Clinical Biochemistry and Haematology Laboratories and Leukaemias - Essay Example

Discuss the role of clinical biochemistry and haematology laboratories in differential diagnosis of Leukaemias of Submission Discuss the role of clinical biochemistry and haematology laboratories in differential diagnosis of Leukaemias The cancers of haematopoietic stem cells are associated with the clonal proliferation of one or more of the stem cell lineages. Particularly associated with the proliferation of white cells are known as leukemias. The increase in the number of the white cells can be demonstrated in either the bone marrow or the peripheral blood, or both. Leukemia can be divided on the basis of presentation and certain decisive factors, which either put them in the category of acute leukemia or the chronic leukemia. These are further subdivided on the basis of biochemical, immunophenotypic, haematological and clinical factors. Common to all types of leukemia is the fact that they occur more commonly in men than in women. Leukemia is a relatively rare malignancy. The overall prevalence of leukemia worldwide is 10 per 100, 000 per annum. The incidence of various types of leukemia varies with age and geographical features (Boon and Davidson 2006). Acute forms of leukemia can afflict any group of age. 80% of the adult acute leukemia is acute myeloid leukemia (Mughal et al 2006). The incidence of acute myeloid leukemia increases steadily after 50 years of age. In contrast, acute lymphoblastic leukemia affects young children in the age group 1 to 5 years of age. AML has highest incidence in Caucasians in north and Western Europe, and North America whereas the incidence is low among Asians (Bennett 2002). Chronic leukemia occurs in middle to old age. Chronic lymphocytic leukemia is the most common leukemia in the west (30% of all). Chronic lymphocytic leukemia is extremely rare in China, whereas in UK 2500 cases of chronic lymphocytic leukemia are diagnosed annually. The prevalence of chronic myeloid leukemia is fairly constant worldwide. About 1-1.5 out of 100,000 adults are affected annually. In UK, it affects about 500 individuals each year (Mughal et al 2002, Baccarani et al 2010). The cause of leukemia is not known and there is no single factor responsible for the occurrence of leukemia. Multiple aetiological factors are believed to play a part in the development and progression of the disease. These include genetic and immunological factors, ionising radiations, cytogenetic drugs and certain viruses. Genetic transmission is evident by the increased incidence in identical twins and higher rates of occurrence in genetic diseases like Down’s syndrome (Boon and Davidson 2006). Ionising radiations break the bonds in DNA strands to induce mutations and increase the chances of leukemia. This has been proven in the survivors of the atomic bomb explosions in Japan (Bennett 2002). According to the findings of a study, children of mothers exposed to ionising radiations during pregnancy have increased risk of leukemia later in life. The risk is directly proportional to the number of exposures (Mughal et al 2006). Exposure to chemicals like benzene and drugs (alkylating agents) also puts an individual at an increased risk of acquiring leukemia. Immunodeficiency diseases like hypogammaglobulinemia are also known to increase the risk. Human T cell lymphotropic virus can cause mutations by the induction of enzyme reverse transcriptase (Mughal et al 2006). Various types of leukemia are classified traditionally into acute and chronic forms. The classification of leukemia is essential as it influences the prognosis and the treatment outcomes. Five main types of leukemia are: 1. Acute myeloid leukemia (AML) 2. Acute Lymphoblastic leukemia (ALL) 3. Chronic myeloid leukemia (CML) 4. Chronic lymphocytic leukemia (CLL) 5. Hairy cell leukemia (HCL) In acute leukemia, bone marrow failure occurs as a result of accumulation of immature blast cells which arise due to uncontrolled proliferation of the stem cells. These present acutely with symptoms of pale skin fatigue and bleeding diathesis (epistaxis, bleeding gums and menorrhagia) and often start with an infection (which is an important differential especially in children). Acute lymphoblastic leukemia is subdivided into two forms, Acute B cell lymphoblastic leukemia (B-ALL) and acute T cell lymphoblastic leukemia (T-ALL). Both types can be differentiated on the basis of biochemical, hematological and immuocytogenetic tests. The symptoms are related to anemia, thrombocytopenia and granulocytopenia. In addition, in ALL the joints become painful and the lymph nodes become swollen, and there is hepatomegaly along with spleenomegaly. CNS involvement is common in ALL, and a spinal tap is done to rule out the differential diagnosis (Siegenthaler 2007). In contrast to the acute leukemia, in chronic leukemia the stem cells differentiate and the proliferation thus results in accumulation of cells that are more mature. These symptoms and clinical correlates prompt to the investigations to make a search for underlying disorders of the haematopoietic system (Boon and Davidson 2006). The biochemical investigations identify certain markers for the activity of the disease. These factors differentiate among various types of leukemia and also affect the prognosis. Biochemical diagnosis of acute leukemia can be done by checking for the biochemical enzyme markers. Seven enzymes have been described that are most relevant and usually checked to rule out the differentials for leukemia. These include 1) terminal deoxynucleotidyl transferase (TdT), 