Clinical Chemistry, Immunology and Laboratory Quality Control

A Comprehensive Review for Board Preparation, Certification and Clinical Practice

Amitava Dasgupta, Amer Wahed

• Publisher: Elsevier
• Year: 2013
• eBook ISBN:
  9780124079359
• Topic: Medicine
• Subtopic: Immunology

All pathology residents must have a good command of clinical chemistry, toxicology, immunology, and laboratory statistics to be successful pathologists, as well as to pass the American Board of Pathology examination. Clinical chemistry, however, is a topic in which many senior medical students and pathology residents face challenges. Clinical Chemistry, Immunology and Laboratory Quality Control meets this challenge head on with a clear and easy-to-read presentation of core topics and detailed case studies that illustrate the application of clinical chemistry knowledge to everyday patient care. This basic primer offers practical examples of how things function in the pathology clinic as well as useful lists, sample questions, and a bullet-point format ideal for quick pre-Board review. While larger textbooks in clinical chemistry provide highly detailed information regarding instrumentation and statistics, this may be too much information for students, residents, and clinicians. This book is designed to educate senior medical students, residents, and fellows, and to "refresh" the knowledge base of practicing clinicians on how tests are performed in their laboratories (i.e., method principles, interferences, and limitations). - Takes a practical and easy-to-read approach to understanding clinical chemistry and toxicology - Covers all important clinical information found in larger textbooks in a more succinct and easy-to-understand manner - Covers essential concepts in instrumentation and statistics in such a way that fellows and clinicians understand the methods without having to become specialists in the field - Includes chapters on drug-herb interaction and pharmacogenomics, topics not covered by textbooks in the field of clinical chemistry or laboratory medicine

Chapter 1

Instrumentation and Analytical Methods

This chapter discusses various techniques used in clinical laboratories, including ion-selective electrodes for measuring electrolytes, colorimetric methods, immunoassays, and more sophisticated techniques such as gas chromatography combined with mass spectrometry and liquid chromatography combined with mass spectrometry or tandem mass spectrometry.

Keywords

spectroscopy; colorimetry; immunoassay; chromatography; mass spectrometry

Contents

1.1 Introduction

1.2 Spectrophotometry and Related Techniques

1.3 Atomic Absorption

1.4 Enzymatic Assays

1.5 Immunoassays

1.6 Nephelometry and Turbidimetry

1.7 Chemical Sensors

1.8 Basic Principles of Chromatographic Analysis

1.9 Mass Spectrometry Coupled with Chromatography

1.10 Examples of the Application of Chromatographic Techniques in Clinical Toxicology Laboratories

1.11 Automation in the Clinical Laboratory

1.12 Electrophoresis (including Capillary Electrophoresis)

Key Points

References

1.1 Introduction

Various analytical methods are used in clinical laboratories (Table 1.1). Spectrophotometric detections are probably the most common method of analysis. In this method an analyte is detected and quantified using a visible (400–800 nm) or ultraviolet wavelength (below 380 nm). Atomic absorption and emission, as well as fluorescence spectroscopy, also fall under this broad category of spectrophotometric detection. Chemical sensors such as ion-selective electrodes and pH meters are also widely used in clinical laboratories. Ion-selective electrodes are the method of choice for detecting various ions such as sodium, potassium, and related electrolytes in serum or plasma. In blood gas machines chemical sensors are used that are capable of detecting hydrogen ions (pH meter) as well as the partial pressure of oxygen during blood gas measurements. Another analytical method used in clinical laboratories is chromatography, but this method is utilized less frequently than other methods such as immunoassays, enzymatic assays, and colorimetric assays that can be easily adopted on automated chemistry analyzers.

Table 1.1

Assay Principles and Instrumentation in the Clinical Chemistry Laboratory

1.2 Spectrophotometry and Related Techniques

Spectroscopic methods utilize measurement of a signal at a particular wavelength or a series of wavelengths. Spectrophotometric detections are used in many assays (including atomic absorption, colorimetric assays, enzymatic assays, and immunoassays) as well as for detecting elution of the analyte of interest from a column during high-performance liquid chromatography (HPLC).

Colorimetry was developed in the 19th century. The principle is based on measuring the intensity of color after a chemical reaction so that the concentration of an analyte could be determined using the absorption of the colored compound. Use of the Trinder reagent to measure salicylate level in serum is an example of a colorimetric assay. In this assay, salicylate reacts with ferric nitrate to form a purple complex that is measured in the visible wavelength. Due to interferences from endogenous compounds such as bilirubin, this assay has been mostly replaced by more specific immunoassays [1]. Please see Chapter 2 for an in-depth discussion on immunoassays.

Spectrophotometric measurements are based on Beer’s Law (sometimes referred to as the Beer–Lambert Law). When a monochromatic light beam (light with a particular wavelength) is passed through a cell containing a specimen in a solution, part of the light is absorbed and the rest is passed through the cell and reaches the detector. If Io is the intensity of the light beam going through the cell and Is the intensity of the light beam coming out of the cell (transmitted light), then Is should be less than Io. However, part of the light may be scattered by the cell or absorbed by the solvent in which the analyte is dissolved, or even absorbed by the material of the cell. To correct this, one light beam of the same intensity is passed through a reference cell containing solvent only and another through the cell containing the analyte of interest. If Ir is the intensity of the light beam coming out of the reference cell, its intensity should be close to Io. Transmittance (T) is defined as Is/Io. Therefore, correcting for scattered light and other non-specific absorption, we can assume transmittance of the analyte in solution should be Is/Ir. In spectrophotometry, transmittance is often measured as absorption (A) because there is a linear relationship between absorbance and concentration of the analyte in the solution (Equation 1.1):

(1.1)

Transmittance is usually expressed as a percentage. For example, if 90% of the light is absorbed, then only 10% of the light is being transmitted, where Ir is 100 (this assumes no light was absorbed when the beam passed through the reference cell, i.e. Io is equal to Ir) and Is is 10. Therefore (Equation 1.2):

(1.2)

If only 1% of the light is transmitted, then Ir is 100 and Is is 1 and the value of absorbance is as follows (Equation 1.3):

(1.3)

Therefore, the scale of absorbance is from 0 to 2, where a zero value means no absorbance.

Absorption of light also depends on the concentration of the analyte in the solvent as well as on the length of the cell path (Equation 1.4):

(1.4)

In this equation, “a” is a proportionality constant termed “absorptivity,” “b” is the length of the cell path, and “c” is the concentration. Therefore, if “b” is 1 cm and the concentration of the analyte is expressed as moles/L, then “a” is “molar absorptivity” (often designated as epsilon, “ε”). The value of “ε” is a constant for a particular compound and wavelength under prescribed conditions of pH, solvent, and temperature (Equation 1.5):

(1.5)

For example, if “b” is 1 cm and the concentration of the compounds is 1 mole/L, then A=ε. Therefore, from the measured absorbance value, concentration of the analyte can be easily calculated from the measured absorbance value, known molar absorptivity, and length of the cell (Equation 1.6):

(1.6)

1.3 Atomic Absorption

Atomic absorption spectrophotometric techniques are widely used in clinical chemistry laboratories for analysis of various metals, although this technique is capable of analyzing many elements (both metals and non-metals), including trace elements that can be transformed into atomic form after vaporization. Although many elements can be measured by atomic absorption, in clinical laboratories, lead, zinc, copper, and trace elements are the most commonly measured in blood. The following steps are followed in atomic absorption spectrophotometry:

The sample is applied (whole blood, serum, urine, etc.) to the sample cup.

Liquid solvent is evaporated and the dry sample is vaporized to a gas or droplets.

Components of the gaseous sample are converted into free atoms; this can be achieved in either a flame or flameless manner using a graphite chamber that can be heated after application of the sample.

A hollow cathode lamp containing an inert gas like argon or neon at a very low pressure is used as a light so...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Preface
• Chapter 1. Instrumentation and Analytical Methods
• Chapter 2. Immunoassay Platform and Designs
• Chapter 3. Pre-Analytical Variables
• Chapter 4. Laboratory Statistics and Quality Control
• Chapter 5. Water, Homeostasis, Electrolytes, and Acid–Base Balance
• Chapter 6. Lipid Metabolism and Disorders
• Chapter 7. Carbohydrate Metabolism, Diabetes, and Hypoglycemia
• Chapter 8. Cardiac Markers
• Chapter 9. Endocrinology
• Chapter 10. Liver Diseases and Liver Function Tests
• Chapter 11. Renal Function Tests
• Chapter 12. Inborn Errors of Metabolism
• Chapter 13. Tumor Markers
• Chapter 14. Therapeutic Drug Monitoring
• Chapter 15. Interferences in Therapeutic Drug Monitoring
• Chapter 16. Drugs of Abuse Testing
• Chapter 17. Challenges in Drugs of Abuse Testing: Magic Mushrooms, Peyote Cactus, and Designer Drugs
• Chapter 18. Testing for Ethyl Alcohol (Alcohol) and Other Volatiles
• Chapter 19. Common Poisonings Including Heavy Metal Poisoning
• Chapter 20. Pharmacogenomics
• Chapter 21. Hemoglobinopathy
• Chapter 22. Protein Electrophoresis and Immunofixation
• Chapter 23. Human Immunodeficiency Virus (HIV) and Hepatitis Testing
• Chapter 24. Autoimmunity, Complement, and Immunodeficiency
• Chapter 25. Effect of Herbal Supplements on Clinical Laboratory Test Results
• Index
• Color Plates