mol unit 2

specimen collection/transport/storage

transport to lab ASAP, Dna and rna can degrade giving false neg bc nothing to detect, consult manufacturer package insert, individual lab guidelines, CLSI

specimen rejection due to

hemolysis (cells blown apart no point in testing), mislabeled/unlabeled, improper collection, chain of custody issue (drug abuse, crime)

most common specimen type for detecting rna and dna

whole blood

edta purple- for dna

hep green- not for molec bc inhibits polymerase

light yellow ACD- used for molecular

bone marrow is used for

isolation of genomic dna hematic stem cells

serum or plasma is used for

testing viral dna or rna

tissue testing

fresh frozen fixed (preserved with forminin, yields lower quality bc protein are attached to fixative) for genomic dna

cheek swabs used for

parental testing

hair is used for

forensic, parentage, need about 50-100

csf, pus, sputum are used for

bacterial, infectious disease, virus

contamination of specimen by

Nucleic acids from person collecting or transporting, Improper storage or transport, Other patient specimens, or Degradation of RNA/DNA Environmental enzymes

contamination of equipment by

Amplification Methods from nucleic acids from other patient samples, QC samples, or the environment, Carryover amplified DNA/RNA from previous samples

good lab practices GLP- separation

physical - 4 separate rooms or areas (Reagent prep, specimen prep, amplification room, detection room), Pre amplif clean-> amplif dirty > post dirty, hoods and cabinets

time- all pre amp in morning, amp and post in afternoon

unidirectional workflow

CLIA requirement for closed lab systems, work one direction

open lab system

lab developed tests, not complete in a kit, prove accuracy with qc and other stuff, potential for aerosols, manual manipulation

closed lab system

fully integrated and automated, pure, minimal or no open tubes and steps, uses cartridges

GLP protocol/practices/controls

Use disposable if possible, never return aliquot back to original, prepare small working volumes and throw away after use, label everything molecular only, use sterile or molecular grade water, positive pressure- prep reagents, neg deal with specimen (take anything contamination away), separate air handling

stages of testing

pre amp- specimen and reagent prep

amp- testing

post- detection of am product, analysis of results, spec storage, report results

QC

ran with every run, qualitative= pos neg sensitivity, PCR based assays= amplification controls, target specific assays= internal controls, mutiples assays= control per analyte

neg QC

contains all reagents but target, checks for contamination

sensitivity QC

does it detect minimum required to pull pos if pos

QC amplification controls

is thermocyc working

QC target internal control

detects genes, organisms, or marker

QC failures

can be pos / amplification, neg, or internal

repeat the test