Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.
1.0 General Introduction
This thesis focuses on the development of contamination parameters for the new Beckman Coulter Ireland Inc (BCII) HbA1c Advanced Reagent. The HbA1c Advanced Reagent is used for the determination of Glycated Haemoglobin (HbA1c) analyte in whole blood samples. Testing of HbA1c is used in conjunction with other laboratory tests and information to determine a patients average blood sugar level over a period of eight to twelve weeks (World Health Organization, 2011) which is used to both diagnose Diabetes Mellitus and monitor patients diagnosed with Diabetes Mellitus. The issue presented was the potential cross contamination of biochemical reagents used to carry out analytical tests and different sample types which these analytical tests are carried out on. This cross-contamination lead to erroneous patient results and incorrect diagnoses of patients. Section 1.1.1 of the introduction briefly overviews the discipline of clinical chemistry and introduces popular analytes used diagnosing, monitoring and screening of different patient sample types. Section 1.1.2 gives an introduction into the different types of measurement techniques which can be used for detecting analytes in clinical chemistry including photometry, enzymatic methods electrochemical and immunological methods used on the AU (Automation Unit) Series of Beckman Coulter Analysers. The background of Diabetes Mellitus is discussed in Section 1.2.1. In Section 1.2.2 the biochemistry of HbA1c and HbA1c’s role in the body is discussed. Section 1.2.3 discusses the importance of HbA1c measurement with clinical and laboratory analysis can lead to the diagnosis and treatment of Diabetes Mellitus. Lastly Section 1.2.4 gives an overview of different methods used to detect HbA1c.
Section 1.3 discusses the hardware and theoretical background to the photometry to the AU series of analysers specifically the DxC700 AU which is the first analyser to have capability to carry out HbA1c Advanced biochemical tests. The HbA1c Advanced reagent and calibrators which are used on the DxC700 AU are discussed in Section 1.4.1 and Section 1.4.2 respectively. The raw materials used in the HbA1c Advanced Reagent and the biochemistry of the reagent are discussed in Section 1.4.3. Section 1.4.4 discusses the HbA1c assay calibration and calculation methods. Section 1.4.5 discusses the National Glycohaemoglobin Standardization Programme reference formulas and the calculation of HbA1c results. Section 1.4.6 consists of a comparison between the Beckman Coulter reagent and competitor reagents.
1.1 Clinical Chemistry
1.1.1 Clinical Chemistry Background
Clinical Chemistry is the study of disease and the related biochemical or chemical mechanism associated. Clinical chemistry focuses on the analysis of body fluids including blood, urine and cerebrospinal fluid. A brief overview of the above analytes is outlined in this section.
Blood including plasma, serum and whole blood samples is the most common specimen type used in clinical chemistry analysis. These specimen types are prepared from whole blood which is drawn from a patient. Whole blood collection is carried out from venous, arterial or capillary blood, concentrations and properties remain relatively unaltered for both extracellular and cellular constituents. In vitro anticoagulation of the whole blood sample stabilizes the sample for a certain time period depending on sample storage. This anticoagulation is achieved using a collection tube containing ethylene diamine tetra-acetic acid (EDTA) which prevents clotting by inhibiting the anticoagulation cascade by binding calcium ions. Coagulation can also be prevented by inhibiting thrombin activity using heparinates or hirudin (“World Health Organization 2002”). Whole blood samples collected in tubes containing anticoagulants are suitable for determination of HbA1c in patients using the HbA1c Advanced Reagent. Serum and plasma samples are prepared by centrifugation of the whole blood sample. Serum samples consist of the liquid portion of the blood with both the cells and clotting factors removed (Tuck et al., 2009). The liquid portion of the blood contains the proteins and other molecules which represent the whole body. Cells and clotting factors are removed from the blood sample by allowing an adequate amount of time for a clot to due to fibrinogen being converted to fibrin. Serum samples require 30 to 60 minutes at room temperature to allow clots to form followed by centrifugation to separate the blood cells from the extracellular components of blood. Plasma includes cellular material providing different analytes (Tuck et al., 2009),Plasma is prepared by collecting a whole blood sample followed by centrifugation to separate extracellular proteins and blood cells. Plasma differs to serum as coagulation does not occur resulting in fibrinogen and clotting being present in the plasma. This results in a sample similar to what is observed in-vivo.
