1.5 collection, receipt, storage, management, processing of specimens Flashcards
1.4.1 principles and procedures of washing, sterilisation and care of glassware and other equipment
- use of glassware/what it was used for
- washing manually or using lab glassware washer
- autoclave for sterilisation - esp prior to washing if it has contacted biological material 120deg 30mins
- drying oven
- chromic acid cleaning solution
- new glassware slightly alkaline so needs acid water soak before first use
1.4.2 make up solutions from written formulae
1) desired concentration, usually g/L or if in M, molecular weight of solute (how many g of substance in a mole)
2) required volume
Total solute= volume x concentration (mol/L=M)
1.4.3 Describe three types of microscopes
- upright; light from below, tip of objective point down (usual, fluorescent)
- inverted: light from above, objective beneath stage and point up, for viewing culture petri dishes
- fluorescent: specimen itself emits light, uses mercury lamp as light source to generate fluorescence in the specimen (excitation light)
Describe the necessary steps for maintenance of a microscope according to NATA 1998.
Specifically mention OH&S requirements and details regarding management of mercury lamp
NATA 1998
- not in a highly illuminated room
- spare objectives in disiccator
- adjustable seat
OH&S visual accommodation 30sec every 10 mins
- clean external surfaces only
- daily mx: clean top plate stage/mounting medium, debris
- periodic mx: check quartz halogen globe for corrosion of metal contacts, remove globe 14weekly and polish
- full service q2-3yearly
mercury lamp
- life is 800hours, log on timesheet
- dont turn on/off within 30mins
- breakage or explosion mercury lamp, evaucate, contain an clean PPE* mercury needs to be aspirated using pipette and covered with zinc dust or sulfar powder* safety
1.4.4. aliquot, dilute, store reagents
- keep inventory incl date of prep/expiry
- datasheet for storage antibodies -20, fluorochrome conj ab 8 deg
- storage container- transfer ab to opaque so don’t get quenched
- aliquot first when arrives so prevent contam/freeze thaw cycles
- optimize dilution
1.4.5 calibration, operation and mx of equipment used in assays
Balances
- q3yearly calibration
- check monthly
service 6-12monthly - zero the balance, calibrate using calib weights (standard weights or known volumes of liquid eg water) using forceps- each weight measure 10times for precision
- record mean, median, CV for each
- calculate SD if >0.02 need to contact provider
1.4.5
Pipettes
- can be affected by temp/humidity
- gravimetric analysis
- distilled water dispensed into vessel in precision analytical balance
- eg. 100microL water weigh 100g, 500microl= 500g etc.
- take 10 measurements at each value calculate mean, sd, cv
- can be done by external companies
1.4.5
Timers
- 2x per year
- verified with national institute of standards and technology traceable stopwatch- linked to atomic clock
- calibrate one timer then use it to calibrate others
- calibrated over 60min interval +/- 1 second
Instrument mx principles
- daily start up: waste bottle empty, rinses replinished, prime lines
- daily shut down: flush line,
- weekly/monthly: rinse system etahnol/isopropanol elim debris, rinse with distilled water
- mx log
How to make 1L 5M solution using solute that has molecular weight 75g/mol
conc x volume = total amount of substance required
5M = 75 g x5
Add this amount and make up to 1L (not add 1L)
How do you make a complex solution of 5L of 50nM Nacl, 10mM Tris HCl (C4H11No3)?
a: 5L of 50mM (50mmol/L) Nacl
Total= conc x volume = 250mmol= 0.25mol
molecular mass 58.44g/mol
58.44 x 0.25mol= 7.78h
B: 5L of 10mM of Tris (10mmol/L) conc x vol= 50mmol= 0.05mol molecular mass (off periodic table) 157.56g/mol 157.56g/mol x 0.05mol= 7.78g
so needs 14.61g NacL and 7.78g Tris-Hcl and add solvent to make up total volume of 5L
How do you make 100ml of 0.5M NaCL from stock solution of 5M?
c1v1= c2v2
5mol/L x X = 0.5mol/L x 0.1L
0.01L (10mL) of stock solution add 90mL of solvent to make 100mL
If precious sample is mislabelled?
What are request criteria?
What should be written on the report if accepted?
Requesting criteria
- requester signature
- three identifiers (name, DOB or URN OR address.)
- location to send report
- test requested/samples to take
- identifier on sample match the form
(Blood bank- not accepted)
To accept
- get collector to identify the sample
- someone needs to take responsibility for the sample
Report
- improperly labelled sample did not meet minimum labelling requirements, care should be taken in interpreting result of x
- details of collector identified sample
- recollection should be arranged if possible
List key information required for specimen collection instructions
where should this info be located/in what format
- request form:
- signature
- two identifieers
- tests requested
- label matches forms
- date and time
specimen collection
- tubes
- handling requirements/mix, temp
- transport requirements
- timing
- patient preparation
- collection details /min volume, number of tubes, site of sampling, order of draw
- if non medicare/sign acknowlegement for billing
electronically on intranet, easily accessible
Quick reference guide at sampling centre
What is the difference between plasma and a serum sample?
Plasma contains debris, cellular material and clotting factors ie platelet poor is ideal <10,000 plts
anticogulant (EDTA/citrate/heparinised tube)
If serum is left to clot for longer than 60mins at room temp may hemolyse
What is Ficoll-Paque and why is it used?
