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What action should be taken if an unlabeled collection tube with a requisition for a factor V Leiden test is received in the laboratory. - Answer Notify the supervisor and reject the specimen. What action should be taken if after PCR, the amplification control has failed to yield a product? - Answer Check the original DNA or RNA preparation. If it is adequate, repeat the amplification. If not, reisolate the nucleic acid. What action should be taken if an isolated DNA sample is to be stored for at least 6 months? - Answer Store at -70 degrees C in a tightly sealed tube. What action should be taken if a bone marrow specimen arrives at the end of a shift and will not be processed for the BCL-2 translocation until the next day? - Answer Place the specimen in the refrigerator What action should be taken if the temperature of a refrigerator set at 8 degrees C (+/- 2 degrees C) reads 14 degrees C? - Answer Recheck the temperature after a few hours. If it does not return to range, notify the supervisor. What action should be taken if a PCR test for BCR/ABL translocation was negative for the patient sample and for the sensitivity control? - Answer Repeat the PCR with the addition of a new sensitivity control What action should be taken if a fragile X result has been properly reviewed and reported? - Answer File the test results, documents, and associated autoradiographs together in the laboratory archives
What action should be taken if a bottle of reagent alcohol with a 3 in the red diamond on its label is to be stored? - Answer Place the alcohol bottle in a safety storage cabinet for flammable liquids. What action should be taken if the expiration date on a reagent has passed? - Answer Discard the reagent. If it can be used for nonclinical purposes, label and store it in a separate area away from patient testing reagents. What action should be taken if the test results are to be faxed to the ordering physician? - Answer Fax the results with a cover sheet containing the proper disclaimers. What action should be taken if among a group of properly labeled containers, there is a container similar to the others, but unlabeled? - Answer Do not use the material in the unlabeled container. Consult the institutional safety representative for proper disposal of the contents. What action should be taken if a blood sample is received in a serum tube for PML-RARA testing by PCR? - Answer Notify the supervisor that the specimen cannot be tested and explain the collection error (wrong/no anticoagulant) What action should be taken if the laboratory is reporting a gene mutation replacing glycine at position 215 with alanine. The results are written 215G --> A in the test report. - Answer Correct the test report to A215G before verification by the laboratory director. What action should be taken if a test result falls outside of the analytical measurement range of the validated method? - Answer If the result exceeds the range, dilute the specimen and retest. If the results are below the range, report the results as less than the lower limit of the assay.
d) A26:01/A26:05/A26:01/A26:15 - Answer C) A02:12. A02 would be low resolution, determined at the serological level. A26:01/A26:05/A26:01/A26:15 is a medium resolution result. Which of the following is a likely haplotype from parents with A25, Cw10, B27/A23, Cw5, B27 and A17, Cw4,B10/A9, Cw7,B12 haplotypes? a) A25, Cw10, B b) A25, Cw5, B c) A23, Cw4, B d) A17, Cw4, B27 - Answer A) A25, Cw10, B27 would be inherited together from one parent. A25,Cw5. B27 may occur as a result of a recombination even, but would be infrequent. Upon microscopic examination, over 90% of cells are translucent after a CDC assay. How are these results scored according to the ASHI rules? - Answer Negative for the presence of the test antigen or a score of 1. Less than 10% of the cells have taken up dye. Cytotoxicity (dye uptake) will only occur in those cells that carry antigens to the test antibodies. A theoretical HLA-A02 allele is a CTC to CTT change at the DNA level (leu --> leu). How is this allele written? a) HLA-A02: b) HLA-A02:01: c) HLA-A d) HLA-A02N - Answer B) Nucleotide substitutions that change the amino acid sequence are indicated by differences in the second group of digits. Those changes in the DNA sequence that do not change the amino acid sequenece (silent or synonymous substitutions) are designated by addition of a third group of digits.
