MOLBIO LAB 5

Test requisition must contain the following:

Type of specimen material; Ordered test; Date and type of collection; Contact of ordering physician

What is usually needed for specimen collection, especially for molecular genetics or parentage testing?

Patient consent forms

What is a possible cause of erroneous or misleading results?

Preanalytical error

What is followed for forensic specimens?

Chain of custody

What should be done for specimens with minimal cellular content (buccal cell suspensions, CSF)?

Centrifugation - collect cells before DNA/RNA extraction (cytocentrifuge)

Which specimens can be used to perform multiple tests?

Snap frozen specimen (-70C)

This requires high MW DNA, and must be intact when subjected to blotting. It does not work on improperly handled specimen.

Southern or Northern blot

Which specimen is preferred for long term storage?

Isolated nucleic acid

What is used for long term nucleic acid storage at ultra-low temperature

Cryotubes

What is the storage and transport condition for blood and bone marrow specimens?

Shipped overnight at room temperature or with ice packs

What is the storage and transport condition for Tissue specimens?

shipped frozen on dry ice

How long can an isolated DNA survive at room temp?

Several months

At what temperature should isolated DNA be stored for a duration of at least 1 year?

Refrigerated temperature (2-8C)

What is the maximum storage time for purified DNA kept at -20C - -70C?

10 years or longer

Storage condition for isolated RNA

room temperature

Why is refrigeration not recommended for isolated RNA unless it is stabilized?

Due to the absence of RNA stabilization

Stability of isolated RNA suspended in Diethylpyrocarbonate- treated water?

At least 1 month

Stability of isolated RNA suspended in Ethanol

-20C for several months

What procedure requires intact cellular structures or cell cultures and only accepts fresh specimens?

FISH (Fluorescence in situ hybridization) and Karyotyping

Specimen Transport Systems

Sterile containers; Swabs; Specialty systems; Proprietary systems; Anaerobic transport systems; Viral Transport System

What specimen collection procedure is preferred for respiratory viral infections?

Oropharyngeal and Nasopharyngeal swab collection

What is the appropriate swab for collecting an oropharyngeal swab specimen?

Sterile dacron/rayon/flocked swab with plastic shaft

What is the appropriate swab for collecting a nasopharyngeal swab specimen?

Sterile dacron/rayon/flocked swab with plastic pliable shaft

This type of swab is comprised of many perpendicularly sprayed nylon fibers, used for NPS.

Flocked Swab

What materials are preferred for the shafts of swabs used in molecular testing?

Plastic shafts (avoid wooden shafts as they can inactivate viruses)

Storage temperature for Universal Transport Medium (UTM)

stored at refrigerated temperature (not exceeding -25C)

What is the ideal color UTM?

clear and light orange

What is the recommended angle for tilting a patient's head during NPS collection according to the CDC?

70 degrees

NPS anatomical location (point of entry)

Base of the nostril near the septum parallel to the palate; Inferomedial aspect of the nostril

How many times should the swab be rotated (NPS)?

2-3 times

How should the UTM be handled after NPS collection?

Place UTM in test tube racks (upright) place in thermo box (4-6 frozen ice packs)

Where should the swab be broken after NPS collection?

Break point or score line of NPS

What physical sensation indicates that the swab has reached the nasopharynx?

The collector meets resistance

Anatomical location for OPS

posterior oropharyngeal wall or posterior oropharynx; tonsillar pillars or tonsils

What motion is done to swab for OPS?

Sweeping or painting motion

What is the preferred anticoagulant for most molecular testing?

EDTA (Lavender top)

What is whole blood, serum, plasma used for in the molecular diagnostics lab?

RNA target studies (HIV or HCV)

How is whole blood handled?

whole blood samples are centrifuged and plasma is removed within 4 hours after phlebotomy

What can inhibit enzyme activity?

Hemoglobin or hemolysis

What is used to remove hemoglobin and anticoagulants?

Buffers; BloodDirect (Novagen); Extract-N-Amp (Sigma); Resins: Chelex

Which anticoagulant should be strictly avoided because it inhibits reverse transcriptase and DNA polymerase?

Heparin

Recommended collection tubes

Tripotassium or Dipotassium EDTA (Lavender top); Acid Citrate Dextrose (Yellow top)

Which collection tube is usually used for trace elements and should be avoided

Dipotassium EDTA (Royal blue topped tubes)

What are the two types of Acid Citrate Dextrose (ACD) solutions and their max blood volumes?

Solution A (10 mL); Solution B (7 mL)

Components of ACD

Sodium citrate; Citric acid; Dextrose

What is the "Black Tiger" pattern/White top tube (PPT) used for?

HIV and HCV analysis; it contains a gel separator

What is the color of the cap of the collection tube used for HIV and HCV analysis?

White top

What is the description of the white top?

Black "tiger" pattern over the appropriate anticoagulant color designation

These collection tubes are designed to stabilize RNA

Tempus RNA Blood tube; PAXgene Blood RNA tube

These collection tubes separate WBCs lysed in Trizol or TriReagent (Sigma)

Std collection tubes

This is stored at -70 to stabilize RNA

lysate

Alternative to std collection tubes

Impregnated paper matrices

Specimen suitable for DNA analysis but not recommended for studies requiring intact RNA

Dried blood spots

Which tissue specimen is best analyzed fresh or frozen (Southern blot or long range PCR)

Solid Tissue

This tissue specimen is subjected to snap frozen using liquid nitrogen

Surgical specimen

This tissue specimen cannot be used for Southern Blot testing due to its lower quality DNA and RNA

Formalin fixed, paraffin-embedded tissues

What are the components of the OCT (Optimal Cutting Temperature) compound and at what temperature is it stored?

A mixture of Polyvinyl alcohol (PVA) and Polyethylene glycol (PEG); Frozen at -80C

Solid tissues: optimal amount

1-2 grams

Sources of Genomic DNA

Bone Marrow; Spleen; Lymph Node

When are tissue specimens used?

If blood or buccal cells are not available (ex: px is deceased)

Prenatal Specimens

Chorionic Villus Sampling; Amniotic Fluid

At what gestational age can the prenatal specimens be collected performed?

Chorionic Villus Sampling (CVS): 10-12 weeks of gestation; Amniotic Fluid: 15-20 weeks of gestation

What is the minimum volume required for prenatal specimens?

CVS: at least 15 mg; Amniotic Fluid: At least 10 mL

This specimen is used for prenatal testing to detect the genotype and fetal type of the fetus prior to delivery; Any molecular diagnostic assay can be applied to this specimen but it requires to be cultured

Amniotic fluid

When should prenatal specimens be processed?

on the same day of arrival

In case of delays, how should prenatal specimens be stored?

at 2-8 C

What must accompany a prenatal specimen to exclude contamination from the mother?

A maternal blood sample

How long should a patient refrain from eating, drinking, or smoking before a buccal cell collection?

30 minutes

Most common sample submitted for molecular testing and less invasive

Whole Blood

Sample of choice for detection of DNA from nucleated cells that are immature (precursor cells)

Bone Marrow

Specimen used for monitoring therapy given to patients with bladder cancer

Urine

Sample of choice for newborn screening

Dried Blood Spot - Capillary blood

Specimen used to isolate DNA and detect colorectal cancer

Feces

Where to collect CSF?

3rd and 4th and 4th and 5th lumbar region of the spine

Synovial fluid collection procedure

Arthrocentesis

What prep should be done prior to collecting buccal cells?

Avoid mouthwash with phenol