Immunocytochemical Staining of a Chronic Myeloid Leukaemia Cell Line - Coursework Example

Haematology xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Name xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Course xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Instructor xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Date Aims This study was aimed at detecting the whether there are specific proteins in cells or even tissues with the use of antibodies referred as primary which binds to certain antigen and antibody antigen which are bound by a secondary, enzyme antibody and finally in the presence of chromogen, and enzymes inform of colored deposits at the sites of antibody antigen binding. Introduction CML is a colonial malignant myeloproliferative disorder which is said to originate from an abnormal haemopoietic stem cell (Lannon, 2006). The abnormal stem cell proliferate over the period of time in a way that when leukemia is diagnosed, the bone marrow is grossly hypercellualr and this being the case, the number of leucocytes increases in the peripheral blood. When this continues, the normal blood cell production in completely replaced by leukemia cells although the somehow function normally (Ilaria, 2004). The method we used is of great important for it inhibits all the mechanisms that implicate the BCR/ABL due to their dependent growth and accumulation of leukemic cells in chronic myeloid leukemia (CML) (Ilaria, 2004). One of the mechanisms exhibited by this methodology is apoptosis. This implies that, the numbers of antiapoptotic molecules which are presentr in CML cells and they can lead to enhanced survival of leukemic cells (Pettit, Moss & Hoffbrand, 2001). CML can become very aggressive especially when the bone marrow produces an excessive number of immature granulocytes which are in other words referred to as blast cells or leukemic blasts (Jacobs, Messick & Valli, 2002).. They expand rapidly crowding the bone marrow and preventing it fro making adequate number of red cells, normal white cells and platelets. This makes those suffering from it become more prone to anemia, bruising and bleeding easily (Dacie & Lewis, 2006). It is worth noting that, leukemic cells show the reciprocal translocation and bcr/abl oncogene. In this case, bcr/abl transforms cytokine dependant cell lines to growth factors independence and acts ocogenic in mice. For at least over two decades, there have been a lot of improvements in the areas of cytometric analysis, immunohistochemestry and molecular diagnostic studies (Lanno, 2006). This has refined and improved diagnostic criteria which formerly were used: cytochemestry and cytogenetic. Throughout this study, we also concentrated on practices and expectations for interpreting biopsies (Lannon, 2006). This records a growth in complexity and dependence of our methodology. However, we cannot rule out the traditional role of histopathology in evaluating the bone marrow biopsy for it remains of great important. We hypothesized that there is a great variation in bcr/abl junction point, meaning that, there is a very big difference in their cell lines (Messick & Valli, 2002). It was noted that some cell lines derived from T cell lineage while other are from B cell or myeloid lineage as determined by the cell surface marker staining. One of the goals of this experiment was therefore to test if the junction effect point effects cellular signaling in the model cell lines (Jacobs, Messick & Valli, 2002). The outcome was then used to show if the lineage of the cell lines is linked to cellular signaling. It is worth noting that, the results are of great importance for any future experiments using model cellular lines to study CML as well as any possible clinical approaches in curbing the epidemic (Jacobs, Messick & Valli, 2002). RESULTS: Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 Fig 9 Black cytoplasmic granules in myeloids cells. DISCUSSION: The hairy cell is a chronic B cell lymph proliferative disorder which is clonal expansions of activated B cells (Lannon, 2006). They have clonal immunoglobulin gene rearrangements and express a monoclonal surface of either IgG or even heavy chains of isotopes (Harmening, 2002). The hairy cell is white blood cells which are characterized by the presence of cytoplasmic vilii, tartrate resistant acid phosphate activity and finally inducible phagocyte properties. It is worth noting that, the linage derivation of the hairy cell was seen to from B cell linage and this is also supported by studies about monoclonal antibody and immunoglobulin gene rearrangement studies. Tumors which had hairy cells appear to correspond to plasma cell tumors. It is also worth noting that; splenectomy is the first line treatment of choice for it corrects the pancytopenia in most affected cells and improves the survival (Jacobs, Messick & Valli, 2002). Hairy cell morphology On light microscopy, these cells have a solitary slight eccentric nucleus which in most cases are round, ovoid or even indented but rarely lobulated (Harmening, 2002). Moreover, the chromatin is dispersed or stippled and nucleoli are not exposed and therefore hard to sight them. In the same case, the many cytoplasms are pale blue gray and rod shaped which is a counterpart of the ribosomal lamella complex which is usually visible on electron microscopy (Harmening, 2002). The common cytoplasmic outline is irregular frayed within fine hair like vilii. Most of the HC have a strong staining acid phosphate reaction resistant to tartrate. Also, the prolymphocytes look like B-prolymphocytic leukemia (B-PILL). In other words, they have substantial round nucleus with a number of prominent nucleus and coarse chromatin pattern. Moreover, they had blue gray cytoplasm with irregular villous surface projections but it is generally negative (Harmening, 2002). Conclusively, the monocytopenia is linked to true HCL and not HCL variants (The EBMT Handbook, 2008). Therefore hairy cell morphology is best supported by phase microscopy which is inclusive of a viable wet preparation of moving cells in suspension for a better picture of the hairy cell. In regard with the results of the study, the established cells