What are its disadvantages?
Neutral highly branched high mass hydrophilic polysaccharide used to isolated human mononuclear cells/flow lymphocytes
anticoag blood diluted layered over F-P and centrifuged 30-40mins
Disadvantages
- toxic material
- labour intensive
- cannot be automated
- time consuming
- can also kill off cells?/compromise cell yield, may need large samples
- selective loss of T/NK cells may occur
- poor recovery of lymphocytes
- may upregulat CD54, 62L, CD11b
(order from bottom to top; erythrocytes, granulocytes, media, mononuclear cells, plasma)
Urine sample for EPP- what are the issues?
Urine protein concentration is much lower than serum so needs concentration
- method ultrafiltiration/gravimtric
- centrifugation ultrafiltration
- multiple applications/inc application time
if underconcentrated then dec sensitivity and may be falsely neg
Ficoll-paque vs Percoll density centrifugation?
Ficoll is a medium with a density of 1,077 g/cm³. Percoll is a seperation solution with a density of 1,131 g/cm³
percoll- colloidal silica particles coated with PVP separates cells purely on density no impact of size of cell, non toxic, isotonic
better purification of lymph from monocytes
What dye can be used on haemocytometer to test for viability, and how does it allow testing for viability?
Trypan blue ‘diazo dye’
viable cells exclude from membrane uptake so appear white/clear
apoptotic/dead cells unable to exclude dye so stain blue
PBMC storage via cryopreservation
cells (generally 1-5x 10^7cells/ml) are pelleted snd resuspended in freezing media of fetal calf serum or DMSO/glycerol
frozen at a rate of -1 deg/min overnight then in -70-90deg freezer
frozen cells transferred into liquid nitrogen
PBMC /cell line thawing - describe the process
Done rapidly by placing vials containing frozen cells in protective container and immerse in 37deg water bath
transfer into laminar flow hood and resuspend in warm cell culture media/10% fetal calf serum added drop wise
wash cells quickly to revmoe dMSO/glycerol which can be cytotoxic
then resuspend in growth medium
What are the principles of density gradient centrifugation?
When centrifugal force and liquid viscosity is fixed, sedimentation rate is proportional to the size of the particle and difference between its density and the density of the surrounding medium
2M HCl solution prepared with total volume 0.35L molecular weight is 36. 46g/mol
What mass on Hcl in g is needed for the solution?
conc x volume = g
2mol/L x 0.35L = 0.7mol
g= 0.7x 36.46
25.5g
0.25L sea water contains 77.4g NacL with mol weight 58.44g/mol
what is the molar conc of NacL in seawater?
conc x volume = g
X x 0.25L = 1.324
X= 5.296M
77.4g= X mol x 58.44 X= 1.324 mol
Preparation of cells from tissue
- tissue prepared asap minimise cell loss
- mechanical separation : mince/seive VS enzymatically
- cell count
- stain mAB
- red cell lysis
- washing
- resuspend
- flow
NBT test and neutrophil isolation from blood
- NBT clear yellow water soluble dye upon reduction forms insoluble blue formazan
- neutrophils placed into reaction w PMA, NBT and autologous plasma at 37
- PMA /NADPH oxidase, activate respi burst in normal neutrophils,
neutrophils isolated from WB using neutrophil isolation media
- WB layered over separation media before centrifugation for 30mins
- 6 distinct bands, pipette layer of neutrophils resuspnd, centfigue and lyse residual RBCs
- cells washed, counted and resuspsneded
Cell sorting methods
- 2 types
1) magnetic bead separation
- magnetic beads conjugated to antibody of interest, ab conjugated beads w sample, apply mag field to column , collect after removal of magnetic field
2) flow cytometry based sorting
- charge assigned to each droplet based on fluorescence quality, enters electric field and eflected depending on charge/fluorescent qualities
- eg FITC stained +, PE stained neg charge
Disadvantages
- losses in yield
- tehcnical expertise
- low throughput
what kind of info available to users of the lab?
- tests available
- collection requirements
- specimen
- -volume
- transport/special precuations
- patient prep
- TAT
- clinical information/significance
- clinical decision points
- reference range
- MOU
- cost/non medicare billing
- factors that can affect performance
- contact for clinical advice
verbal add on test
1.5.22 NPAAC
need to document nature and time fo test
also receive confirmatory documentation of request
Key info on lab reports/ lab records? NPAAC requirement
- 3 identifiers of patient
- gender
- type of specimen
- type of test
- date and time collected
- date and time received by processing lab
- clinical status of patient (if fasted)
- collector name (if blood group)
- specimen characteristics that may affect interpretation
- informed consent when required
- name of requester
Name some tests with special requirements in Immunology for storage
- IgD, tryptase, ECP - immediate -20deg storage
- c1nh citrate plasma tube, immediately -20deg, ideally needs to arrive on ice
- CH50 immediately -70deg
- c3nef immediately at -70deg
- cryoglobulins- 37 min 60mins, clot then centrifuged and separated if blood allowed to cool then cryo will pp and be lost w the clot
- flow lymph subsets, function, HLA B27- test within 48hours of collection
- HIV VL- store -70deg
- urine epg store 4deg, conc x25-100
- csf if/igG needs paired sample collected withn 48hours
- TB gold- 16-24 incubation at 37 prior to storage,
