Clinical Chemistry
Introduction
Clinical chemistry is one key critical area in medical laboratory technology. In other books, it is referred to as medical biochemistry, clinical biochemistry or chemical pathology. Essentially, clinical chemistry is a branch of clinical pathology that deals with analysis of different body fluids for both therapeutic and diagnostic purposes (Bishop et al 7). This discipline originated in the early 1900 where it was exclusively used to deal with diseases found in urine and blood. Clinical chemistry is one of the methods that utilize different instruments and apparatus to determine whether the results of any bodily fluid analysis are positive or negative. Some of the common methods that were utilized in the earlier phases included immunoassays, enzyme activity, spectrophotometry and electrophoresis (Bishop et al 33). Soon enough, other methods such as chromatography set in making the analysis and identification of different results very easy.
All biochemical conditions occurring in the body will always involve an increase or decrease in the amount of body fluids. Some of the elevated elements present in the body include enzymes, metals, white blood cell or other substances present in the body. All of these substances present in the body can be detected by the various instruments and apparatus used in clinical chemistry. In most cases, analysis of the products to be tested from the body is done on plasma and serum. Serum refers to the yellow watery part of the blood that lacks blood cells (Yang et al., 1507). One common method that has been used in serum preparation has been the use of centrifugation which involves separation of substances basing on their densities. Plasma is the same as serum but lacks clots. It is obtained during the first process of centrifugation. Though, it is significant to note that the type of tests will solely depend on the type of sample that was used.
Tests in clinical chemistry are classified into various categories. These include general chemistry or routine chemistry tests, special chemistry tests, toxicology tests, urinalysis, fecal analysis, clinical endocrinology, and therapeutic drug monitoring. General chemistry deals with the commonly blood chemistries that deal with liver and kidney function test. Special chemistry deals with techniques that relate to manual testing and electrophoresis. Clinical endocrinology deals with hormones and diagnosis of all related endocrine disorders. Toxicology involves study of drugs and all chemicals while therapeutic drug monitoring involves measurement of medication. Urinalysis involves analysis of effusions as well as cerebrospinal fluid while fecal analysis involves detection of all the gastrointestinal disorders.
Clinical chemistry covers a huge range of areas including direct tests mentioned above as well as panel tests. However, numerous instruments stand out in the analysis of most experiments in clinical chemistry. Analysis of serum, plasma and blood components would require use of certain instruments or use of certain medical laboratory techniques including centrifugation, spectrophotometry, chromatography, electrophoresis and immunoassays. All these methods use certain important properties to detect specific components in clinical chemistry. The basic understanding of most of these factors dictates the level of acceptance of results that will be obtained from most of the tests. Most clinicians have to understand then basic working behind most of these components as well as how to know the specific point at which most of the instrumental methods are applied. In recent years, new analytical techniques used in clinical chemistry have been revolutionized to make the process much easier compared to past process. This paper will look at specific techniques used in clinical chemistry especially when it comes to analyzing and incorporating analytical techniques.
Analytical Techniques in Clinical Chemistry
Centrifugation is one if the commonly used analytical method that is commonly used in clinical chemistry especially when it relates to analyzing blood samples. Blood is made up of various components with the two main components being blood cells and plasma (Foster 157). Analysis of biochemical changes in clinical chemistry requires exact determination of the amount of blood cells, plasma and serum (Lippi 172). Most of these substances that make blood have different densities. Centrifugation involves the separation of these substances depending on their density. Substances which are heavier tend to be found at the bottom while those found at the top are lighter. In the case of blood, blood cells and other components are denser and as a result they are found at the bottom of the centrifuge (Lippi 172). Plasma and serum will be the supernatant. In most cases, all tests that involve analysis of blood components would generally begin with the centrifugation process before any other form of analysis is conducted (Minder 1). The separation of blood by use of centrifuges will depend on some critical factors. These factors include the relative centrifugation force, temperature, length of collection tubes, centrifugation time. The volume of plasma or serum to be analyzed will depend with the volume of the tube. Relative centrifugal force is defined as the resistance of both the blood and tube components due to factors such as gravity and limits of the rotor and centrifuge (Minder 1). Change in temperature of the analyte under investigation affects its stability. The current guidelines require the centrifugation time for all serum components to be used for analysis to be at least ten minutes while plasma was to have a centrifugation time not less than 15 minutes (Minder 1). The speed at which the centrifuge should operate would also vary with the amount of revolutions per second. Generally, serum uses a force of 2000g while plasma uses a force of about 3000g (Minder 1). Centrifugation as a method has been used to increases the Turn Around time (TAT) in most of the clinical settings across the world. Consequently, new methods have been utilized which decrease the TAT to less than 30 seconds. Centrifugation is also the choice model when it comes to reducing removing sediments from urine as well as measuring the volume fraction of the amount of red blood cell commonly known as hematocrit (Minder 1). It is also used to separate free binding elements from other component in immune-procedure and in protein binding. Centrifugation is less commonly used in separation of lipoproteins as well as separating cellular components from cells. It also plays a critical role when it comes to extracting DNA from both plant and animal samples. For the machine to be utilized effectively, it requires that enough training be provided to the staff handling the machine. It also requires that the staff be fully aware of how the variables generally affect the functioning of the apparatus.