Urine samples used for laboratory tests using either random urine specimens or timed collections (samples collected at specific time intervals). Timed urine tests can be used to test for patients with potential proteinuria in patients with chronic kidney disease by screening for urinary albumin levels and urine creatinine levels (Martin, 2011). Cerebrospinal fluid (CSF) is a biologic fluid formed mainly in the ventricular choroid plexus and is distributed within the subarachnoid space the ventricular system and the basal cisterns (Jurado and Walker, 1990). CSF plays a vital role in normal brain function. Variation in CSF’s composition can result in a negative impact to normal brain function. CSF samples are collected using a lumbar puncture which is an invasive technique which involves the introduction of a needle below the termination of the spinal cord giving access to the subarachnoid space (Wright et al., 2012). Comparison of glucose concentrations in serum samples compared to glucose concentrations present in CSF samples is a valuable tool in the diagnosis of neurological conditions(Moosa et al., 1995).
Clinical chemistry methods are used in the diagnosis, prognosis screening and monitoring of disease in patients. In diagnosis clinical chemistry tests provide a physician confirmation of either the presence or absence of a patient disease when patient history is taken into account. The HbA1c Advanced Reagent is released with a diagnostic claim for Diabetes Mellitus. Clinical Chemistry tests may also provide prognostic information. For example the measurement of urinary creatinine and urinary albumin over time can reflect the progression of renal disease (Martin, 2011). Monitoring patient diseases over the course of an illness can give an indication of treatment effectiveness. HbA1c is monitored every 8 to 12 weeks (World Health Organization, 2011) to determine a patients glycaemic control. HbA1c monitoring is carried out using the HbA1c Advanced Reagent. Screening of patient samples involves using clinical chemistry to determine if a clinical condition is present sub-clinically, e.g. screening for the disease hyperthyroidism which is characterized by a low Thyroid Stimulating Hormone levels and normal levels of circulating thyroid hormones. Hyperthyroidism causes include Graves disease, multinodular goiter and solitary thyroid nodule (Helfand, 2004).
1.2 The Role of HbA1c in the body
1.2.1 HbA1c: A Biochemistry Background
Measurement of HbA1c in whole blood is used with clinical tests including both oral glucose tolerance tests and fasting plasma glucose tests to determine glucose concentration of a patient. HbA1c measurement compared to oral glucose and fasting glucose allow for long term monitoring of glycaemic control over a period of eight to twelve weeks due to the Lifecyle of red blood ` cells.
Hemoglobin is an oxygen carrying protein present in erythrocytes. A normal human erythrocyte is roughly 6 to 9 micrometres in diameter and biconcave in shape. Pre-cursor stem cells called hemocytoblasts form erythrocytes. In the maturation process this stem cell produces daughter cells, these daughter cells form large amounts of haemoglobin before losing their intercellular organelles including nucleus mitochondria and endoplasmic reticulum. Erythrocytes are therefore incomplete cells which are unable to reproduce. Erythrocytes are destined to survive for roughly 120 days in normal patients. Erythrocytes main function is to carry haemoglobin. This haemoglobin is dissolved in high concentrations in the cytosol of the erythrocytes Haemoglobin is spherical and is 5.5 nm in diameter weighing 64,500 Daltons. Haemoglobin is a tetrameric protein which contains four heme prosthetic groups. One prosthetic group is associated with each polypeptide chain. Adult haemoglobin consists of two globin types two identical α chains which consist of 141 amino acid residues each and two identical β chains which consist of 146 amino acid residues each. The tertiary structure of the α and β subunits are quite similar. This similarity in structure occurs although the α and β subunits only share less than half or the same amino acid sequence (Nelson and Cox, 2008 pp 158).