A candidate for kidney transplant has a PRA of 75%. How might this affect eligibility for immediate transplant? - Answer Transplant candidates with % PRA activity of more than 50% are considered to be highly sensitized. Finding a crossmatch-negative donor will be difficult in this case. What is the relationship between alleles HLA-A10 and HLA-A26(10)? - Answer HLA-A10 is the parent allele of HLA-A26(10). Number designations of new alleles of a previously defined allele with broad specificity (parent allele) are followed by the number of the parent allele in parenthesis. A CDC assay yields an 8 score for sera with the following specificities: A2, A28, and A2, A28, B7, and a 1 score for serum with an A2 specificity. What is the HLA-A type? - Answer This is type HLA-A2. The high cytotoxicity (reading>6) in the wells containing A28 indicates that the cells being tested have cell surface antigens matching the A28. The results of an SSP-PCR reaction are the following: lane 1, one band; lane 2, two bands; lane 3, no bands. If the test includes an amplification control multiplexed with the allele-specific primers, what is the interpretation for each lane? - Answer Lane 1 is the expected result if the test allele and is not present; lane 2 indicates the presence of the allele; lane 3 is not interpretable as the amplification control is absent. What are the two important checkpoints in the cell division cycle that are crossed when the regulation of the cell division cycle is affected? - Answer The transition from unreplicated to replicated DNA (G1 to S) and from replicated DNA to cell division (G2 to M) are regulated checkpoints in the cell division cycle. An EWS-FLI-1 translocation was detected in a solid tumor by RT-PCR. Which of the following does this result support? a) Normal tissue
What enzyme is responsible for continued sequence changes in the immunoglobulin heavy chain gene after gene rearrangement has occurred? - Answer Activation-induced cytidine deaminase alters C residues so that they base-pair with A instead of G residues, resulting in different amino acid substitutions in antibody proteins. Why are translocation-based PCR tests more sensitive than IgH, IgL, and T-cell receptor gene rearrangement tests? - Answer Gene rearrangements are not tumor- specific. Therefore, there will be a background from normal cells in gene rearrangement tests performed with consensus primers. Translocations are tumor- specific. The absence of background allows for more sensitive detection of tumor cells. Gene rearrangements may be detected with increased sensitivity using patient-specific primers that will detect only the gene rearrangement in the tumor cell population. A PCR test for the Bcl-2 translocation is performed on a patient with suspected follicular lymphoma. The results show a bright band at 400 bp (amplification control) and another at about 300 bp for this patient. The negative control has only the 400 bp band. How would you interpret these results? - Answer The results are positive for the presence of the Bcl-2 translocation, given that the 300 bp translocation specific product is within the expected size range with the primers used. Which of the following misinterpretations would result from PCR contamination? a) False-positive for the t(15;17) translocation b) False-negative for the t(15;17) translocation c) False-negative for a gene rearrangement - Answer A) False positive for the t(15;17) translocation. Contamination would result in a false-positive pattern. After amplification of the t (12;21) breakpoint by RT-PCR, the PCR products were loaded and resolved on an agarose gel. What might be the explanation for the gel being blank (no bands, no molecular-weight standard)? - Answer If the gel is
blank, then the ethidium bromide or SyBr green detection dye was omitted from the procedure. After amplification of the t(12;21) breakpoint by RT-PCR, the PCR products were loaded and resolved on an agarose gel. What might be the explanation for only the molecular-weight standard being visible? - Answer The visible molecular-weight standard indicates that the electrophoresis process was adequate. The absence of sample bands suggests an error in the sample preparation or amplification. After amplification of the t(12;21) breakpoint by RT-PCR, the PCR products were loaded and resolved on an agarose gel. What might be the explanation for the molecular-weight standard being visible and bands being present in every lane at 200 bp, even in the reagent blank lane? - Answer Bands in every lane at 200 bp, even in the reagent blank lane, is an indication of contamination. What is observed on a Southern blot for gene rearrangement in the case of a positive result? a) No bands b) Germline bands plus rearranged bands. c) Smears d) Germline bands only - Answer B) Germline bands plus rearranged bands. The rearranged bands represent the gene rearrangement in the monoclonal population of tumor cells. Cyclin D1 promotes passage of cells through the G1 to S cell division cycle checkpoint. What test detects translocation of this gene to chromosome 14? a) t(14;18) translocation analysis (BCL2, IGH) b) t(15;17) translocation analysis (PML/RARA) c) t(11;14) translocation analysis (BCL1/IGH)
b) Full Fragile X Disorder c) Mitochondrial Point Mutations - Answer B and C) Full Fragile X disorder and Mitochondrial point mutations. With modern methods, both full fragile X disorder and mitochondrial point mutations are detectable by PCR. A patient was tested for Huntington disease. PCR followed by PAGE revealed 25 CAG units. a) This patient has Huntington disease b) This patient has a 1/25 chance of contracting Huntington disease. c) This patient is normal at Huntington locus d) The test is inconclusive - Answer C) This patient is normal at the Huntington locus. In the Huntington repeat expansion, the sequence CAG expands from 9 to 37 repeats to 38 to 86 repeats. The Factor V Leiden mutation can be detected by a) PCR-RFLP b) SSP-PCR c) Invader technology d) All of the above methods - Answer D) All of the above methods. There are several methods available for the detection of Factor V Leiden mutation. The most frequently occurring mutation in the HFE gene results in the replacement of cysteine (C) with tyrosine (Y) at position 282. How is this expressed according to the recommended nomenclature? - Answer C282Y MELAS is a disease condition that results from an A to G mutation at position 3243 of the mitochondrial genome. This change creates a signal ApaI restriction site in a PCR product, including the mutation site. What would you expect from a PCR-RFLP analysis for this mutation on a patient with MELAS?
a) A single PCR product resistant to digestion with ApaI b) A single PCR product that cuts into two fragments upon digestion with ApaI. c) A single PCR product only if the mutation is present. d) Two PCR products - Answer B) A single PCR product that cuts into two fragments upon digestion with ApaI. D16S539, an STR, was analyzed in a family where the father was affected with a single gene disorder and the mother was unaffected. They had four children, three boys and one girl. One boy and one girl were affected. The result showed that the father had the 6,8 alleles, the mother had the 5,7 alleles. The affected children had the 5,6 and 6,7 alleles and the unaffected children had the 5,8 and 7,8 alleles. If D16S539 is located on chromosome 16, where is the gene for this disorder likely to be located? To which allele of D16S539 is the gene linked? - Answer Linkage with a specific D16S539 allele locates the gene on chromosome 16. The gene is linked to allele 6 of the D16S539 locus. How might one perform a DNA analysis for the presence of the disorder? a) Analyze D16S539 for the 6 allele by PCR. b) Sequence the entire region of the chromosome where D16S539 was located. c) Test as many STRs as possible by PCR. d) Use invaged technology to detect the unknown gene mutation. - Answer A) Linkage of this allele with the affected allele of the gene. Exon 4 of the HFE gene from a patient suspected of having hereditary hemachromatosis was amplified by PCR. The G to A mutation, frequently found in hemachromatosis, creates an RsaI site in exon 4. When the PCR products are digested with RsaI, which of the following results would you expect to see if the patient has the mutation? a) None of the PCR products are digested with RsaI. b) There will be no PCR product amplified from the patient DNA.
What chemical change in DNA is associated with imprinting? - Answer Addition of methy groups to DNA nitrogen bases (methylation) silences genes and is associated with imprinting. What accounts for the different phenotypes of Prader-Willi syndrome, del(15) (q11q13), and Angelman syndrome, del(15)(q11q13)? - Answer Dissimilar imprinting on paternal and maternal chromosomes is thought to account for phenotypic differences, depending on which chromosome 15 carries the deletion. Which of the following genes would be analyzed to determine whether an isolate of Staphylococcus aureus is resistant to oxacillin? a) mecA b) gyrA c) inhA d) vanA - Answer MecA. Staph aureus developed resistance to antibiotics that target its penicillin-binding protein (PBP1) by replacing PBP1 with PBP2a encoded by the mecA gene. PBP2a found in methicillin-resistant S. aureus (MRSA) has a low binding affinity for methicillin. Which of the following is a genotypic method used to compare two isolates in an epidemiological investigation? a) biotyping b) serotyping c) ribotyping d) bacteriophage typing - Answer Ribotyping. There are many laboratory methods that can be used to determine the relatedness of multiple isolates, both phenotypic and genotypic.
For which of the following organisms must caution be exercised when evaluating positive PCR results because the organism can be found as normal flora in some patient populations? a) Neisseria gonorrhoeae b) HIV c) Chlamydophila pneumoniae d) Streptococcus pneumoniae - Answer Streptococcus pneumoniae. Although PCR is specific for S. pneumoniae, the clinical significance of a positive PCR assay is questionable because a significant portion of the population (especially children) is colonized with the organism and PCR cannot discern between colonization and infection. Which of the following controls are critical for ensuring that amplification is occurring in a patient sample and that the lack of PCR product is not due to the presence of inhibitors? a) reagent blank b) sensitivity control c) negative control d) amplification control - Answer Amplification control. A PCR assay was performed to detect Bordetella pertussis on sputum obtained from a 14-year old girl who has had a chronic cough. The results revealed two bands, one consistent with the internal control and the other consistent with the size expected for amplification of the B. pertussis target. How should these results be interpreted? a) False-positive for B. pertussis b) The girl has clinically significant B. pertussis infection. c) B. pertussis detection is more likely due to colonization
both type of drug(s) as well as duration. Methods available in the laboratory for HCV genotyping are PCR with RFLP analysis and reverse hybridization (Inno- LiPA, Bayer), and direct DNA sequencing (HCV DupliType, Quest; TRUGENE, Bayer; and ViroSeq, Celera) A positive result for HPV type 16 indicates a) high risk for antibiotic resistance b) low risk for cervical cancer c) high risk for cervical cancer - Answer C) High risk for cervical cancer. Of the sexually transmitted types of HPV, 12 to 15 oncogenic genotypes have strong association with cervical cancers and are considered high-risk (HR) HPV types. Which of the following is used to type molds? a) Sequence-specific PCR b) Microarray c) ITS sequencing d) Flow cytometry - Answer C) ITS sequencing. Molds are typed by PCR and sequencing of Internal Transcribed Spacer (ITS) regions or 28S rRNA. Which of these would be used for a surname test: Y-STR, mini-STR, mitochondrial typing, or autosomal STR? - Answer Y-STR An ancient bone fragment was found and claimed to belong to an ancestor of a famous family. Living members of the family donated DNA for confirmation of the relationship. What type of analysis would likely be used for this test? Why? - Answer Mitochondrial DNA typing might be indicated because the small circular, naturally amplified mitochondrial DNA is more likely to be attained from the old sample and lineage across several generations can be determined using the maternal inheritance of mitochondrial type.
What are two biological exceptions to positive identification by autosomal STR? - Answer Identical twins and clones have identical nuclear DNA profiles. A partial STR profile was produced from a highly degraded sample. Alleles matched to a reference sample at five loci. Is this sufficient for a legal identification? - Answer Five loci are not sufficient for a legal identification. What is a SNP haplotype? What are tag SNPs? - Answer Single nucleotide polymorphisms that are inherited together in a block without recombination between them comprise a SNP haplotype. SNP haplotypes are identifiable by two or three representative SNPs or tag SNPs within the haplotype. Which of the following is an example of linkage disequilibrium? a) Seventeen members of a population of 1000 people have a rare disease and all 17 people have people have the same haplotype at a particular genetic location on chromosome 3 b) Five hundred people form the same population have the same SNP on chromosome 3. - Answer Example A Why are SNPs superior to STR and RFLP for mapping and association studies? - Answer SNPs are more numerous in the genome and, therefore, offer higher resolution for mapping of precise genome locations. A dideoxy sequencing electropherogram displays bright (high, wide) peaks of fluorescence, obliterating some of the sequence peaks. What is the most likely cause of this observation? How might it be corrected? - Answer The likely cause is the presence of unincorporated labeled dideoxynucleotides or dye blobs. Cleaning the sequencing ladder with spin columns, ethanol precipitation, or bead binding will correct this problem.
Compare and contrast EIA with western blots for detection of protein targets - Answer EIA is a liquid handling method and more easily automated than western blot. EIA is performed with immobilized antibodies exposed to test fluid. In western blot, antibodies or antigens are resolved by electrophoresis before exposure to test fluid. On a size exclusion column, large molecules will elute __________ (before/after) small molecules. - Answer BEFORE jMALDI methods separate ions by: a) molecular volume b) mass c) charge d) mass and charge - Answer D What is a heteroduplex? - Answer A heteroduplex is a double-stranded nucleic acid with one or more noncomplementary bases. Exon 4 of the HFE gene from a patient suspected to have hereditary hemachromatosis was amplified by PCR. The G to A mutation, frequently found in hemachromatosis, creates an Rsal site in exon 4. When the PCR products are digested with Rsal, what results (how many bands) would you expect to see if the patient has the mutation and no other Rsal sites are naturally present in the PCR product? - Answer Digestion of Rsal would produce two bands if the patient has the mutation if no other Rsal sites are naturally present in the PCR product. Which of the following methods would be practical to use to screen a large gene for mutations? a) SSP-PCR
b) SSCP c) PCR-RFLP d) DGGE e) FP-TDI - Answer D) DDGE would be practical to use to screen a large gene for mutations as it can scan several hundred base pairs at a time. SSCP is limited by the size and GC content of fragments that can be accurately analyzed. The remaining methods are designed to detect known point mutations. What is the effect on the protein when a codon sequence is changed from TCT to TCC? - Answer There would be no effect on the protein as both TCT and TCC code for serine. What chromosomal location is indicated by 15q21.1? - Answer This location is on the long arm of chromosome 15, region 2, band 1, sub-band 1 During interphase FISH analysis for the t(9;22) translocation, one nucleus was observed with two normal signals (one red for chromosome 22 and one green for chromosome 9) and one composite red/green signal. Five hundred other nuclei were normal. What is one explanation for this observation? - Answer Since only 1 of 500 cells showed the composite signal, this observation may be due to overlap of the chromosome on the slide, rather than a translocation. The presence of a translocation, however, cannot be ruled out. Is 47; XYY a normal karyotype? - Answer No. There are two Y chromosomes Write the numerical and structural chromosomal abnormalities represented by this genotype: 47, XY, +18 - Answer Trisomy 18