Spectrophotometry is another example of a technique that is used daily in clinical chemistry. The technique is based on the principle of Beer Lambert laws that relate concentration of a substance being measured to absorbance (Lakowicz: Behera). In essence, it states that concentration of a substance is directly proportional to the absorbance of a substance. Spectrophotometry commonly measures products by the use of a spectrophotometer. It detects the substances that have structures similar to benzene ring. These substances would normally absorb light of a certain wavelength. Once light has been passed through and the concentration determined, clinical chemists can look at the results and determine whether or not the concentration of the substance being measured in an individual is within or above the required limits. Standards are commonly used to determine if the values being measured are within the accepted levels. The standards contain substances that provide the true value of what is being measured. Analysis of most of these substances using a spectrophotometer is used as indicative values.
Spectrophotometers are predominantly used when analyzing protein substances present in serum or plasma. The total amount of proteins in a serum sample is determined by this method because proteins absorb light at a particular wavelength. Since samples provided will have different measurements, a standard curve is normally drawn using the values that have been obtained from the standards. The value obtained in the experiments, for example during analysis of total protein in a sample is then interpolated and determined from the curve. Spectrophotometry is also utilized in molecular biology techniques that involve the extraction of DNA to determine the purity of substances being measured (Didelot et al 815). It does this by measuring the amounts of RNA, DNA and proteins available in the sample. When values are found to be higher than anticipated, one of the products is in excess. Such results reveal that there has been possible contamination of the samples. Generally, all samples in a clinical chemistry lab that can absorb light at particular wavelengths can be determined use of spectrophotometers although this are not confirmatory methods since other tests will also be conducted confirm the results.
Different chromatographic methods are used in clinical chemistry. This method involves the separation of substances depending on their interaction with the mobile and stationary phase. The mobile phase is normally a gas or liquid that is moving while the stationary phase is the substance that is immobile (Peter et al 220). When the two surfaces interact, substances will separate depending on their interaction with both the two phases. Those that interact more with the stationary phase are left behind while those that interact faster with the other phase move out faster. As a result, separation of different substances is achieved.
Chromatography has become the method of choice since it involves detection of substances at higher sensitivity as compared to other methods of analysis. This method has other advantages compared to the currently utilized methods including its high selectivity rates, versatility and low cost of production. One type of chromatography that is constantly being used to examine most tests is the high performance liquid chromatography that is sometimes couples to the mass spectrometer to increase it selectivity (Yang and Sihe 96). Several examples are provided to show the important role that this machine plays in the analysis of substances in clinical chemistry. HPLC is used to analyze the presence of immunosuppressant drugs. Most of these drugs are provided to patients who are undergoing treatment after transplant (Kumar et al 1622). The immune system of individuals responds differently to different drugs that are commonly utilized. Some will respond quite well while others will respond differently. Some of the drugs that are being utilized may not be pure as earlier anticipated and would therefore reduce the likelihood that an individual gets better much faster. HPLC can be used to determine the purity of most of these drugs due to its high selectivity and sensitivity.
Secondly, chromatography is constantly used to monitor use of illegal drugs by different athletes and sportspersons. Gas chromatography is another method that is commonly used in clinical chemistry. It analyses for the presence of illegal products in blood through a method known as headspace analysis. As a method, it only requires the presence of a small amount of sample that is easily vaporized. The illegal substance plus the amounts will then be detected from blood. It can also be used to detect the type of alcohol present in an individual’s bloodstream especially when it involves cases where patients have taken too much alcohol that they have been admitted (Crunelle et al 2). This method is also selective and will only identify all products present in the sample. The only issue with gas chromatography is that it requires substances that easily vaporize. Thin layer chromatography commonly abbreviated as TLC is used to analyze products that are either amino acids or protein in nature. This method represents one of the easiest methods that could be used to analyze all substances present in a sample. Similar to the above two methods, it also utilizes standards to determine the amount of analyte of interest in a sample.