Glucose is the brains principal source of energy in the human body. Glucose is monosaccharide. A monosaccharide is a carbohydrate consisting of a single sugar unit. Glucose has a molecular formula of C6H12O6. Glucose is broken down to carbon dioxide and water through cellular respiration in the presence of oxygen carbon dioxide and water (Cummings, 2011 pp 68)The concentration of glucose in blood and urine change with the timing of both meals and exercise these measurements do not accurately reflect the average blood glucose of an individual over a period of hours or days. This can result in dangerous increases going un-detected. Average glucose concentration can be determined by looking at its effect on haemoglobin. Transporters present in the erythrocyte membrane equilibrate both plasma and intercellular glucose concentrations. This results in haemoglobins constant exposure to glucose concentration present in patients’ blood. A non-enzymatic reaction called glycation occurs between the primary amino groups in haemoglobin at the amino terminal valine or the ε-amino groups of Lysine (Nelson and Cox, 2008 pp 241-242). This reaction results in the formation of a Schiff Base intermediate products. This haemoglobin forms quite rapidly. These Schiff base rearranges to form a stable ketoamine which is essentially an irreversible reaction (Bunn et al., 1975). The rate of this glycation is determined and proportional to the concentration of glucose and is an accurate reflection of average blood glucose over a period of eight to twelve weeks (World Health Organization, 2011).
In 1993 the Diabetes Control and Complications Trial (DCCT) completed a nine-year study which showed the risk for development and progression of chronic complications associated with Diabetes Mellitus is linked to the degree of glycaemic control which is measured using HbA1c as a marker. This landmark study resulted in the establishment of specific diabetes treatment goals using HbA1c as a marker for mean blood glucose. Standardization of HbA1c results to the DCCT method would allow for individual clinical laboratories to provide diabetic patients with test results which could be related directly to both mean blood glucose levels and risks for development and progression of chronic diabetes complications. Early efforts to standardize Glycohaemoglobin results among laboratories was based on the use of a universal calibrator. This proved feasible for certain methods but did not work for a number of existing methods due to the preparative processes which calibrators quality control materials and proficiency testing material can result in an appreciable difference in comparison from patient samples. It was concluded that any further Standardization efforts to the DCCT method would be carried out at manufacturer level. In 1996 the National Glycohaemoglobin Standardization Programme (NGSP) HbA1c Standardization program. The NGSP program is based on performing split sample method comparisons with an NGSP certified SRL’s (Secondary Reference Laboratories) .SRL’s work directly with HbA1c assay manufacturers including Beckman Coulter to standardize methods by providing reference samples for comparison studies and method certification (NGSP).
1.3 Diabetes Mellitus
Diabetes Mellitus are a group of metabolic diseases which are characterized by hyperglycaemia, defects in insulin secretion and or action. Chronic Hyperglycaemia results in damage dysfunction and failure of the eyes, kidneys, nerves, heart and blood vessels. Diabetes development occurs due to several pathogenic processes including the autoimmune destruction of β-cells of the pancreas which results in insulin deficiency and resistance to insulin action(American Diabetes Association, 2004).
1.3.1 Insulin and Diabetes
Insulin is a small protein which consists of two polypeptide chains joined by two disulphide bonds. Insulin is a hormone which regulates the function glucose metabolism. Synthesis of insulin occurs in the pancreas. Insulin is synthesized as an inactive single chain precursor preproinsulin. Preproinsulin contains an amino terminal signal sequence which directs preproinsulins passage to secretory vessels. Removal of the 23-amino acid signal sequence by proteolysis and the subsequent formation of three disulphide bonds results in the formation of proinsulin. Storage of proinsulin occurs in the secretory granules of pancreatic β-cells. Insulin secretion is triggered in the presence of elevated blood glucose levels. Proinsulin is converted to active insulin by specific proteases which cleave two peptide bonds to form the mature insulin molecule(Nelson and Cox, 2008 pp906).