2) adenosine deaminase, 3) 5-nucleotidase, 4) purine nucleoside phosphorylase, 5) acid phosphatase, 6) esterase, 7) hexosaminidase isoenzymes. The most powerful among these is terminal deoxynucleotidyl transferase (TdT). Normally, TdT is only evident in small number of lymphocytes within the bone marrow and thymus cells. In acute lymphocytic leukemia, TdT activity is greatly increased, i.e. 85-95%. However in contrast to this, among the patients with acute myeloid leukemia TdT activity is present in only 5-15% of patients. This makes TdT a very important biochemical marker for differentiating ALL from AML. TdT is also important when differentiating T-ALL from B-ALL. TdT is highly positive in T-ALL approaching 90-100%, where as in B-ALL it is almost always negative (Drexler et al n.d). A study by Patric C Kung concluded TdT to be a rapid and specific test for the detection of acute lymphoblastic leukemia as well as chronic myelogenous leukemia. Among 77 patients of ALL, 73 were found to have significant TdT activity. As for CML, 24 out of 72 patients were confirmed to have TdT in the blast cells (Kung et al 1978). The three enzymes of the purine pathway, adenosine deaminase, 5-nucleotidase and purine nucleoside phosphorylase are measured to check the functioning of this pathway as inherited deficiency of these enzymes leads to immunodeficient states. Various subtypes of leukemia can be differentiated by quantifying the levels of these enzymes. 5-nucleotidase is either low or normal in acute myeloid leukemia. In acute lymphoblastic leukemia, both subtypes (T-ALL, B-ALL) show decreased levels of this marker. Adenosine deaminase is not a helpful marker in differentiating AML from ALL because the levels do not show any clear cut demarcations. ALL is usually associated with high adenosine deaminase activity, which is highest for T-ALL. AML may be associated with moderate to high increase in its activity. The levels of purine nucleoside phosphorylase are usually very low in both types of acute lymphoblastic leukemia as compared to normal. AML patients exhibit purine nucleoside phosphorylase activity that is almost same as normal, but when compared with the low activity found in ALL, this level will be considered as high (Drexler et al n.d) Acid phosphatase and hexosaminidase are the two lysosomal enzymes. Acid phosphatase does not completely differentiate AML from ALL, not does it differentiate among various subtypes of ALL. It is more of a qualitative differentiation rather than quantifying the levels to discriminate and divide these types. After isolating its isoenzymes by gel electrophoresis, an isoenzyme was found that was resistant to inhibition by tartrate. This isoenzyme is particular for hairy cell leukemia (HCL). By focusing the isoelectric changes in the isoenzyme profiles, various subtypes of ALL can be identified as they correspond to the changes in isoenzyme patterns. Hexosaminidase has three isoenzymes A, B and I. Activity of these isoenzymes can be assessed and it differentiates among other less common forms of leukemia. Its activity is high only in some cases of AML. Neither B-ALL nor T-ALL is associated with abnormal increase in its activity. Estrase is also a hydrolase but it is more specific for its substrates. Their sensitivity or resistance to inhibitors (sodium fluoride) and particular cytochemical staining patterns make these a valuable marker for disease analysis. Gel electrophoresis and then focusing of isoenzymes offer clear distinction between AML and ALL. ALL is characterized by the incomplete expression of the isoenzyme marker (Drexler et al n.d). In case of chronic myeloid leukemia, biochemical analysis can also be targeted to detect the increased tyrosine kinase activity in the BCR-ABL fusion gene. Due to increased tyrosine kinase activity, it continues to phosphorylate several substrates and ultimately the loss of growth control mechanisms lead to the cancerous growth (Sawyers 1999). The basic haematological investigations include complete blood count, bone marrow examination and the examination of the peripheral blood film. An accurately prepared blood slide and bone marrow slide are essential for the diagnosis. A number of stains are used in the diagnosis of leukemia to stain the immature malignant cells. These include Sudan black B and myeloperoxidase which stain myelomonocytes, ,chorioacetate estrase which is the marker for blast cells, myeloblasts show a speckled pattern when stained with periodic acid schiff (PAS), tartrate resistant acid phosphatase for hairy cell leukemia (Wiernik 2001). In acute leukemia, there is anemia and the white cell count is increased. However, most often it remains below 100x109. Thrombocytopenia is usually severe. Characteristic ‘blast cells’ are seen on the blood film (Boon and Davidson 2006). Blast cells are the cells with raised nuclear to cytoplasmic ratio. Unexplained leukocytosis should always lead to the examination of blood smear to check for blast cells. The low counts should also lead to the similar examination as the presentation can be of acute leukemia, hairy cell leukemia or other malignancies infiltrating the bone marrow. Blood smear is one of the initial investigations that not only help diagnosis and rule out differentials but also guide further investigations and diagnostic workup. In cases of acute promyelocytic leukemia, blood smear offers rapid detection for appropriate management (Bain 2005). The reliability of the blood smears can be increased by labeling the films with alkalaine phosphatase/ Antialkaline phosphatase (APAAP) technique. This technique helps to detect effectively the cellular markers of leukemia that are assessed routinely (Erber et al 1986). The most important haematological investigation is the bone marrow examination as this also provides the sample for cytology, genetic and immunological tests. In acute leukemia, bone marrow is hypercellular and blast cells are seen (above 20%). In AML, ‘auer rods’ are seen in the cytoplasm of the cells (Boon and Davidson 2006). Electron microscopy is helpful in detecting acute lymphoblastic leukemia and acute myeloblastic leukemia. Ultra-structural cytochemistry can differentiate between the two, as lymphoblasts and myeloblats are clearly visible and appreciated (Dvorak et al 1981). In chronic myeloid leukemia, blood counts reveal anemia which is normocytic and normochromic. Thrombocytopenia is often the presenting feature .The mean leukocyte count is 220x109 and the average platelet count 445x109. Blood film examination reveals white blood cell precursors at various stages ranging from myeloblasts to mature neutrophils. The most dominant cell type however is neutrophils. Overall, myeloblasts are usually less than 10%, however during the blast transformation stage there is a significant increase in their number. Other types of cells include nucleated red cells, basophils and eosinophils. The level of neutrophil alkaline phosphatase is very low. The level of vitamin B12 is shown to be high. Also, uric acid and Lactate dehydrogenase are found to be high. Bone marrow examination is undertaken to reveal the Philadelphia chromosome in CML and to undertake studies to look for BCR-ABL fusion gene (Boon and Davidson 2006, Baccarani et al 2010). Chronic lymphocytic leukemia can be divided in typical and atypical types. The diagnosis is dependent on initial atypical findings and further investigations to look for cytogenetic aberrations and rule out other forms of disease. Several markers are there that can be assessed in the laboratory, but none of these is exclusive for CLL. The diagnosis thus relies on the combined picture taking into account all the features present and integrating them into a scoring system (Matutes et al 2007). Peripheral film shows excess of mature lymphocytes. They are monoclonal B cells expressing CD19 and CD23 antigens. Reticulocyte count is checked and direct Coomb’s test is done as there is a risk of developing autoimmune haemolytic anemia. Levels of immunoglobulin are tested to assess the severity of immunosuppression. Bone marrow examination is usually not required (Boon and Davidson 2006). Hairy cell leukemia is a B cell malignancy and is far more common in men than in women. Blood count reveals monocytopenia, neutropenia, characteristic ‘hairy cells’. The hairy cells are seen in peripheral blood as well as on bone marrow examination. The acid phosphatase reaction in these characteristic cells is highly resistant to the action of tartrate (Boon and Davidson 2006). Also, low cholesterol levels have consistently proved to be associated with the risk of leukemia. The study by Naik analyzed the relationship between leukemia and hypocholesterolemia in patients with acute myeloid leukemia by detecting blast cell maturity. In patients with leukemia, levels of cholesterol, HDL and LDL are found to be consistently low. This is attributed to high level of LDL receptor activity in the cancer cells. Patients with hairy cell leukemia also have decreased levels of cholesterol (Naik et al 2006). Flow cytometry has evolved to be an important adjunct in the haematological laboratory for the diagnosis differential work up of various types of leukemia. The differences in antigen profiles and cellular characteristics are analyzed through flow cytometry as variations patterns and intensity of the antigen expression. It effectively differentiates myeloid and lymphoblastic types of leukemia. Isolation of blast cells can effectively be undertaken by CD45/ side scatter gating (AML). Isolation of cells is essential obtain a pure sample. Standard type of forward and side gating does not provide a pure sample as there is overlap between various types of cells. Mature bone marrow cells express CD45 and thus CD45 side scatter gating provides a sample that is pure and is separated into cellular components for obtaining the results showing marrow infiltration by blast cells or other malignant cells (Brown et al 2000, Jennings et al 1997). Thus, measuring the levels of biochemical enzyme markers has provided a quantitative advancement in the diagnosis and differential work up of various types of leukemia. By measuring the levels of enzymes, the genetic expressions of the malignant cells can be detected. Not only are these markers useful in diagnosis and ruling out differentials, but also in identifying the stages of the disease and cell lineages and act as an essential tool in routine leukemia investigations now. Basic hematological tests serve as the initial investigations that point to the malignancy of the haematopoietic system. These tests are simple and are usually a part of diagnostic work up of many other conditions. Any abnormalities of blood cells (blood count, morphology) should be assessed thoroughly. The test results of these investigations rule out other diseases allow other more relevant and required investigations and also help formulate the treatment plan. This has allowed for the leukemia to be diagnosed early, as evident by an increase in the number of people with CLL and CML being diagnosed in early asymptomatic phase (Wiernik 2001). Bibliography 1. BACCARANI M., & DREYLING M. (2010). 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