Substitution Information and Courses from MediaLab, Inc.

Scenario 4: The donor returns from the restroom with a sufficient specimen. It is very warm to the touch. The collector is unable to obtain a reading from the temperature strip. Collector’s response: The collector completes the collection and prepares the specimen for shipment. The collector explains the situation with a supervisor. If the supervisor concurs that an observed collection is in order, the collector next tells the donor that a new collection will be conducted under direct observation. The collector explains that because the temperature of the specimen was not within the acceptable range (90-100º F/32-38º C) there is suspicion of substitution or adulteration. A new CCF is initiated. The collector marks on the CCF that the collection is observed and notes under Remarks why it is observed. The collector also notes the control number of the suspect collection. The observed specimen along with the suspect specimen are both shipped to the laboratory in separate plastic tamper-resistant bags.

The reason to chose a particular molecular method can be influenced by disease detection, monitoring or therapy in certain patient populations. Molecular methodologies can be used to identify alterations or variations or changes in DNA sequencing that can cause disease. Sequence alterations that are known to cause disease are termed mutations. These changes or mutations can be applied to areas of the clinical lab such as infectious disease, paternity, genetic testing, and pharmacogenetics. Some of the more common alterations are:Deletion: a missing nucleotide or other portion of DNA sequence Insertion: an extra DNA nucleotide or other portion of DNA sequence Missense: a nucleotide or sequence substitution that codes for a different amino acidNonsense: a nucleotide substitution that ends in early termination of the protein manufacturing process; usually due to a stop codon.The most common alteration is a single base change or single nucleotide polymorphism (SNP)

Several mutations of the HFE gene have been described. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.