Figure 1: Formation of mature insulin from preproinsulin
Insulin release is dependent on the glucose level present in the blood supply to the pancreas. The pancreas produces the peptide hormones glucagon, insulin and somatostatin via specialized cell clusters located in the pancreas, the islets of Langerhans. Each different cell type of the islets of Langerhans produces a single hormone α cells produce glucagon, β cells produce insulin and δ cells produce somatostatin. A rise in blood glucose levels results in GLUT2 transporters carry glucose into the β cells. Glucose is immediately converted to glucose-6-phosphate by hexokinase IV (glucokinase) and enters the glycolysis pathway. Glycolysis involves the breakdown of glucose to pyruvate. As a result of the higher rate of glucose catabolism ATP increases resulting in the closing of ATP-gated K+ in the plasma membrane. This reduced efflux of K+ results in the depolarization of the membrane. This membrane de-polarization triggers the opening of voltage gated Ca2+ which results in a cytosolic increase in Ca2+ triggers insulin release by exocytosis. Exocytosis is a process by which a cell transports secretory product through the cytoplasm to the plasma membrane in vesicles. The ATP gated K+ channels are central to β cell regulation of insulin secretion. These channels are octamers composed of four identical Kir6.2 subunits and four identical SUR1 subunits. The formation of a cone around the K+ channel by the four Kir6.2 subunits allow the functionality of a selectivity filter and an ATP gating mechanism. When a rise in ATP occurs, which indicates a rise in blood glucose levels the K+ channels close resulting in plasma membrane depolarization and the consequent insulin release(Nelson and Cox, 2008 pp 923-924).
Figure 2: Glucose Regulation of Insulin secretion by β pancreatic cells.
Insulin stimulates glucose uptake by both muscle and adipose tissue. Where glucose is converted to glucose-6-phosphate. Insulin released from the pancreas travels to the liver in the bloodstream. Insulin in the liver then activates the oxidation of Glucose-6-phosphate to pyruvate through glycolysis and subsequently the oxidation of pyruvate to Acetyl- CoA. This Acetyl- CoA if not further oxidised for energy production is used in the synthesis of fatty acids. Fatty acids once synthesised are exported from the liver as triacylglycerol’s of plasma lipoproteins to adipose tissue. Insulin stimulates triacylglycerol’s synthesis in adipocytes. These fatty acids are derived due to excess glucose levels taken up from blood by the liver. Insulin in effect converts excess blood glucose to two storage forms: glycogen and triacylglycerol’s(Nelson and Cox, 2008 pp922).
Deficient action of insulin on target tissues results in abnormalities in the metabolism of carbohydrates, fat and protein in diabetes. Inadequate insulin secretion and diminished tissue responses to insulin at one point or numerous points in the hormone action pathways occurs due to Deficient Insulin Action. Insulin action defects and insulin secretion defects coexist in patients with Diabetes and is unclear if either defect alone is the primary cause of hyperglycaemia. The vast majority of diabetes cases fall into two etiopathogenic categories Type 1 diabetes and Type 2 diabetes. Type 1 diabetes is caused by an absolute deficiency of insulin secretion. Type 2 diabetes is caused by a combination of resistance to insulin action and compensatory insulin secretion which is inadequate. In type 2 diabetes a degree of hyperglycaemia which is sufficient to cause both functional and pathological changes in various target tissues with the absence of clinical symptoms. Abnormalities in carbohydrate metabolism can be determined using clinical tests during this asymptomatic period(American Diabetes Association, 2004).
1.3.2 Type 1 Diabetes Mellitus
Type 1 diabetes usually presents with an absolute insulin deficiency due to destruction of pancreatic β cells(Lambert et al., 2004). Two forms of type 1 diabetes has been identified type 1A and type 1B. Type 1A diabetes results from a cell mediated autoimmune attack on β cells (Redondo et al., 2001). Type 1B diabetes rarer with no identifiable cause known. Type 1B diabetes occurs in patients who possess varying degrees of insulin deficiency ranging from sporadic hyperglycaemia to ketoacidosis in patients of African and Asian descent (7). Eisenbarth (8) in 1980’s proposed the current model to describe the development of the immune form of type 1 diabetes. Although knowledge on the topic has greatly advanced since then the basic building blocks of this model is still relevant (Redondo et al., 2001).This model suggests that individuals are born with a certain susceptibility to develop type 1 diabetes. For certain individual’s susceptibility is higher than others(Daneman, 2006).