All other important chromatographic techniques that are frequently utilized in clinical chemistry include affinity chromatography, ion exchange chromatography, partition chromatography, and adsorption chromatography. All of the above methods will depend on the nature of the stationary phase being utilized. Currently, the stationary phase can either be normal or reverse. Recent advances have seen the incorporation of a mass spectrometer which increases the efficiency of either gas or liquid chromatography by detecting substances at the parts per billion levels. This means that minute details in a sample can be easily detected via this approach. Initially, the HPLC and Gas chromatography were utilized it detects substances at parts per million (Lehmann et al 919)
Electrophoresis refers to the separation of substances by use of electric current. Different substances present in the body have charges (Giri 190). Examples of such substances include proteins and DNA. There are two methods of electrophoresis that are currently utilized in clinical chemistry. They include capillary electrophoresis and agarose gel electrophoresis. Capillary electrophoresis is currently utilized in serum protein analysis (McCudden 451). Agarose gel electrophoresis is also utilized to separate serum protein (McCudden 451). Serum contains different immuno-globulins of varying masses. There are five types of globulins present in any individual. These are immunoglobulins A, G, D, E and M (Kang 28). These immunoglobulins play specific roles in the body. Certain disease conditions are associated with varying types of immunoglobulins. Some increases in the number of immunoglobulins immediately during their onset while others are associated with a decrease in a certain type of immunoglobulin. All of the above immunoglobulins have different masses and contain charges (Kang 28). It becomes nearly impossible to separate these immunoglobulins by utilizing some of the methods mentioned above. Electrophoresis stands out as the only effective methods that can separate all of the above products in one go and identify the any form of disease that is associated with any condition (Hungria 5705). This makes it an effective tool in analysis of immune factors that play a critical role in the body of an individual. Electrophoresis is also used in determining the size of DNA fragments that may be required for analysis.
Immunoassays represent another method that is used to analyze some important factors present in the immune system. It solely bases its principles on the formation of the antigen antibody complex (Lequin 2415). Analyte is the molecule detected by the complex. Once a positive result has been provided with the analyte of interests, a coloring agent is applied and fluorescence is detected it provides to be an indicator of results. Failure to form the color at the end of the reaction means that both the antigen or antibody of interest is not present and as a result the result might be false (Lequin 2415). This is not the case if the method being utilized involves competition between the antibody and antigen of interest. There are several examples that help illustrate the effects of the above mechanism. The first method involves the use of pregnant kits that have been used to show that patients are pregnant. This method exclusively relies on the use of kits that are founded or based on the immunoassay. The kit is normally lined with antibodies representing Human chorionic gonadotrophin hormone commonly abbreviated as HCG. Immediately, an individual becomes pregnant the body releases antigens associated with HCG and as a result antigens and antigens come into contact and the fluorescent material that has been added is able to fluoresce indicating a positive outcome (Chang 14443). If the patient is not pregnant there is a likelihood that no antigen is produced and as a result, there would not be the formation of the fluoresce (Greene et al 220). This kit is commonly used to show the difference when an individual becomes pregnant.
Lessons
Laboratory analytical techniques play a critical role when it comes to areas in clinical chemistry. As shown above most of the methods require an understanding of what is happening before analysis of any component in the human system can be initiated. Essentially, all of the above techniques have been utilized to improve or make the process to become much easier as compared to the past where a lot of time could be utilized to determine the main cause of a certain ailment or illness in the body. Some of this method such as HPLC and GC can be used to analsyse more than one sample at the same time increasing the likelihood of a keratin ailment being treated. Basically, when the method described above have made the treatment process to be much better as compared in the past cases and as a result more clinics have embraced the use of technology in dealing with issues associated with clinical chemistry. Diagnostic method such as immunoassays has come up to exclusively ensure that some sensitive tests can be conducted in different environments. Essentially, analytical techniques have improved clinical chemistry.
Works Cited
Behera, Siladitya, et al. “UV-visible spectrophotometric method development and validation of assay of paracetamol tablet formulation.” Journal of Analytical & Bioanalytical Techniques 2012 (2012).
Bishop, Michael L., Edward P. Fody, and Larry E. Schoeff, eds. Clinical Chemistry: Principles, Techniques, and Correlations. Lippincott Williams & Wilkins, 2013.
Chang, Chia-Chen, et al. “Colorimetric detection of human chorionic gonadotropin using catalytic gold nanoparticles and a peptide aptamer.” Chemical Communications 50.92 (2014): 14443-14446.
Crunelle, Cleo L., et al. “Hair ethyl glucuronide levels as a marker for alcohol use and abuse: a review of the current state of the art.” Drug and alcohol dependence 134 (2014): 1-11.
Didelot, Audrey, et al. “Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.” Clinical chemistry 59.5 (2013): 815-823.
Foster, Kimberly, et al. “Evaluation of a Centrifuge with Rapid Turnaround Time for the Preparation of Plasma Samples for Measurement of Common STAT Markers on the ACS: 180® System.” Clinical laboratory 46.3/4 (2000): 157-160.
Giri, K. V. “AGAR ELECTROPHORESIS Part I. A Simple Clinical Method for the Analysis of Serum Proteins.” Journal of the Indian Institute of Science 38.3 (2013): 190.
Greene, Dina N., et al. “Limitations in qualitative point of care hCG tests for detecting early pregnancy.” Clinica chimica acta 415 (2013): 317-321.
Hungria, Vania TM, et al. “Comparison of Kappa & Lambda Freelite to Total Kappa & Lambda Immunoassays for the Detection of Monoclonal Gammopathies, Both As Standalone Tests and Alongside Serum Protein Electrophoresis.” Blood 124.21 (2014): 5705-5705.