lines expressed the Bcr-Abl proteins and are oncegenic for syngeneic animals. A look at standard conditions the two cells showed comparable amounts of the bcr-abl protein, however, there were big differences in the number of characteristics (Harmening, 2002). Considering the morphological aspect, some cells were the more homogenous. In this case, they were largely represented by leukemic blastic cells which have a high concentration of AgNORs which are markers which showed high proliferative activity. In some cells, there were more polymorphic and had big cells within their population (The EBMT Handbook, 2008). Moreover, the leukemic myeloid leukemia cells also manifest as small cells with scant cytoplasm and minimal differentiation. Expression of myeloid markers with the absence of co-expression of a substantial number of lymphoid associated markers is one of the important aspects that should be considered when it comes to diagnosing myeloid leukemia (Harmening, 2002). It is worth noting that the consistent translocation of c-abl gene to the ph chromosome in all cytogenetic subtypes of positive CML, in conjunction with the detected abl RNa species in leukemic cells, giving an implication of the presence of oncogene in the development of CML. This on the other hand entails that the abl-encoded protein in leukemic cells has a higher molecular weight compared with its normal tissue counterpart (Lannon, 2006). Some clone chromosomes were detected to be negative cells. This can be observed when cells suffering from CML are addressed with imatinib. This is due to the fact that, they gradually achieve cytogenetic response to the treatment. The patterns realized in the myeloid and lymphoid cell lines suggest that, there is are traces of homing receptors on the human cells which retain the ability to recognize tissue specific adhesion molecules in the mouse tissue. However, there are diverse interactions of leukocyte integrins which are referred to as lymphocytes function associated molecules. Consequently, there are three proteins, p190, p120 and p230 which have different tyrosine kinase activity and different in vitro transformation characteristic. All of them have the capability of changing both myeloid and lymphoid cell lines (Lannon, 2006). The proliferation activity of these cells also differs in great ways in regard to with their tryrosine kinase activity. It is a point worth noting that, in a murane bone marrow transplantation model, the three forms of bcr-abl have the capacity to induct CML like myeloproliferation syndrome (Gajewski et al, 2004). An analysis of proviral integration has shown that this disease which is very similar to CML is polyclonal and also involves B-lymphoid lineages (Harmening, 2002). This being the case, the primitive multi-potent stem cell implicated as the target cells shows positive results. This is support by a test whereby a disease similar to CML was induced in mice by implanting bone marrow cells transducer by Bcr-Abl encoding retroviral factors. After several days of the transplant, the disease manifested itself by cachexia and visible poor grooming (Jacobs, Messick & Valli, 2002). Conclusion CML is its chronic state is a myelopriliferative disease which is characterized by an over proliferation of a well differentiated leukocytes and blast in the peripheral blood. In this experiment, we did not observe the any maturation of implanted neoplastic cells. However, there were a substantial number of myeloid cells in the mouse sample. It is a matter of fact that, ABL gene is the human homologue which is in v-abl oncogene and is carried by leukemia virus is aid to encode a non receptor tyrosine kinase. Human Abl is characterized by 145kd proteins with 2 isoforms which arise after the first exon is suppressed. It has been found that, leukemia is a stem cell disorder which result from progenitor cells. It is characterized by the clonal expansion of hematopoietic stem cells which are as a result of the gene translocations of BCR gene on chromosome 9 and ABL chromosome 22. In the test, there are some cells that were found to be resistant. This is attributed to a substrate for the transportation of protein and transportation of the cell from the use of imatinib. Moreover, alpha 1 acid-glycoprotein binding to the imatinib can also be a cause. The relationship between the drug and the protein decreases the intercellular levels of drugs. Another cause of resistance is the gene amplification of BCR/ABL due to the fact that, the amplification is linked with an increase in level of ph chromosome and a decrease of imatinib effect. The detection of tumor suppressor gene is found in many cases of blast crisis. Moreover, the growth pattern for these cells in DC culture and progressive proliferation and differentiation resulting from an increase in total cell culture period is different from the growth pattern of CML cells from the chronic phase. This therefore means that, the ph positive stem cells in the chronic phase do not have the potential for unlimited proliferation under these culture conditions Reference Dacie, J.V. and Lewis, S.M (2006), Practical Haematology, 10th Ed X3., Churchill Livingstone, ISBN:0443066604. Gajewski ,J., L, Foote M, Tietjen J, Nelson B, Simons A, Champlin R. (2004) Blood and marrow transplantation compensation: perspective in payer and provider relations. Biol Blood and Marrow Transplant; 10: 427–432. Harmening, D. M. (2002) Clinical Haematology and Fundamentals of haemostasis, fourth edition, ISBN: 0803617321 5th edition not published yet 2009 Jacobs, R. M., Messick, J. B., Valli, V. E. (2002) Tumors of the hem lymphatic system. In: Tumors in Domestic Animals, Fourth Edition, ed. Meuten, D. J., pp. 119-198, Iowa State Press. Lannon, C.,M. (2006) Principles for the development and use of quality measures. Pediatrics 2008; 121: 411–418. Ilaria, R. L., Jr. (2004) Animal models of chronic myeloid leukemia. Hematol. Oncol. Clin. North Amer. 18, 525-543. Pettit, J., Moss, P., Hoffbrand,A.V. (2001) Essential Haematology, 6th Ed Wiley, ISBN: 978-1 4051-9890-5. 5th edition – 7 in stock in library. .The EBMT Handbook, (2008), 5th Edition, Hematopoietic Stem Cell Transplant. Read More