Susceptibility to type 1 diabetes is inherited principally in the HLA genotypes DR and DQ and to a lesser degree in a host of other genetic loci which are defined as IDDM susceptible genes. The HLA locus is believed to confer roughly 50% of an individual’s genetic susceptibility to develop type 1 Diabetes. Two other genes confer roughly 15% of an individual’s susceptibility to develop type 1 diabetes 1. Insulin-VNTR (IDDM2) and CLTA-4 with minor contributions made from other genes(Anjos and Polychronakos, 2004). Susceptibility genes are thought to play an important role in immune response regulation (Daneman, 2006).
Type 1 diabetes development requires the exposure of an individual to one or more environmental triggers that result in an alteration of immune function, resulting in the initiation of β- cell destruction. Environmental triggers include viruses (e.g. enteroviruses, congenital rubella and coxsakie) (Robles and Eisenbarth, 2001), environmental toxins or foods including early exposure to cow’s milk proteins cereals or gluten (Thorsdottir and Ramel, 2003).
In Diabetes susceptible individuals the abnormal activation of the T-cell mediated immune system results in an inflammatory response occurring at the islets of Langerhans coupled with a humoral (B- cell) response which results in the production of antibodies to β-cell antigens. Islet cell antibodies were the first antibodies to be described but later have been succeeded by more specific autoantibodies to insulin glutamic acid decarboxylase and the protein tyrosine phosphatase(Devendra et al., 2004). Clinical onset of type 1 diabetes can be preceded by the presence of one or more of these antibodies types for years to decades. The presence of these antibodies and their persistence increases the likelihood of progression to clinical disease(Barker et al., 2005). No evidence exists to suggest that these antibodies play an active role in pathogenesis of Diabetes in humans(Daneman, 2006).
The continuing destruction of the β cells results in the progressive loss of insulin-secretory reserve. This results in loss of first phase insulin secretion in response to an intravenous glucose tolerance test, this is followed by clinical onset of diabetes due to insulin secretion levels falling to below a critical level and finally in the majority but not all type 1 diabetes cases a state of total insulin deficiency(Devendra et al., 2004). A remission period takes place soon after diagnosis and insulin therapy initiation when limited endogenous insulin secretion is restored to exhausted β cells which have not yet been destroyed and initial hyperglycaemia associated with insulin resistance is lessened (Sochett et al., 1987). Generally, β-cells are destroyed quicker when diabetes onset occurs at a younger age, this results in the likelihood of a long remission period is lessened (Sochett et al., 1987). As a result, older individuals are more likely to respond to immune interventions which are aimed at preserving residual insulin secretion soon after diagnosis. Evidence which supports the hypothesis of type 1 diabetes being linked to autoimmune pathogenesis is the susceptibility of individuals with type 1 diabetes to autoimmune conditions including Graves’ disease, Addison disease and coeliac disease (Barker et al., 2005; Kordonouri et al., 2005; Skovbjerg et al., 2005). While recent knowledge has helped to boost knowledge regarding the pathogenesis of type 1 diabetes but no unifying theory of disease causation has been determined (Daneman, 2006).
3.2.2 Type 2 Diabetes
Type 2 Diabetes is characterised by pancreatic β-cell dysfunction and insulin resistance in target organs which is characterised by insulin deficiency. The rise of obesity, aging populations and sedentary lifestyles have quadrupled the incidences of type 2 Diabetes between 1980 and 2004 (NCD Risk Factor Collaboration, 2016). In 2015 the number of diabetes cases was estimated at 415 million people, 90 percent of these cases presented as type 2 diabetes (Atlas, 2015.). It is projected diabetes cases will increase to 642 million by 2040 (Atlas, 2015.)
Insulin is a key hormone involved in blood glucose regulation, generally normal levels of glucose are maintained by insulin action and insulin secretion. Normal pancreatic β-cells have the ability to adapt to changes in insulin action e.g. a decrease in insulin action is counteracted by increased insulin secretion. Type 2 diabetes occurs when β-cell function insulin function compared to insulin sensitivity is relatively low. This β-cell dysfunction is critical in the pathogenesis of type 2 diabetes. Progression of decreasing insulin action the β-cells compensates by increasing the cell function (Stumvoll et al., 2003). However mild glucose increases will be observed during this period. While this increase is small it is damaging due to glucose toxicity which in itself is a cause behind β-cell dysfunction. Theoretically even with a unlimited β-cell reserve insulin resistance will result in hyperglycaemia and type 2 diabetes (Stumvoll et al., 2005).
