does not refer directly to clonal lymphocytes, so that it could even be a monoclonal B lymphocytosis. But this is going too far.

A question that I think can be complicated for a MIR examiner. We are told of a leukocytosis at the expense of lymphocytes with a B-CLL phenotype. It seems that there are no other alarming data. No clinical data are given (so we assume that the patient is asymptomatic). No cytopenias... Conclusion: FOLLOW UP. The main hesitation with the answer should be with option 1. Why do I consider it [HIDDEN]? Because the TP53 mutation, although it establishes prognosis, is not indicated at diagnosis, but when treatment is going to be initiated (first line and prior to successive lines in case they are needed). It would be possible to go too far in this question in which the percentage given does not refer directly to clonal lymphocytes, so that it could even be a monoclonal B lymphocytosis. But this is going too far.

A 67-year-old patient who in the last 6 months, in two routine analyses, presents progressive lymphocytosis. In the last one, hemoglobin 15.4 g/dL; leukocytes 18.5 x103/μL with 82% of mature lymphocytes that by flow cytometry express CD5/CD19/CD23 antigens and platelets 240 x103/μL. What attitude do you think is correct?

Study of TP53 mutations to establish prognosis.

Bone aspirate/biopsy to confirm the diagnosis.

PET/CT to establish the therapeutic attitude.

New clinical and analytical control in 6 months.

[Chronic lymphocytic leukemia].

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that accounts for approximately 30 % of adult leukemias and 25 % of Non-Hodgkin lymphomas (NHL). It is the most common form of leukemia in the western world (incidence 3-5/100 000). Elderly people are mainly affected, median age at diagnosis is around 70 years and there is a slight predominance in men. The etiology of the disease is unknown. The initial symptoms are nonspecific. Cervical lymphadenopathy and splenomegaly followed by general fatigue are seen most commonly. Other possible symptoms include night sweats, fever, loss of weight (so-called B symptoms) and frequent infections. Several patients develop autoimmune complications as autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP). To confirm the diagnosis more than 5000 B-lymphocytes/µl need to be present. The expression of the typical surface markers CD5, CD19, and CD23 has to be confirmed by flow cytometry. Imaging studies as X-ray of the chest, ultrasound of the abdomen, or CT scan are used to assess the degree of lymphadenopathy or organomegaly. A bone marrow biopsy is not mandatory for the diagnosis. According to the European Binet staging system, CLL is divided into 3 stages (A, B and C). Patients in Binet stage A have 0 to 2 areas of node or organ enlargement with normal levels of hemoglobin and platelets. Binet stage B patients have 3 to 5 areas of node or organ enlargement and normal or slightly decreased levels of hemoglobin and platelets. Binet stage C patients have anemia (hemoglobin < 10 g/dl) and/or thrombocytopenia (platelet counts < 100 000/µl), with or without lymphadenopathy or organomegaly. As there is no survival benefit associated with early intervention, asymptomatic patients with early stage CLL (Binet stage A and B) are usually not treated but are followed on a "watch and wait" principle. Treatment indications include stage Binet C or signs of an active disease as rapidly progressive lymphadenopathy or organomegaly together with physical limitation, B symptoms that cannot be tolerated, rapidly deteriorating blood values, or rapidly increasing leukocyte counts. The patient's physical condition has major impact on the treatment decision. Currently immunochemotherapy with fludarabine, cyclophosphamide and the CD20-antibody rituximab (FCR) is the standard of care in previously untreated and physically fit CLL-patients. An alternative regimen is the combination of bendamustine and rituximab (BR). Physically compromised patients can be treated with the oral drug chlorambucil or with bendamustine with or without rituximab. Due to high morbidity and mortality, allogeneic stem cell transplantation is limited to a small group of patients and should be discussed in a high-risk situation, such as 17p deletion, lack of response to standard therapy or early relapse.

[Chronic lymphocytic leukemia--the old and the new].

Chronic Lymphocytic Leukemia (CLL) is the most frequent lymphoproliferative disease and leukaemia in western countries. CLL occurs more frequently in men than women, the median age at diagnosis is 65 years. CLL is defined as a persisting chronic lymphocytosis > 5 G/l with classical morphological features (small lymphocytic cells with round nuclei, dense chromatin and small cytoplasmic rim) and a classical immunophenotype (CD5+, CD19+, CD20+, CD23+); however, deviations from classical morphology are frequent. In cases with classical diagnostic features in the peripheral blood, a bone marrow biopsy is not necessary for diagnosis. Prognostic features comprise the stage of the disease according to the Rai or Binet systems, laboratory markers such as LDH, beta-2-microglobulin, lymphocyte doubling time and CD38 expression by flow cytometry (and ZAP-70 expression if available) as well as the status on IgV(H) hypermutations and cytogenetic analysis. Up to 2/3 of patients do not need treatment at the time of diagnosis and can initially be followed using a watch and wait strategy. If therapy becomes necessary, initial standard treatment still is chlorambucil. Later, purine analogues and Alemtuzumab are treatment options for refractory or relapsing disease. Therapies with antibodies such as Alemtuzumab and Rituximab in combination with purine analogues are currently under clinical investigation. With recurrent or atypical infections, hypogammaglobulinemia should be searched for and immunoglobulins should be substituted if necessary. However, their prophylactic use is not recommended.

[Chronic lymphatic leukemia].

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. Median age at diagnosis is around 70 years. To confirm the diagnosis more than 5000 B-lymphocytes/µl need to be present. The expression of the typical surface markers CD5, CD19, CD20 and CD23 has to be confirmed by flow cytometry. A bone marrow biopsy is not mandatory for the diagnosis. Before start of treatment the assessment of 17 p deletion and/or TP53-mutational status is recommended. Treatment indications include stage Binet C or signs of an active disease as rapidly progressive lymphadenopathy or organomegaly together with physical limitation, B symptoms that cannot be tolerated, rapidly deteriorating blood values, or rapidly increasing leukocyte counts (Lymphocyte doubling time less than 6 months). The patient's physical condition has major impact on the treatment decision. Currently immunochemotherapy with fludarabine, cyclophosphamide and the CD20-antibody rituximab (FCR) is the standard of care in previously untreated and physically fit patients. An alternative regimen is the combination of bendamustine and rituximab (BR) or ofatumumab. Physically compromised patients can be treated with the oral drug chlorambucil in combination with an anti-CD20 antibody. Due to high morbidity and mortality, allogeneic stem cell transplantation is limited to a small group of patients and should be discussed in a high-risk situation, such as 17 p deletion and/or TP53-mutation, lack of response to standard therapy or early relapse. Recently several new chemo-free treatment options have been introduced within clinical trials. Among them are monoclonal antibodies, most of them targeting the CD20 molecule: besides the licensed drugs rituximab and ofatumumab, obinutuzumab, in combination with chemotherapy, has recently shown high clinical efficacy in front-line treatment of elderly patients with CLL. Novel agents have been designed to block aberrant signaling from the B-cell receptor. Ibrutinib acts by inhibiting the Bruton's tyrosine kinase (BTK) while idelalisib represents a first-in-class specific inhibitor of the phosphoinositol-3 kinase (PI3K) delta isoform. Another class of drugs with potential impact for chemo-free treatment strategies in CLL is the BH3-mimetic inhibitor of the Bcl-2 family of pro-survival proteins, ABT-199. Given all these novel agents and targets, chemo-free or at least chemo-reduced concepts may become reality in the near future for our patients suffering from CLL.

[CD43 in B-cell chronic lymphocytic leukemia].

CD43 (other names: sialophorin, leukosialin, sialoglycoprotein of white blood cells) is an integral cell membrane mucin. In population of peripheral B cells CD43 occurs only on activated B cells and CD5 positive B cells. These last cells create neoplasm population in patients with B-cell chronic lymphocytic leukemia (B-CLL). Anti-CD43 monoclonal antibodies are used routinely in investigations of tissue fragments in cases of non-Hodgkin's lymphoma, whereas we did not find publication on theme of CD43 expression on peripheral blood B cells in patients with B-cell chronic lymphocytic leukemia. Wherefore advisable appeared estimation CD43 expression on B-CLL cells and comparison it with expression of typical B-CLL markers--such as CD5 and CD6. Immunological phenotype of peripheral blood and bone marrow lymphocytes has been evaluated using flow cytometry (Cytoron Absolute Ortho-Diagnostic Systems) and two-color staining. Twenty six untreated patients with B-CLL were studied. Because on well-known correlations between CD43 expression and metastasis potential of tumor, patients were divided on two groups differing score of total tumor mass (score TTM). Score TTM was evaluated according to criterion of Jaksic and Vitale. Twelve patients whose TTM score was equal or lower than 9 and median lymphocytosis was 24.6 x 10(9) in microliter were included in group I. 14 patients whose TTM score was higher than 9 were included in group II. Median lymphocytosis in these patients was 152.6 x 10(9) in microliter. The median percentage of CD43+/CD19+ cells in peripheral blood was 62.6% in the group I, and 75% in the group II (p < 0.05). Median fluorescence intensity (MFI) of CD43 antigen was 87.7 in the I group comparing to 77.4 in the group II. So one observed tendency to lowering MFI during tumor growing but the difference was not significant (p = 0.25). In peripheral blood during progression of disease more clearly than CD43+ cells increased percentage of CD5+ and CD6+ cells. The median percentage of CD19+/CD5+ cells was 62.7% in the group I, 82.4% in the group II and the difference was significant (p < 0.002). The difference in the median percentages CD6+/CD19+ cell 71.8% in group I and 84.3% in the II one were also significant (p < 0.03). MFI of CD5 and also CD6 antigens did not change in course of disease. Moreover, examination of CD43 and CD5 expression in marrow additionally to blood study were performed in 12 cases (6 from group I, 2 from group II and 4 new not included). The median percentage of CD43+/CD19+ cell was 35.1% in blood and 43.7% In marrow, in contrast to these results was the median percentage of CD19+/CD5+ cell, which was higher in peripheral blood (70.4%) than in bone marrow (60.9%). The results of this study indicate that CD43 is present on peripheral blood B-CLL cells. Moreover, percentage of these cell increases during progression of disease however more weakly than percentage of CD5 and CD6 positive cells. Expression of CD43 is independent from expression CD5 and CD6 and diminishes during tumor mass increasing, what can depended from releases exocellular domains of CD43. CD43+ cell from B-CLL patients have a tendency to accumulation in tissues what is illustrated by higher percentage of CD43+ cell in bone marrow than in peripheral blood.

An unknown chromosomal aberration in a patient with chronic lymphocytic leukemia: Extra isochromosome 4q.

The genetic characterization of chronic lymphocytic leukemia (CLL) has made significant progress over the past few years. Chromosomal abnormalities are detected in up to 80% of patients. Determination of new chromosomal disorders is important in the pathogenesis and treatment facilities. A patient was diagnosed with CLL Stage 2 on 2012 and followed since then by hematology clinic. She was 63 years old. Mature, small lymphocytes, and smudge cell was found in the patient's peripheral blood smear. Bone marrow (BM) biopsy made and hypercellularity showing infiltration of atypical cells with CD5+, CD20+, and CD23+ were determined. Hypoplasia is detected in myeloid/erythroid series, and Stage 2 reticular fibers proliferation were detected. The patient was followed up without medication. While follow-up of patient's white blood cell: 57300, hemoglobin: 5.36, and PLT: 99700 are determined in May 2014. According to the patient's flow results, CD5+, CD23+, and FMC7+ were detected. Mature, small lymphocytes and smudge cell was found in the patient's peripheral blood smear. In ultrasonography imaging, multiple laps were found in the abdomen and multiple neck lymph nodes were detected. The patient BM aspiration was performed in 2014, and hypercellularity was found to contain 54% of atypical lymphocytes in the BM. Fluorescence in situ hybridization (FISH) analysis made two times in 2014. At first, FISH analysis patient's rate of 18% in RB1/13q14.2/13qter revealed a deletion of the gene regions. Patient's FISH result was reported as normal (for RB1/13q14.2/13qter) after 5 months at second analysis. Cytogenetic analysis is made from the patient's BM at the same time. According to the results of karyotyping and FISH, 47, XX, isochromosome 4q (+i4q) is determined. According to literature, extra isochromosome 4q is reported by our case for the first time in CLL. She was diagnosed with Stage 4 CLL and FISH treatment was initiated. Our patient showed disease progression compared to previous results. Hence, we offer that this evidence can be considered regarding triggering the disease's progression or as a result of disease progression i4q was occurred.

CD5 negative lymphocytosis mimicking typical B-chronic lymphocytic leukaemia. Description of 26 cases.

We report 26 elderly patients (median age 68.3 years) who met diagnostic criteria for B-cell chronic lymphocytic leukaemia (B-CLL) but whose lymphocytes lacked CD5 expression. Haematological and clinical features of this CD5- series were compared with those of 333 CD5+ B-CLL patients from the same institute. No significant differences were observed regarding peripheral blood (PB) and bone marrow (BM) lymphocytosis, Hb level, platelet count, incidence of adenomegaly, hepatomegaly or splenomegaly or diffuse BM pattern. Due to an absence of nodal enlargements or to general clinical condition, lymph node biopsy was performed in only three patients, while spleen histology was examined in two cases following splenectomy. All histological results confirmed the clinical diagnosis of CLL. The distribution of the CD5- subjects according to the different staging categories proposed by Rai, Binet and Mandelli was similar to that of CD5+ subjects. Ten patients received standard chemotherapy with Chlorambucil (CHL) and Prednisone (PDN). All achieved partial remission, although one of these patients later died of disease progression; 80 months after diagnosis. We conclude that rare cases of CD5- lymphocytosis fulfilling all criteria for B-CLL may occur. Haematological and clinical features at presentation and the response to conventional treatment with Chlorambucil support our hypothesis of considering this disease as a less frequent subgroup of B-CLL.

Oncology

Diagnosis and staging Diagnostic and staging investigations depend on the size and type of malignancy. Blood cancer Blood investigations including hemoglobin, total leukocyte count, platelet count, peripheral smear, red cell indices. Bone marrow studies including aspiration, flow cytometry, cytogenetics, fluorescent in situ hybridisation and molecular studies. Lymphoma Excision biopsy of lymph node for histopathological examination, immunohistochemistry, and molecular studies. Blood investigations include lactate dehydrogenase (LDH), serum uric acid, and kidney function tests. Imaging tests such as computerised tomography (CT scan), positron emission tomography (PET CT). Bone marrow biopsy. Solid tumors Biopsy for histopathology and immunohistochemistry. Imaging tests like X-ray, ultrasonography, computerised tomography (CT), magnetic resonance imaging (MRI) and PET CT.

Blastic transformation after splenectomy in a patient with nonvillous splenic marginal zone lymphoma with p53 overexpression: a case report.

A 61-year-old man with no subjective symptom was admitted to our hospital for further examination of the causes of anemia (hemoglobin, 9.5 g/dL) and thrombocytopenia (platelets, 9.2 x 10(4)/microL), which had been pointed out in a medical checkup half a year previously. A bone marrow examination showed 73% lymphoid cells. Immunophenotyping of these cells were CD19+CD20+CD3-CD5-CD10-CD23-, and light chain restriction (kappa) was positive by fluorescence-activated cell sorting analysis. A computed tomography scan showed mild splenomegaly. To confirm the diagnosis histologically, we performed a splenectomy. Finally, we diagnosed the patient's disease as nonvillous splenic marginal zone lymphoma (SMZL). A month after the splenectomy, the white blood cell count was remarkably increased to 7 x 10(4)/microL with the blastic transformation of lymphoid cells. We first treated the patient with fludarabine and then with the CHOP regimen (cyclophosphamide, hydroxydaunomycin, vincristine [Oncovin], and prednisone), but the disease was so refractory that the patient died of the disease 13 months after the splenectomy. Immunohistochemical staining and a molecular examination for p53 were carried out with specimens from the splenectomy. We found overexpression of the p53 protein in lymphoid cells and a point missense mutation in codon 280 at exon 8 that changed AGA (Arg) to AGT (Ser). This case may indicate the existence of a more aggressive subset of SMZL, suggesting a reconsideration of the roles of splenectomy and p53 overexpression in the diagnostic and therapeutic approaches to patients with SMZL.

[Monoclonal B-cell lymphocytosis: from literature to laboratory practice].

Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic condition characterized by the presence of less than 5,000 monoclonal B-cells per microliter and the absence of clinical signs or symptoms of a B-cell lymphoproliferative disorder. Most MBL cases involve B cells presenting an identical phenotype to CLL (CLL-like MBL) with a Catovsky-Matutes score of 3 to 5 and share the same chromosomal abnormalities than CLL. Depending on the absolute B cell count, one may distinguish low-count CLL-like MBL (<500 B cells/μL) which have no evidence of progression, no reduction in overall survival, no increase in infection risk and do not require any specific follow-up. Patients with clinical CLL-like MBL (>500 B cells/μL) have a 1% to 2% per year risk of progression to CLL requiring therapy, a higher risk of infectious complications and mortality implicating an annual follow-up by hematologist. MBL may also express other less common phenotypes and are named atypical MBL in case of CD5 antigen expression (Catovsky-Matutes score: 1-2) and non-CLL-like MBL for CD5 negative cases (Catovsky-Matutes score: 0-2). Their poorer prognosis implicates imaging studies, bone marrow biopsy and cytogenetic analysis in addition to physical examination in order to rule out non-hodgkinien lymphoma, and require a more frequent follow-up. This review focuses on key concepts in the classification, diagnosis, monitoring and biology of MBL in laboratory practice.

Chronic lymphocytic leukemia presenting as gingival swelling and tooth mobility.

Chronic-lymphocytic-leukemia (CLL) is the most prevalent leukemia in developed countries, caused by monoclonal proliferation of CD5+ B-cells and accumulation of mature-appearing-neoplastic lymphocytes in blood, bone marrow, and secondary lymphoid organs. Oral manifestations of CLL are infrequent and rarely reported in literature. We report a new case of a 67-year old man who presented with the complaints of tooth mobility and gingival swelling. Extra-oral examination was remarkable for cutaneous pallor and bilateral cervical lymphadenopathy involving the submandibular, and deep cervical lymph nodes on both sides of the neck. Complete blood count revealed normal red blood cell count (4.25 × 106/μl), normal platelet count (136 × 103/μl) and increased white blood cell count (25.3 × 103/μl). Differential white blood cell count showed marked lymphocytosis (88%), and blood film revealed the presence of leukocytosis, with small mature-looking lymphocytes, and mild thrombocytopenia. A flow cytometry immune-phenotyping revealed that 55% of peripheral blood cells were monoclonal B-lymphocytes expressing CD19, CD20, CD23, CD200, CD22, CD5, CD38, CD11c, sIgD and Kappa light chain confirming the diagnosis of CLL. Oral healthcare professionals should consider systemic causes, such as CLL, in the differential diagnosis of generalized tooth mobility and gingival swelling, particularly in patients with associated symptoms such as lymphadenopathy, fever, weight loss, and general fatigue.

Lymphocytosis -- Evaluation

Additional testing: Flow Cytometry: Peripheral blood flow cytometry is essential to determine the proliferation of monoclonal cells. It is a costly test and should not be ordered routinely on all patients with lymphocytosis. Certain features on peripheral blood smear or on review of CBC which prompt a physician to order flow cytometry include: The presence of lymphoblasts on the peripheral blood smear, suggesting ALL- This should also prompt a referral to a tertiary care center to obtain further workup. The presence of other abnormal lymphocyte morphology on PBS as detailed above ALC more than 30000 cells/microL Persistent unexplained lymphocytosis for more than one month Abnormalities in other cell lines including anemia and thrombocytopenia Presence of lymphadenopathy and/or hepatosplenomegaly in the right clinical context where reactive causes have been ruled out Flow cytometry patterns are beneficial in delineating clonality and differentiating clonal disorders as follows: CLL: lymphocytes are CD5+, CD23+, CD20 (dim), CD10-, cyclin D1-, weak sIg (surface immunoglobulin), FMC -, CD200 + MCL: lymphocytes are CD5+, CD23-, CD10-, CD20+, sIg +, cyclin D1 +, FMC +, CD200- FL: lymphocytes are CD5-, CD10+, CD20+, sIg+ and often BCL2+, BCL6+ MZL: lymphocytes are CD5-, CD10-, CD20+, sIg+, cyclin D1- HCL: lymphocytes are CD5-, CD10-, CD20+, sIg+, CD11c+, CD25+, CD103+ T-LGL: lymphocytes are CD3+, CD8+, CD16+, CD56+ Fluorescence in situ hybridization (FISH), karyotype, and mutation analysis: FISH, karyotype, and certain mutation analysis help diagnose and risk stratification of hematologic malignancies, especially CLL and lymphomas. The FISH can not only help to ascertain the clonal nature of lymphocytosis but also helps to confirm the diagnosis of certain lymphomas. Examples include: FL: characterized by t(14,18) MCL: characterized by t(11,14) HCL: characterized by the presence of BRAF mutation CLL: different karyotypic abnormalities including del 17p, del 11q, trisomy 12, and del 13q14.

Concomitant Presence of Two Distinct Clones of Chronic Lymphocytic Leukemia and Plasma Cell Myeloma in a Patient.

A 74 years old male patient, presented with history of generalized weakness, fatigue, loss of appetite and breathlessness on exertion for past one and a half months. On examination, he was found to have significant pallor and generalized lymphadenopathy (cervical, axillary and inguinal). The skeletal survey showed punched out lytic lesions in skull and pelvic bones. The peripheral smear examination showed lymphocytosis with absolute lymphocyte count of 25,000/μL. The bone marrow aspirates revealed a hypercellular marrow with 74 % lymphocytes & 14 % plasma cells, suggestive of chronic lymphoplasmacytic disorder. The bone marrow biopsy had two morphologically distinct populations of lymphocytes & plasma cells. The immunohistochemical markers on bone marrow biopsy showed hat plasma cells were positive for CD138 with kappa light chain restriction. Flow cytometry showed B cell population with CD19/CD5 co expression, CD5/CD23 coexpression, were positive for CD22, CD20 and negative for FMC-7 and lambda light chain. In addition, plasma cells were also identified as CD45 negative cells and showed CD38/CD138 co-expression with variable CD19 and CD56 positivity. Serum protein electrophoresis revealed M band, serum immunofixation electrophoresis corresponded to IgA -Kappa. The final diagnosis of chronic lymphocytic leukemia with concomittant presence of plasma cell myeloma was concluded. This case imparts an important message to look for presence of coexisting entities in a single specimen and highlights the benefits of testing both plasma cell and B-cell compartments when the clinical features are not entirely consistent Flow cytometry together with protein electrophoresis can help to clinch difficult and rare dual diagnosis. These cases are rare and pose therapeutic challenge.

[Monitoring minimal residual disease in patients with hairy cell leukemia in complete remission after treatment with 2-chlorodioxyadenosine].

Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine (2-CdA) induces in 85% patients complete remission. Complete remission is defined as the condition when signs of activity of the disease are absent, splenomegaly and lymphadenopathy are absent, the hemoglobin concentration is > or = 120 g/l, the absolute number of granulocytes is > or = 1.5 x 10(9)/l and the number of thrombocytes is > or = 100 x 10(9)/l. In complete remission in the peripheral blood, bone marrow aspirate and bioptic samples obtained by trephin bone marrow core biopsy, using standard staining (hematoxylin-eosin and May-Grünwald-Giemsa's method), no leukemic cells are present. When more sensitive methods are used (immunophenotyping, immunohistochemistry or molecular genetic methods), a persisting leukemic population can be detected which is described as minimal residual disease (MRD). For detection of MRD the authors used immunohistochemical examination of bone marrow with DBA.44 antibodies. As leukemic cells they described those which produced intense cytoplasmic and membrane positivity with antibody DBA.44 and corresponded morphologically to hairy cells. For evaluation computer analysis of the picture LUCIA-M was used. The infiltration grade was examined on three areas of standard size (3 x 65,265 micron 2) and expressed in percent. A total of 45 trepanobioptic specimens from 21 patients were examined who achieved after treatment with 2-CdA complete remission. In all samples suitable for evaluation the presence of leukemic cells (MRD) was detected with a median of 3% and a range of 1% to 18%. With induction of complete remission correlates also the low serum level of the soluble receptor for IL-2 (sIL-2R). In a female patient after 24 months of treatment with 2-CdA the grade of leukemic infiltration rose from 1% to 12% and during the 36th month to 50% DBA.44+ leukemic cells. The incipient relapse in this patient was not associated, despite marked infiltration of bone marrow, with failure of hematopoiesis and a marked rise of sIL-2R.

High-Grade Epstein-Barr Virus-Negative Biphenotypic Lymphoma with Expression of B- and T-Cell Markers and Leukemia Presentation: Case Report and Literature Review.

Lymphomas are presently categorized according to their origin from B or T lymphocytes. The co-expression of CD3 in B-cell lymphomas or CD20 in T-cell lymphomas has been rarely reported. Immature and less often mature lymphomas may incorporate the rearrangements of both B- and T-cell antigen receptor genes (dual genotype or bigenotype). Lymphoma cells with a sole genotype hardly concurrently express both B- and T-cell markers (biphenotypic lymphomas). We describe a 63-year-old female who was presented with obstructive jaundice and epigastric pain of 10 days. Initial CBC revealed 43×103/μL white blood cells, 11.2 g/dL hemoglobin, and 88x103/μL platelets. CT abdomen revealed hepatomegaly and suspected pancreatic mass with large retroperitoneal lymph nodal mass. Peripheral smear showed 56% lymphoid cells with blast morphology. The bone marrow (BM) aspirate smear was infiltrated by 83% immature-looking cells. BM biopsy showed interstitial to diffuse extensive infiltration by primitive-looking cells, positive for pan-B-cell antigens CD20, CD79, and PAX5 as well as the T-cell antigen CD4, CD5, CD3, while negative for all immaturity markers (CD34, TdT, and CD1a). In situ hybridization for Epstein-Barr virus (EBV)-encoded small RNA (EBER) was negative. Flow cytometry on BM aspirate showed an abnormal population (50%) expressing the B-cell antigens (CD19, CD20, CD79, CD22) and CD10, and showed lambda light chain restriction as well as the T-cell antigens cCD3 and CD4 with partial CD5. The analysis showed, also, another abnormal population of lambda restricted monotypic B cells (8%) with dimmer CD45 (blast gate) and showed the same immunophenotype (expressing the B-cell antigens), but negative for CD10, cCD3, CD5, and CD4. Conventional cytogenetic revealed complex karyotype. Molecular studies revealed rearrangements of the immunoglobulin heavy chain region consistent with a clonal B-cell population. TCR gene rearrangement analysis was equivocal concerning clonality but was not conclusive for clonal T-cell disease. Our final diagnosis was peripheral blood and BM involvement by EBV-negative high-grade lymphoid neoplasm (in leukemic phase with blast morphology) and an ambiguous immunophenotype with a differential diagnosis that may include the rare entity of bigenotypic lymphoma or an unusual case of high-grade B-cell lymphoma with aberrant expression of T-cell markers (biphenotypic lymphomas).

Antigen Expression on Blast Cells and Hematological Parameters at Presentation in Acute Lymphoblastic Leukemia Patients.

To analyze the expression of various antigens on the leukemic blasts and to determine the hematological parameters, in Acute Lymphoblastic Leukemia (ALL) patients at presentation. Observational study. King Edward Medical University, Lahore and Hameed Latif Hospital, Lahore, from February 2013 to March 2014. A total of 50 newly diagnosed and untreated patients of ALL were selected from Mayo Hospital and Hameed Latif Hospital. These patients included both genders and all age groups. Hemoglobin, total leukocyte count and platelet count were determined on hematology analyser-Sysmex-Kx-2I. Blast cell percentage was estimated on Giemsa stained blood smears. Immunophenotyping was done on bone marrow samples by 5 colour flowcytometery on Beckman Coulter Navious Flowcytometer. An acute leukemia panel of 23 antibodies was used. The data was entered and analyzed in SPSS version 22. Of the 50 ALL patients, 36 (72%) were B-ALL and 14 (28%) T-ALL. There were 18 (36%) children and 32 (64%) adults. T-ALL included 22% of the childhood and 31% of the adult cases. Immunophenotypic analysis showed that CD19, CD79a and CD20 were B-lineage specific markers whereas cCD3, CD3 and CD5 were T-lineage specific. CD10 was the most sensitive marker for B-ALL and CD7 was the most sensitive marker of T-ALL. TdT was expressed in 92% B-ALL and 71% T-ALL cases, CD34 in 58% and 43% cases and CD45 in 83% and 100% respectively. High leukocyte count (> 50 x 109/L) was present in 58% cases. Hemoglobin was < 10 g/dl in 74% patients and platelet count was below 20 x 109/Lin 12% patients. Leukocyte count, hemoglobin, platelet count and blast cell % did not show a significant difference in the two ALL immunotypes. The frequency of T-ALL is higher in childhood as well as adult ALL in our population compared to the Western literature. Antigenic expression of the blast cells also shows some interesting differences. A large number of our patients present with high leukocyte count which is a known factor associated with poor prognosis.

A 61-year-old man presented with myopathy, neuropathy, and inflammatory dermatitis responsive to chronic lymphocytic leukemia treatment.

The prevalence of paraneoplastic neurologic syndrome in cancer is 0.01%. Neurological syndromes can be seen in chronic lymphocytic leukemia (CLL) and mostly present as either leukemic infiltration of the central nervous system (CNS) or progressive multifocal leukoencephalopathy. To our knowledge, this is the first reported case of combined sensory-motor neuropathy, myopathy, and dermatitis in a patient with CLL. A 61-year-old African American man presented with acute dysphagia, rapidly progressive proximal limb-girdle weakness, and dermatitis. He had a white blood cell (WBC) count of 14,600/mm(3), hemoglobin of 11.4 mg/dL, and a platelet count of 165,000/mm(3). Lymphocytes comprised 15% of the total WBC with an absolute lymphocyte count of 2100/mm(3). Metabolic profile was unremarkable except for a serum creatine phosphokinase (CPK) level of 1056 mg/dL. Serum protein electrophoresis, serologic studies for autoimmune, genetic diseases, and paraneoplastic syndromes were all negative. Electrodiagnostic studies revealed sensorimotor neuropathy with mixed axonal and demyelinating features. Muscle biopsy revealed discrete areas of interstitial fibrosis juxtaposed to areas of intact muscle without any inflammation. At that point, a bone marrow biopsy was done because of anemia and slightly elevated mean corpuscular volume of 103. Bone marrow biopsy revealed minimal involvement with CD5/CD19-positive CLL. Flow cytometry demonstrated monoclonal CD5/CD19/CD20/CD23-positive cells, with dim kappa expression, and negative FMC-7 and CD3. This case doesn't meet the criteria for CLL/small lymphocytic lymphoma. However, considering the possibility of paraneoplastic phenomenon for his symptoms, it was decided to start the patient on CLL-directed therapy with Rituximab and Cyclophosphamide. After only two cycles, the patient experienced a dramatic improvement in his muscle strength with disappearance of the rash. This case highlights a unique clinical picture of inflammatory dermatitis with electromyography and biopsy findings suggestive of myopathy and combined sensorimotor neuropathy with response to CLL-directed therapy. Also the symptoms started before peripheral lymphocytosis which masked the diagnosis for over a year.

[Mantle cell lymphoma as a diagnostic and therapeutic problem].

The authors present the characteristics of a group of 23 patients with mantle cell lymphoma. In the group only a slight predominance of men over women was found (1.1:1), the median age was 63 years. Twenty-one (91%) of the patients were diagnosed in stage IV (Ann Arbor). In all these patients the bone marrow was affected. In 19 of them immunoflowcytometric analysis revealed the typical clone of B lymphocytes (CD5 positive)/CD 23 negative). The majority of patients had at the time of diagnosis a large tumourous mass with massive splenomegaly (61%), hepatomegaly (57%) and bulky disease (52%). The node was excised in 17 patients, but in four patients (24%) during the first session the diagnosis was not assessed correctly. In the laboratory findings an inclination to anaemia, thrombocytopenia, lymphocytosis and in particular to high levels of serological indicators of activity of the disease dominated--lactate dehydrogenase, beta-2-microglobulin and serum thymidine kinase. All patients were treated by chemotherapy. Complete remission was achieved by the date of evaluation in one patient (4%), partial remission in seven patients (30%) but 48% patients did not respond to first line treatment. Nine patients of the group died, their median of survival was 14 months (0-24), the median of the follow up of the remaining patients was 133 months (2-31). Two female patients had large-dose treatment with subsequent administration of autologous stem cells. The first one is after 370 days of treatment in complete remission, the second one developed a relapse 100 days after the procedure. From the results and analysis of the literature ensues that mantle cell lymphoma is one of the aggressive malignant B-lymphoproliferations with a very adverse prognosis and it deserves therefore special diagnostic and intense therapeutic attention.

A rare occurrence of hairy cell leukemia in the Thai population: a case report.

Hairy cell leukemia (HCL) has been mainly reported from the Western countries. Herein we describe a case of HCL diagnosed in a Thai patient. A 36-year-old man presented with abdominal discomfort, frequent gum bleeding and significant weight loss for 2 months. Physical examination revealed moderate anemia, petechial hemorrhage on the extremities and an enlarged spleen down to the umbilicus. No hepatomegaly or lymphadenopathy was detected. Complete blood counts revealed a hemoglobin (Hb) of 6.6 g/dL, a white blood cell (WBC) count of 1.6 x 10(9)/L (neutrophil 16%, lymphocyte 71%, monocyte 11%, atypical lymphocyte 1%), and a platelet (PLT) count of 17 x 10(9)/L. Abnormal large mononuclear cells with villous projections were seen in the blood smear. Although bone marrow (BM) aspiration resulted in a dry tap, abnormal lymphocytes with villous projections could again be identified in the touch preparation. Flow cytometric analysis showed a distinct population above the normal lymphocyte region on CD45/SSC gates with a strong expression of CD19, CD20, CD22, CD25, CD11c, and kappa. CD5, CD23, CD10, CD4, and CD8 were all negative. BM biopsy was consistent with HCL. The patient was treated with splenectomy followed by 8 cycles of fludarabine and cyclophosphamide chemotherapy. At 21 months after diagnosis, the patient was doing well with a Hb of 16.9 g/dl, a WBC count of 6.8 x 10(9)/L, neutrophil 49.9%, lymphocyte 39.6%, monocyte 8.6%, and a PLT count of 329 x 10(9)/L). No abnormal lymphoid cells were detected in the blood smear. This present report represents the first Thai HCL case that was immunophenotypically confirmed by flow cytometry and successfully treated at Siriraj Hospital.

[Detection of PNH clones using flow cytometry in aplastic anemia and paroxysmal nocturnal hemoglobinuria].

To detect and quantify by flow cytometry (FC) PNH clones in paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA) patients. We have performed a flow cytometric analysis to determine the granulocyte expression of CD55 and CD59 from 29 patients with AA and 11 patients with PNH. In the 11 PNH patients the study showed 58 +/- 34% and 56 +/- 32% (mean +/- SD) CD55(-) y CD59(-) granulocytes. A good correlation was found between the results of FC and haemolysis. The follow-up study showed PNH clone progression in one case and stability in 5 cases. Among 11 AA patients studied at diagnosis, two presented a population of CD55(-) granulocytes (14% and 48%) with CD59 normal, this defect disappeared in both patients after immunosuppressive therapy. The FC study revealed PNH clones in 7 cases among the 26 analyzed after treatment (23 with ATG and/or CyA), in 3 cases with negative Ham's test (in two this became positive 6 and 12 months later). The mean values obtained in these 7 patients with PNH-AA syndrome were 26 +/- 15% y 36 +/- 30% (mean +/- SD) CD55(-) and CD59(-) granulocytes. The median time from diagnosis to detection of PNH phenomenon was 83 months. In the follow-up study, 4 cases had stability, one case had a decrease and one a progression of the abnormal clone. In a retrospective analysis, among the 7 patients with PNH-AA syndrome, 5 had a partial response after the initial treatment. The FC on granulocytes is a useful method to diagnose and characterize PNH. This test is good for early detection of PNH clones in AA patients at initial diagnosis and in long term survivors. In both diseases it permits measuring the extent of the abnormal clone and its follow up. The extent of the defect is more related to haemolysis than the haematopoietic deficiency. PNH development seems to be more frequent in AA patients with incomplete response after immunosuppressive therapy and in some cases the defect could be latent at the time of diagnosis.

The sorafenib anti-relapse effect after alloHSCT is associated with heightened alloreactivity and accumulation of CD8+PD-1+ (CD279+) lymphocytes in marrow.

We studied three FLT3 ITD acute myeloid leukemia (AML) patients who relapsed after allogeneic haematopoietic stem cell transplantation (alloHSCT) and received multikinase inhibitor (MKI) sorafenib as part of salvage therapy. MKI was given to block the effect of FLT3 ITD mutation which powers proliferation of blast cells. However, the known facts that sorafenib is more effective in patents post alloHSCT suggested that this MKI can augment the immune system surveillance on leukaemia. In the present study, we investigated in depth the effect of sorafenib on the alloreactivity seen post-transplant including that on leukaemia. The patients (i) responded to the treatment with cessation of blasts which lasted 1, 17 and 42+ months, (ii) developed skin lesions with CD3+ cell invasion of the epidermis, (iii) had marrow infiltrated with CD8+ lymphocytes which co-expressed PD-1 (programmed cell death protein 1 receptor, CD279) in higher proportions than those in the blood (163±32 x103 cells/μl vs 38±8 x103 cells/μl, p<0.001). The Lymphoprep fraction of marrow cells investigated for the expression of genes involved in lymphocyte activation showed in the patients with long lasting complete remission (CR) a similar pattern characterized by (i) a low expression of nitric oxide synthase 2 (NOS2) and colony stimulating factor 2 (CSF2) as well as that of angiopoietin-like 4 (ANGPTL4) (supporting the immune response and anti-angiogenic) genes, and (ii) higher expression of fibroblast growth factor 1 (FGF1) and collagen type IV alpha 3 chain (COL4A3) as well as toll like receptor 9 (TLR9) and interleukin-12 (IL-12) (pro-inflammatory expression profile) genes as compared with the normal individual. The positive effect in one patient hardly justified the presence of unwanted effects (progressive chronic graft-versus-host disease (cGvHD) and avascular necrosis of the femur), which were in contrast negligible in the other patient. The anti-leukemic and unwanted effects of sorafenib do not rely on each other.

Atypical chronic lymphocytic leukaemia with t(11;14)(q13;q32): karyotype evolution and prolymphocytic transformation.

In order to define better the cytological and clinical features of atypical B-cell chronic lymphocytic leukaemia (B-CLL) with t(11:14)(q13;q32), sequential morphologic immunological and cytogenetic studies were performed in seven patients belonging to a series of 72 consecutive cases presenting with a diagnosis of CLL or atypical CLL according to the FAB criteria. Cytologic diagnosis in these seven patients with t(11;14) was typical CLL in two cases presenting with < 10% large lymphocytes (LL) and prolymphocytes (PL) and atypical CLL in five cases in which LL and PL comprised between 10% and 55%. The diagnosis was supported by histologic findings on bone marrow biopsy (five cases) or splenectomy specimens (two cases). A progressive increase of peripheral LL and PL was observed, resulting in a switch of FAB diagnosis over a 6-60-month period from typical CLL into atypical CLL in two cases and from atypical CLL into prolymphocytic leukaemia in five cases. Immunophenotyping showed a mature B-cell phenotype with CD19, CD22, CD24 positivity and CD10 negativity in all patients. A bright-staining pattern for surface immunoglobulins (SIg) was detected in 6/7 cases, CD5 positivity in 6/7 cases, and CD23 positivity in 1/7 cases. The FMC-7 monoclonal antibody was positive in > 40% cells in 5/6 cases. Chromosome changes in addition to t(11;14) were seen in five cases; in two cases unbalanced translocations involving the 3q21 chromosome region, resulting in partial trisomy for the long arm of chromosome 3, were detected early in the course of the disease. Karyotype evolution that was associated with disease progression occurred in 3/6 assessable patients. Comparison of these findings with similar data from 65 B-CLL patients without t(11:14) showed that atypical morphology, switch of FAB diagnosis during the course of the disease, and karyotype evolution were more frequently seen in cases with t(11;14) (5/7 v 15/65 cases, P = 0.015, 7/7 v 7/65 cases, P < 0.0001, and 3/6 v 5/45 assessable cases, P = 0.04, respectively). The frequency of positivity for CD23 and bright SIg staining differed significantly in the two groups. It is concluded that t(11;14) identifies a cytologically atypical subset of B-CLL, characterized by frequent cytologic and cytogenetic evolution and by a distinct immunological profile, sharing some biological features with mantle cell lymphoma.

Epstein–Barr virus-associated lymphoproliferative diseases

receptor, and, in EBV+ HLH cases, circulating EBV. In the latter cases, histological examination of lymphatic, bone marrow, liver, neuronal, and other involved tissues show infiltrations of small EBV+ T cells, scattered small bystander EBV+ B cells, reactive histiocytes, reactive macrophages, and, in ~70% of cases, hemophagocytosis, i.e. ingestion of erythrocytes, leukocytes, platelets, and/or their precursor cells by histiocytes and macrophages. (Evidence of hemophagocytosis is not critical for the diagnosis of HLH.) The EBV in infected lymphocytes is in its lytic cycle rather than any latent phase. Criteria consistent with the diagnosis of HLH, as developed by the Histiocytic Society (2004), include finding five of the eight following signs or symptoms: fever ≥38.5 °C; splenomegaly; low blood levels of any two of the following, hemoglobin (<10 mg/L), platelets (<100,000/μL), or neutrophils <1,000/μl; either one or both of the following, blood fasting triglyceride levels >265 mg/dL

[DBMP-85 was effective at diagnosis and LVP was effective at relapse in a case of acute mixed leukemia].

A 16 year-old boy was admitted to our hospital in April 1985, because of bilateral submandibular swellings. Hematological examination revealed Hb was 7.3 g/dl, WBC was 89,000/microliters (76% blast), and platelet was 154,000/microliters. His bone marrow was hypercellular and consisted with 91% blasts. Myeloperoxidase staining was positive for 38% of blasts. Auer rods were seen in some of blasts. Thus, the diagnosis was M1 according to FAB classification. Cytogenetic studies of 20 marrow cells were performed and all cells had 46, XY, -1, -7, 3q-, 7q-, 17q+, +2mar. Eighty five percent of blasts expressed HLA-DR and 43% of blasts expressed CD2 and CD13 simultaneously. Thus, this leukemia was considered as the hybrid type of acute mixed leukemia by surface marker analysis. DBMP-85 regimen, the chemotherapy for AML, was started after admission and complete remission (CR) was attained in June 1985. After 4 courses of post remission chemotherapy, he discharged in December 1985 and was followed at our outpatient clinic without chemotherapy. His disease was relapsed in June 1986, and the combination chemotherapy with mitoxantrone, etoposide and Ara-C was applied to him but failed to attain CR. Then, LVP protocol, the chemotherapy for ALL, was started and CR was achieved. The blasts at relapse had morphologically myeloid features, and expressed HLA-DR, CD2 and CD13 as well as at diagnosis. Cytogenetic studies at relapse showed some karyotype except gaining 12p- anomaly. Therefore, same blasts were considered to emerge at relapse. Our case suggests that LVP therapy may be effective for AML expressing myeloid and lymphoid surface markers.

Chronic lymphocytic leukemia

Diagnosis The diagnosis of CLL is based on the demonstration of an abnormal population of B lymphocytes in the blood, bone marrow, or tissues that display an unusual but characteristic pattern of molecules on the cell surface. CLL is usually first suspected by a diagnosis of lymphocytosis, an increase in a type of white blood cell, on a complete blood count test. This frequently is an incidental finding on a routine physician visit. Most often the lymphocyte count is greater than 5000 cells per microliter (µl) of blood but can be much higher. The presence of lymphocytosis in an person who is elderly should raise strong suspicion for CLL, and a confirmatory diagnostic test, in particular flow cytometry, should be performed unless clinically unnecessary.

[Identification of splenic marginal zone lymphoma from B lymphoproliferative disorders by flow cytometry].

The splenic marginal zone lymphoma (SMZL) is a relatively rare chronic B lymphoproliferative disease, which primarily manifest increase of peripheral blood lymphocyte count and/or scale, and splenomegaly, while the peripheral superficial lymph nodes are often not swollen. Therefore, the splenectomy are usually needed to confirm the diagnosis, but the majority of patients could not accept such management, resulting in early difficult diagnosis. This study was purposed to explore the more prior way for diagnosis based flow cytometry (FCM). Six patients with suspected diagnosis of SMZL were used as research objects, 10 healthy bone marrow donors and 10 cases of chronic lymphocytic leukemia (CLL), 3 cases of hairy cell leukemia (HCL), 3 cases of lymphatic plasma cell lymphoma/Waldenströ's macroglobulinemia (LPL/WM) were selected as control. The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45, CD5, CD10, CD19, CD20, CD22, CD23, CD25, CD103, CD11c, CD123, κ,λ, Cyclin D1, and combined with bone marrow cell morphology. The results indicated that 6 cases of suspected SMZL showed a large increase of lymphocytes and splenomegaly. Because absence of peripheral lymphadenopathy, 6 patients did not suffer from lymph node biopsy, only 1 patient underwent diagnostic splenectomy. The immunophenotypes of bone marrow in patients and controls were analyzed by FCM, as a result, except for the healthy donors, varying degrees of abnormal mature B cell clones were found in bone marrow of all patients, and the further differentiation from other B-cell tumors was performed through CD5, CD10 expression and combination with other B-cell phenotype. All 6 cases of SMZL patients expressed CD19(+) and CD20(+), but CD10 expression was negative, 4 patients expressed CD5(-), 2 patients expressed CD5(+). The expressions of CD23, CD38, ZAP-70, CD11c, CD103, CD123, Cyclin D1 were negative. The morphological examination of bone marrow cells showed velutinous abnormal lymphocytes. Combined with clinical characteristics, 6 patients were diagnosed as SMZL, 1 patient suffered from splenectomy because of concurrent hypersplenism, and this postoperative pathologic examination confirmed the patient with SMZL. Ten cases of CLL mainly expressed CD5, CD23; 3 cases of HCL had more typical morphology of "hair like" in addition to CD11c, CD103 and CD123 positive; 3 cases of LPL/WM had significantly increased light chain restriction expression, IgM, plasmacytoid lymphocytes. It is concluded that the FCM immunophenotype analysis can be used as a powerful tools for clinical diagnosis of SMZL.

[The limited possibility of using a simplified approach to detect minimal residual disease by the flow cytometry technique in children with precursor B-lineage acute lymphoblastic leukemia].

Minimal residue disease (MRD) is a state in which the tumor cells remain in the patient in the amounts unrecognizable with the standard cytological techniques. Flow cytometry is one of the basic methods for evaluation of MRD in precursor B-lineage acute lymphoblastic leukemia (PBLALL). The so-called simplified three-color analysis using the combination of CD19/CD10/CD34 antibodies has been proposed to detect MRD in the midcourse of induction therapy. Four-to-nine-color is presently used to identify MRD. One hundred and thirty-four bone marrow samples taken at different stages of therapy in 55 children with PBLALL were examined to estimate the possibility of using the flow cytometry technique using the 3-color simplified approach to determining MRD. The results of the simplified and standard approaches were compared in the samples stained with 6-8 monoclonal antibodies in the combinations that always included CD19, Cd10 and CD34. The comparison revealed that MRD had been incorrectly identified by the simplified method in 8.0, 17.6, and 75.8% of the patients on therapy days 15, 36, and 85, respectively. In addition, the content of residual tumor cells with respect to the threshold values more frequently proposed to stratify patients was found to be incorrectly calculated in some true positive samples. Thus, when the simplified approach was applied using the results of MRD detection to stratify the patients into risk groups, 16.0, 27.4, and 81.8% of the samples would yield incorrect information on therapy days 15, 36, and 85, respectively. Thus, the simplified approach to identifying MRD is most applicable on day 15 of therapy; however, there may be mistakes in this point of observation. This method used on day 36 more frequently yields incorrect results and is inapplicable on day 85.

Monoclonal B-cell lymphocytosis

MLB falls into three phenotypes that are distinguished based on the cell surface marker proteins which they express viz., the CLL/SLL, atypical CLL/SLL, and non-CLL/SLL phenotypes. These markers are: CD5, CD19, CD20, CD23, and immunoglobulins (Ig) (either Ig light chains or complete Ig, i.e. light chains bound to Ig heavy chains. Distinguishing between these phenotypes is important because they progress to different lymphocyte malignancies. The following table gives the markers for the three MBL phenotypes with (+) indicating the expression (either dim, moderate, or bright depending or the intensity of their expression), (−) indicating the absence of expression, and na indicating not applicable as determined using fluorescent probes that bind the marker proteins. Detection of fluorescent probe binding by the cells requires the use of flow cytometry preferably employing 6 to 8 different fluorescent probes that bind to different markers on 5 million cells from the patient's blood. The

B-cell prolymphocytic leukemia

Immunophenotype This technique is used to study proteins expressed in cells using immunologic markers. In B-PLL patients there is strong expression of surface immunoglobulin – a membrane-bound form of an antibody, b-lymphocyte surface antigens CD19, CD20, CD22, CD79a and FMC7, and weak expression of CD5 and CD23. Due to the similarities among lymphoproliferative disorders, it is often difficult to diagnose patients. Immunophenotyping helps distinguish B-PLL from similar diseases, one of its key identifiers is the absence in expression of the surface antigens CD10, CD11c, CD25, CD103 and cyclin D1 – an important regulator of cell-cycle progression. A case has been described as CD20+, CD22+, and CD5-. It can also be CD5+. Another case was described as CD45+, CD19+, CD20+, CD5+, HLA-DR+, CD10-, CD23+/-, CD38+ and FMC7-.

[Minimal residual disease monitoring by flow cytometry in children with acute lymphoblastic leukemia].

The cells that have avoided the action of antitumor drugs may be retained after remission achievement during induction therapy and consolidation. A combination of these cells is given the name minimal residual disease (MRD). Multicolor flow cytometry has recently attracted considerable interest as the most promising method for measuring the content of residual tumor blasts. This technique is based on the detection of the so-called leukemia-associated immunophenotype (LAIP), i.e., a tumor-specific combination of the expression of membrane and cytoplasmic markers. Flow cytometry may be successfully used to monitor MRD in 90-95% cases of acute lymphoblastic leukemia (ALL) and in 80-85% of patients with acute myelocytic leukemia. The sensitivity of flow cytometry, which is real for routine flow techniques, is a possibility of identifying one cell among 10(4)-10(5) cells. Multicolor flow cytometry (that involves the simultaneous analysis of the expression of a few markers) is the most reasonable tool for MRD monitoring. The monoclonal antibody panels recommended by different groups of investigators for MRD monitoring in B-lineage ALL include antibodies to the pan-B-cell antigen CD19, markers of different stages of differentiation of B-lineage precursors of CD10, CD34, and CD20 and leukemia-associated markers different for each panel, such as CD22, CD38, CD58, CD45, TdT, CD13, CD33. The hyperexpression of CD10, CD34, CD19, TdT, the decreased expression of CD38, CD45, CD22, CD19, the simultaneous expression of markers of different stages of differentiation of B lymphocytes, such as CD10 and CD20, and the lymphoblast coexpression of myeloid markers of CD13, CD33, CDS15 are the most frequently described immunophenotype aberrations in B-lineage ALL. The selection of combinations of markers for MRD monitoring in children with T-ALL is based on the simultaneous expression of combinations of the antigens characteristic for early stages of differentiation of normal T lymphocytes, namely TdT and cytoplasmic CD3. Some authors consider the use of CD99 versus TdT to be most appropriate. There is recent evidence that MRD-positive patients have a higher cumulative risk for recurrences as compared with those without residual blasts. Moreover, the longer the tumor cells are retained during therapy, the worse the prognosis is. Thus, for choice of the adequate intensity of antitumor therapy, it is necessary to qualitatively and quantitatively assess MRD by multicolor flow cytometry at different stages of therapy.

A case of chronic lymphocytic leukemia with massive ascites.

An 81-year old woman with a history of chronic lymphocytic leukemia (CLL) was admitted with night sweats and abdominal distension. A complete blood count showed hemoglobin 5 g/dL, white blood cell (WBC) count 28.5×10(9)/L and platelets 38.4×10(9)/L. Peripheral blood smear examination showed a large number of smudge cells and lymphocytosis composed of mature-looking lymphocytes with clumped nuclear chromatin. Computed tomography scan demonstrated enlarged cervical, axillary, paraaortic, retroperitoneal and mesenteric lymph nodes with concomitant omental thickening and ascites. Also, the liver and the spleen were enlarged in the presence of multiple ill-defined hypoechoic areas in the latter. Histopathological analysis of the cervical lymph node biopsy was consistent with CLL. Bone marrow examination showed diffuse infiltration of the marrow with small lymphocytes. Analysis of the ascitic fluid revealed an exudate with WBC 1220 cells/mL. Cytocentrifuge preparation of the ascitic fluid showed small mature lymphoid cells containing hyperchromatic nuclei with coarsely granular chromatin. On flow cytometric analysis of the ascitic fluid, expression of CD5, CD19, CD20, CD22, CD23, CD45 and HLA-DR was compatible with a diagnosis of CLL, in accordance with the results of the peripheral blood analysis. The patient was treated with chemotherapy consisting of cyclophosphamide, vincristine and prednisolone but died within one month after development of non-chylous ascites.

[Analysis and significance of hematopoietic progenitor B cells in patients with acute leukemia].

Normal hematopoietic B progenitor cells are similar with acute B lymphoblastic leukemia (ALL) cells in terms of morphology and immunophenotypes which easily result in misdiagnosis of diseases. This study was purposed to explore the importance of B progenitor cell (BPC) level in differential diagnosis of hematologic diseases. A total of 664 specimens including 87 specimens from patients with non-malignant hematologic diseases as control and 577 specimens from AL patients in different progressive stage were analyzed. Out of 577 specimens 26 were collected from ALL patients, 261 were collected from B-ALL, 290 were collected from AML. The relation of different clinical status (new diagnosis, remission, relapse), age and degree of leukemia cell involvement with hematopoietic BPC level were analyzed through identification of CD34/CD10/CD19/CD45 antibody combination and quantification of hematopoietic BPC. The results indicated that (1) CD45 distributed from positive to weak positive, and with very low side scatter. The early hematopoietic BPC expressed CD34⁺, along with increasing of cell maturation, the CD34 expression gradually disappeared, while CD19 and CD10 showed positive in whole stage of hemaropoietic BPC, and early CD10 highly was expressed. (2) the mean percentage of hematopoietic BPC was 1.36% in control group, 0.60% in T-ALL, 1.39% in B-ALL and 0.80% in AML; the detected rate of hematopoietic BPC in control, T-ALL, B-ALL and AML were 87.4%, 61.5%, 83.5%, 75.9%, respectively; the mean percentage of hematopoietic BPC was 0.37% at new diagnosis, 1.66% in remission and 0.55% in relapse. (3) along with increase of age, the hematopoietic BPC level generally disclined. (4) specimens >5% hematopoietic BPC were mainly found in remission stage of leukemia patients. It is concluded that the hematopoietic BPC are present in malignant and non-malignant hematologic diseases. The changes of hematopoietic BPC level correlate with disease state, age and leukemia cell involvement. The increased hematopoietic BPC level are observed most often in the patients with remission after themotherapy. It should be carefully to diagnose and discriminate between malignant and benign cells with double positive CD19 and CD10. Use of multiparametric flow cytometry and optimal antibody combination are important for discriminating hematopoietic BPC from minor residual disease and accuratly diagnosing diseases and evaluating curative effectiveness.

Case report: rare case of infiltration of small lymphocytic B-cell lymphoma in the thyroid gland of female patient with B-cell chronic lymphocytic leukemia (CLL-B/SLL-B).

The article presents a case of 57-year-old woman with the infiltration of rare small lymphocytic B cell lymphoma in the thyroid gland. Initially, the patient was followed-up due to chronic lymphocytic B-cell leukemia diagnosed on the basis of histopathological examination of cervical lymph node. Eight months later, general symptoms occurred along with lymphocytosis and exacerbation of lesions in lymph nodes, and therefore, chemotherapy was started according to COP regimen. After four chemotherapy cycles, further progression of the disease was observed during chemotherapy. Computed tomography (CT) performed at that time showed generalized lymphadenopathy and the presence of an irregular area in left thyroid lobe. On palpation, the thyroid was asymmetrical, with enlarged left lobe and palpable lymph node packages on the left side of the neck. The levels of thyroid hormones and anti-thyroid antibodies were normal. Ultrasound examination of the thyroid gland showed non-homogeneous hypoechogenic structure of the left lobe and complete focal remodeling. Cytological examination of left-lobe lesion obtained during fine needle aspiration biopsy showed multiple small lymphoid cells, suggestive of small lymphocytic lymphoma. To confirm this diagnosis, flow cytometry of the biopsy material sampled from the left lobe was performed showing B cellimmunophenotype: CD19+/CD20+/CD22 dim/FMC-7, CD23+/CD5+, sCD79b-+, CD38-, CD10-, kappa and lambda-/weak reaction. The results of flow cytometry of the thyroid bioptate and blood were nearly identical, confirming leukemic nature of the infiltration in left thyroid lobe. Cytogenetic findings included the presence of 17p deletion (TP53 gene). The patient received immunochemotherapy with alemtuzumab. The progression of the disease occurred in the sixth week of therapy. The treatment was discontinued after 8 weeks due to worsening of patient's general status. The patient died 15 months after the diagnosis.