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Laboratory methods

Last updated: May 5, 2021

Summarytoggle arrow icon

The laboratory methods explained here are used to screen for and confirm medical conditions. For additional laboratory methods see “Pathology techniques.”

Preanalytical phase

The preanalytical phase encompasses the selection of relevant diagnostic tests and the collection and transport of samples. Specificity and sensitivity are important factors that should be considered when selecting a diagnostic test (e.g., many screening tests have a high sensitivity but low specificity). See also “Evaluation of diagnostic tests” in “Epidemiology.”

Preanalytical-phase errors (i.e., errors during sample collection or transport) should be considered if actual test results differ significantly from expected results.

Analytical phase

The analytical phase comprises sample processing and the generation of results.

Postanalytical phase

The most important steps of the postanalytical phase include:

  1. Ensuring the correct allocation of results to patient data
  2. Plausibility check
  3. Conveyance of results
  4. Medical interpretation based on the combination of test results, patient history, and physical examination findings

Results from laboratory studies should always be interpreted in conjunction with the medical history, clinical examination, and other diagnostic tests.

  • A technique used to detect DNA, RNA, and proteins that involves transferring the DNA, RNA, or proteins onto a membrane. The sample of interest is then visualized using marked detector molecules (e.g., radiolabeled DNA or chemiluminescent detector RNA).
  • The blotting techniques most commonly employed in laboratory medicine are Southern, western, and northern blot.
  • All blotting techniques follow a similar process:
    1. DNA, RNA, or protein molecules contained in the sample are separated by gel electrophoresis and/or electric charge, depending on their size (small molecules travel faster than bigger molecules).
    2. The separated molecules are transferred from the gel onto a membrane.
    3. The DNA, RNA, or protein molecule of interest is detected by using a labeled, highly specific oligonucleotide probes, antibodies, or x-rays.
Comparison of blotting techniques
Northern blot Southern blot Western blot

Southwestern blot

Sample
Detection
Use
  • Confirmation of the presence of a protein in a sample and rough estimation of its amount

SNoW flakes DRoP: Southern blot, Northern blot, and Western blot are used on DNA, RNA, and Proteins respectively.

  • Sample: RNA
  • Principle of detection: annealing of marked detector RNA or DNA to the target RNA fragment
    • 32P-DNA or -RNA
    • DNA or RNA labeled with a chemiluminescent dye
  • Procedure
    • The RNA sample is cleaved by enzymes and separated by gel electrophoresis (commonly on agarose gels).
    • Separated and cleaved RNA is transferred (blotted) to a membrane.
    • The membrane is incubated with labeled probes of RNA or DNA.
    • Labeled probes recognize and anneal to the complementary strand if it is present on the membrane.
  • Result: double-stranded RNA
    • One unlabeled strand (cleaved RNA sample)
    • One labeled strand that can be visualized using one of the following techniques:
      • When radiolabeled detector DNA/RNA is used, an x-ray film is placed against the western blot. This film develops when exposed to the label, creating dark regions that correspond to the target protein band.
      • In case of chemiluminescent DNA/RNA, a CCD camera that captures a digital image is used.
  • Uses

References: [2]

Overview

Direct ELISA

  1. The patient's sample supposedly containing the protein of interest (i.e., the antigen) is added to a well of microtiter plates with a buffered solution.
  2. The specific antibody-enzyme conjugate is added to the solution.
  3. A substrate for the enzyme is added
  4. Spectrometry is used to detect the generated chromophore

Indirect ELISA

Sandwich ELISA

  1. A surface plate is coated with capture antibodies (not the patient's antibodies).
  2. The sample is added to the coated plate where the captured antibodies bind the antigen of interest.
  3. Specific (labeled) antibodies for the antigen are added. ; if the antigen is present, the antibody binds to the antigen.
  4. A substrate for the enzyme is added (color, fluorescent, or electrochemical changes are due to the reaction between substrates and enzymes).
  5. Spectrometry, fluorescence, or electrochemical studies are performed to assess for the amount of antigens present.

Uses

References:[3]

Overview

Procedure

A PCR usually consists of 25–50 cycles, which are divided into three phases.

  1. Denaturation
    • The sample is heated; at a target temperature of 90–98°C (194–208°F) for 20–30 seconds.
    • High temperature breaks the hydrogen bonds between complementary base pairs; of the double-stranded DNA and produces two single DNA strands.
  2. Hybridization
    • The sample is cooled; at a target temperature of 50–65°C (122–149°F) for 20–40 seconds.
    • The following is added to the sample:
    • Primers bind the 3′ ends of the DNA sequence that needs to be amplified.
  3. Elongation and amplification
    • The sample is heated; at a target temperature of 70–80°C (158–176°F); 72°C (162°F) is most commonly used for DNA polymerase.
    • DNA polymerase uses dNTPs to elongate the primers, thereby replicating the sequence of the sample DNA strands.
    • The process is repeated until it yields an amplification to 106 –1010 copies of the original DNA fragment (approximately 25–50 cycles).

Uses

Reverse transcription polymerase chain reaction (RT-PCR)

  • Procedure
    1. A sample mRNA is converted to complementary DNA (cDNA) by reverse transcriptase.
    2. cDNA is amplified by the standard PCR procedure (see above).
  • Use: : detection and quantification of mRNA levels in a sample

Real-time polymerase chain reaction (also known as quantitative PCR, or qPCR)

  • Procedure
    • A PCR technique that utilizes fluorescence (e.g., intercalating dyes or DNA probes) for monitoring amplification of targeted DNA during the PCR via computer software (in a graph).
    • Melting temperatures specific to the amplified fragment allow for high specificity.
    • There are many types, including quantitative reverse transcription PCR (RT-qPCR) and semiquantitative real-time PCR.
  • Use
    • Allows monitoring of a targeted product at any point throughout the amplification
    • Rapidly detect nucleic acids for diagnosis (e.g., RT-qPCR for SARS-CoV2)
    • Easily quantify gene expression (e.g., via comparing to a standard curve of serial dilutions)
    • Quantify and genotype viruses

Molecular biological methods are used in the (prenatal) diagnosis of inherited disorders, e.g., to detect gene mutations. They are also used in the diagnosis of infectious diseases (e.g., diphtheria), in forensics, and in tumor diagnostics. Specimens include DNA from nucleated blood cells or, in prenatal diagnostics, chorionic villi.

Genetic markers

Numerous loci vary dramatically in a population. Repetitive sequences of various lengths are present in several noncoding regions of the genome. These repetitive sequences differ in sequence motif length. The frequency of repetition differs in each individual. Polymorphisms in DNA form the basis, e.g., for the diagnosis of diseases and allow individuals to be identified.

Detection of gene mutations (DNA diagnostics, molecular genetic testing)

DNA diagnostics is suitable for the direct or indirect detection of a gene mutation that results in disease. It can also be used to exclude the presence of a gene mutation.

Direct detection

Various methods can be used in the direct detection of gene mutations.

Indirect detection (genetic linkage analysis)

Indirect DNA diagnostics are performed if direct detection is not possible.

  • Prerequisite: The disease occurs in several family members and the suspected locus is known.
  • Principle: analysis of genetic markers associated with the mutated gene and comparison of the patient's genotype with that of unaffected family members
  • Result: There is no direct detection of a gene mutation, but a probability of the presence of a certain mutation that causes disease (genetic risk score) can be calculated. The validity of indirect DNA diagnostics depends on the pattern of inheritance and the number of family members being investigated.

Identification of chromosomes

Karyotyping can be used to visualize chromosomes for examining chromosome numbers and for an overview of potential structural changes within a chromosome. Staining is used to visualize the special banding patterns that are characteristic for every chromosome.

  • Karyotyping
  • Banding pattern: transverse bands of various widths and distribution, which can be induced depending on the preparation and staining technique
    • Preparation
    • Banding techniques
      • Staining with quinacrine (fluorescent bands; not used routinely in diagnostics)
      • Giemsa banding (standard banding technique, results in dark G bands with transcriptionally inactive chromatin and bright, transcriptionally active R bands)
    • Analysis: assessment of an average 10–15 metaphase chromosome pairs in 1250x magnification
  • Karyotype

Fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization is a method used for the staining of specific DNA sequences by a fluorescence-labeled DNA or RNA probe, e.g., to stain chromosomes in karyograms, in tumor diagnosis, or to map specific genes on chromosomes in metaphase.

DNA microarray (array comparative genome hybridization, CGH)

Mainly used to simultaneously examine expression levels of multiple genes or to genotype many regions at the same time.

  • Procedure
    1. Preparation of the sample
      • A sample (m)RNA and control (m)RNA are converted to complementary DNA (cDNA) by reverse transcriptase.
      • cDNA is then labeled with a fluorescent dye, one color for the sample that is being tested, another color for the control (e.g., green for the control, red for the sample being tested).
      • mRNA is removed and samples are combined.
    2. Preparation of the chip
      • Thousands of genetic sequences of nucleic acid (DNA or RNA) probes are attached to a chip (e.g., glass, silicon).
      • Both patient and control DNA is applied to this chip and hybridizes to the probes on the chip.
      • The chip is mounted to a scanner that can detect complementary binding of probes and sample sequences.
      • Each region of the chip stands for a known genetic sequence.
      • The higher the expression of the gene in one sample, the more intense the fluorescence.
  • Uses

References:[4]

Overview

CRISPR gene editing is a technique in genetic engineering that employs the prokaryote CRISPR/Cas9 defense system directed against foreign gene sequences to modify the genomes of living organisms (e.g., adding or deleting genes in DNA sequences).

  • CRISPR (clustered regularly interspaced short palindromic repeats)
  • Cas9 (CRISPR-associated system 9)
    • Enzyme (endonuclease) that produces single or double-strand breaks at a specific nucleotide sequence guided by a site-specific RNA (targeted DNA double-strand break)
      • In prokaryotes, the guide RNA consists of crRNA and tracrRNA.
      • For laboratory use, a single guide RNA is specifically designed and synthesized.
    • The cas9 gene sequence is found adjacent to the CRISPR sequence.
  • tracrRNA (transactivating crRNA): RNA sequence that is partially complementary to crRNA; also binds to Cas9 (needed for Cas9 maturation)

Adaptive prokaryotic immune response

  1. Foreign DNA is incorporated into own DNA at the CRISPR locus (acquisition).
  2. The CRISPR locus is transcribed together with foreign DNA and forms the primary transcript.
  3. The primary transcript binds tracrRNA and is processed to form a crRNA-tracrRNA hybrid, which contains a foreign genetic sequence.
  4. crRNA-tracrRNA hybrid forms a complex with Cas9.
  5. Now foreign DNA that contains the sequence complementary to the one contained by the crRNA/tracrRNA/Cas9 complex can be recognized and cleaved.

CRISPR/Cas9 in gene editing

For gene editing purposes, tracrRNA and crRNA are combined into one molecule, the single synthetic guide RNA (sgRNA) that is complementary to the DNA sequence of interest (target DNA). There are three major variants of Cas9 used in CRISPR gene editing:

  • Wild-type Cas9
  • Cas9D10A
    • Cleaves only one DNA strand (nickase activity)
    • More specific because it does not activate NHEJ but only the high-fidelity HDR pathway
  • Nuclease-deficient Cas9
    • Mutations in nuclease domains prevent cleavage but not binding.
    • Can be used to activate or silence genes by creating fusion proteins of Cas9 with effector proteins

Potential applications of CRISPR/Cas9

Some of the potential applications of CRISP/Cas9 system (not currently used in clinical medicine) include:

Definition

A screening test that detects and quantifies the types of hemoglobins present in a sample by separating them based on their electrical charge.

Applications

Procedure

  • A sample of the patient's hemoglobin is obtained by hemolyzing a sample of blood using a hemolysate reagent.
  • The hemoglobin sample is added to the gel electrophoresis buffer.
  • An electric field is applied to the buffer that causes the different hemoglobin types to separate according to their electrical charge.
  • A stain is applied to the gel to make the charged molecules visible.
  • Hemoglobin is negatively charged at an alkaline pH and migrates on the gel towards the anode, forming a band.
  • The degree of negative charge of the hemoglobin molecule determines the migration speed and distance from the cathode to the anode.

A FaSt Car can go far (A > F > S > C).

Expected findings

Please also see “Hemoglobin patterns” in thalassemia, sickle cell disease, and hemoglobin C disease.

RNA interference (RNAi)

Cre-Lox system

Molecular cloning [5]

  • Definition: experimental production of recombinant DNA molecules within host organisms (e.g., bacterial hosts)
  • Process
    1. Isolation of eukaryotic target mRNA
    2. Production of complementary DNA (cDNA) using reverse transcriptase
    3. Insertion of cDNA fragments into a cloning vector (e.g., bacterial plasmids carrying the antibiotic resistance genes)
    4. Transformation of the produced recombinant plasmid into bacteria
    5. Selection of bacteria that contains the plasmid (e.g., via antibiotic exposure when cloning antibiotic resistance genes)
  • Result: : synthesis of multiple copies of target cDNA (cloned DNA)
  1. Lequin RM. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin Chem. 2005; 51 (12): p.2415-2418. doi: 10.1373/clinchem.2005.051532 . | Open in Read by QxMD
  2. Microarray. https://www.nature.com/scitable/definition/microarray-202. Updated: January 1, 2018. Accessed: July 19, 2018.
  3. Foundations of Molecular Cloning - Past, Present and Future. https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future. . Accessed: June 24, 2020.
  4. Blotting, Southwestern. https://meshb.nlm.nih.gov/record/ui?name=Southwestern%20Blot. Updated: June 22, 2015. Accessed: June 25, 2020.
  5. Jia Y, Nagore L, Jarrett H. Southwestern Blotting Assay. Methods Mol Biol. 2016 : p.85-99. doi: 10.1007/978-1-4939-2877-4_5 . | Open in Read by QxMD
  6. Jorde L, Carey J, Bamshad M. Medical Genetics . Elsevier ; 2015
  7. Nussbaum RL, McInnes RR, Willard HF. Thompson & Thompson Genetics in Medicine. Elsevier Health Sciences ; 2016