Insulin resistance is present when the biological effects of insulin are less than expected for both the suppression of endogenous glucose production primarily in the liver and glucose disposal in skeletal muscle (Dinneen et al., 1992). Production of endogenous glucose is accelerated in patients with type 2 diabetes (Weyer et al., 1999). This acceleration of endogenous glucose production occurs in the presence of hyperinsulinemia in the early and intermediate disease stages, hepatic insulin resistance as a result is the driving force of hyperglycaemia of type 2 diabetes (Stumvoll et al., 2005)
Insulin resistance is strongly linked with both obesity and physical inactivity. Several mechanisms which are involved in mediating this interaction have been identified. A number of circulating metabolic fuels such as non-esterified fatty acids(NEFA), hormones and cytokines originate in the adipocyte and play a role to modulate insulin action. Increased mass storage of triglyceride in either visceral or deep subcutaneous adipose depot results in large adipocytes which are resistant to the ability of insulin to supress lipolysis. This results in an increased level of NEFA and glycerol increased and circulating both of which can aggravate insulin resistance in both skeletal muscle and the liver (Boden, 1997). Excessive fat storage ectopically in non-adipose cells plays an important role in insulin resistance (Danforth Jr, 2000). Examples include increased intramyocellular lipids associated with skeletal muscle insulin resistance in certain circumstances (43).
Insulin activates its pleiotropic metabolic responses as it binds to and activates specific plasma membrane receptors using tyrosine kinase activity (51). Cellular substrates of this insulin receptor kinase most notably the insulin receptor substrate (IRS) are tyrosine phosphorylated on several sites which act as binding scaffolds for adaptor proteins. This process leads to the downstream signalling cascade (52). Insulin results in the activation of a series of lipid and protein kinase enzymes which are linked to the translocation of glucose transporters to the cell surface and the synthesis of glycogen, protein mRNA’s and nuclear DNA which all play a role in both cell proliferation and cell survival (Stumvoll et al., 2005).
In cases of insulin resistance at least one of the following molecular mechanism which result in the blocking of insulin signalling are likely to be involved. The positive effects of tyrosine phosphorylation of the receptors and the IRS proteins on downstream responses are opposed by the dephosphorylation of tyrosine side chains carried out by cellular protein tyrosine phosphatases and serine and threonine residue phosphorylation which often occur in tandem (53). Serine and threonine phosphorylation of IRS1 reduces its ability to function as a substrate for tyrosine kinase activity of the insulin receptor and inhibits coupling of the IRS to its major downstream effector systems(Stumvoll et al., 2005). Signal downregulation can occur also through both the internalisation and loss of the insulin receptor from the surface of the cell and IRS protein degradation (55).
Increased concentrations of both inflammatory cytokines (eg TNF-α and interleukin 6) and NEFA are released as a result of expanded visceral adipose tissue has an adverse effect in the insulin signalling cascade (57,58). NEFA inhibits insulin stimulated glucose metabolism in the skeletal muscle and results in the stimulation of gluconeogenesis (generation of glucose from non-carbohydrate carbon sources) in the liver (59,60). NEFA also activates cellular kinases including atypical protein kinase C isoforms by increasing diacylglycerol levels which in turen can activate the inflammatory inhibitor kB kinase (IKK).and c-jun N- terminal kinase. This results in an increase of serine/threonine phosphorylation of IRS1 reducing downstream IRS1 signalling (61-63). TNF-α enhances adipocyte lipolysis which results in a further increase in
If you need assistance with writing your own dissertation, our professional dissertation writing service is here to help!Find out more
Cite This Work
To export a reference to this article please select a referencing stye below:
Related ServicesView all
DMCA / Removal Request
If you are the original writer of this dissertation and no longer wish to have your work published on the UKDiss.com website then please: