Site/Area of application / Biochemical Genetics (Metabolic Laboratory)
Index code / SLF2BGM016 Ver 3.3
Superseded documents / BISJCP208 versions 2 and 3
Implementation date of this version / 20/08/2012
Approver of content of SOP/OWNER / Janet Mitchell
Reason for change / Relocation of laboratory to block 46
Keywords for search on EQMS / White cell cystine, leucocyte cystine, white cell pellet, cystinosis
.
Signature
Section / Page Number
1. Clinical Relevance/Purpose Of Procedure / 2
2. Principle of procedure / 2
3. Personnel / training requirements / 2
4. Specimen requirements / 3
5. Equipment / 3
6. Health and Safety/Risk assessment / 3-4
7. Reagents / 4-5
8. Calibration / 5
9. Quality control / 5
10. Computer / telepath codes / 5
11. Procedure or methodology ( expand as required) / 5,6,7,8
12. Uncertainty of Measurement / 8
13. Reference range / Action limits / 8
14. references / 8
15. Appendices / 8
16. Training record
1. CLinical relevance/purpose of procedure
White cell cystine is used to diagnose and monitor the treatment of the inborn error cystinosis.
One function of lysosomes is to break down proteins into amino acids for re-use by the cell. These amino acids are then exported from lysosomes via specific transport proteins. Patients with Cystinosis have an inactive lysosomal transport protein for cystine. This causes an accumaulation of cystine which disrupts the integrity of the lysosomal membrane leading to cell damage by release of proteolytic enzymes. Tissues and organs of low cell turnover/regenerative ability are the most vulnerable to this damage. Proximal renal tubule cells are particularly vulnerable with significant renal impairment being seen by 1 to 2 years of age. This will cause polyuria, polydypsia, failure to thrive and ricketts. A generalised aminoaciduria, glycosuria and phosphaturia is also observed (Fanconi syndrome).
White cells contain lysosomes and are therefore used as a source of cells for diagnosis or monitoring. Cystagon (cysteamine bitartrate) binds with cysteine which is in equilibrium with cystine. The resulting disulphide has a similar size and shape to lysine and is exported from lysosomes via the lysine transport protein. This systemic treatment is for all affected organs, although eye drops are used to avoid cystine crystal formation in the eye. The lower the white cell cystine, the better the treatment, although there have been some reports of side effects in over-treated individuals.
2. principle of procedure
A lithium heparin blood sample is mixed with ACD-Dextran solution in a conical bottomed tube. The tube is placed in wet ice and red cells allowed to settle over 45 minutes. The white cell rich supernatant is then removed into another conical tube, and centrifuged at 450g in a pre-cooled (4oC) centrifuge for 10 minutes. At this stage the red cells are layered in the bottom of the tube with white cells on top. The plasma-like supernatent is then removed, and the cells are re-suspended in normal (0.9%) saline. Distilled water is added to produce hypotonic lysis of the red cells which is accelerated by gentle vortexing for 45 seconds. Without delay, the white cells must then be ‘rescued’ by addition of 3.6% saline with mixing. This restores the mixture to isotonicity. The sample is then re-centrifuged at 450g and 4oC. The white cells will now be on the bottom of the tube with red cells haemolysed above the cells. A thin band of red cells is often seen above the white cells. These are removed by repeating the hypotonic lysis step once more, leaving a pure mixed leucocyte pellet. Finally, 150 mL of distilled water is added to cause white cell lysis. This process is slow, so an ultrasonic probe is used to fully lyse the cells and organelles (including lysosomes). 50 mL of 12 % sulphosalicylic acid is then added to precipitate the protein, and the sample frozen at -20 oC until stage 2 of the assay is performed.
3. personnel / training requirementS
Advanced BSW
BMS
Senior BMS
Clinical Scientist
4. specimen requirements
Sample type : Lithium heparin blood, 5 mL, mix gently but thoroughly immediately after venepuncture to avoid micro-clots. Try to use a tube which can be filled - reduces mechanical damage to cells during transit.
Minimum volume : 2 mL
Sample timing: The sample is taken as a trough level, i.e. just prior to the next dose of Cystagon whilst trying to maintain the normal dosage pattern.
Transport: Must be processed using this procedure by the end of the next calendar day after venepuncture. Sample should not be taken if it will arrive at a weekend or Bank Holiday.
Stored at : Room temperature
4. equipment
PALL Corporation (Gelman) FP Vericel 450 filters, Part No: 66480, 0.45 μm, 47 mm diameter filters (Supplier: VWR)
Ice crusher
15 mL plastic conical bottomed tubes with screw cap.
MSE Soniprep 150 ultrasonic disintegrator.
6. health and safety/risk assessment
Further guidance relating to laboratory accommodation, personal protective equipment and other general safety considerations is available in the Pathology Safety Manual [PHS039].
See COSHH and procedure risk assessments [SLC5CBG014].
Unless otherwise specified all laboratory work must be performed at containment level 2 [PHS007]
Hand and eye protection must be worn when handling blood and serum/plasma.
Laboratory procedures that may give rise to infectious aerosols must be conducted in a microbiological safety cabinet [PHS006].
High risk samples – Laboratory work must be performed at containment level 2+.
Additional precautions as described in [PHS007]
· Gloves and disposable apron must be worn
· Eye protection must be worn where splashing is assessed as a risk
· Analysis must only be undertaken by experienced BMS staff
· Analysis must be undertaken as a discrete task
· Access by other staff to the area should be restricted
All reagents are disposed of in accordance with the Waste Management procedures outlined in section 15 of the Health & Safety Manual [PHS011]
Any spillages should be dealt with according to the spillage procedures outlined in section 13 of the Pathology health & safety manual [PHS009]
Material, Etc.. / Source / Main Hazard Information / Precautions / Hazard SymbolsSoniprep 150
Ultrasonic disintegrator
(used for lysing white cells) / Sanyo Gallenkamp / The ultrasound used in this
machine can cause hearing damage, and damage to other
body parts
(eg. bones in fingers, skin, etc). / Always wear ear protectors.
ALWAYS close and lock the instrument door BEFORE operating it.
Ask other people to leave the room. /
DANGER
Ultrasound Noise
5-Sulphosalicylic
Acid / Sigma
Cat No:
S-2130 / Harmful by ingestion and if inhaled as dust.
Irritating to skin and eyes / Wear gloves and eye protection.
Wash off body surface with water. /
Harmful
Corrosive
7. reagents
ACD-Dextran Solution.
Weigh out the following into a 250 mL measuring cylinder:-
6.0 g of Dextran (Sigma Cat No: D-4876)
4.2 g of D(+)Glucose (Sigma Cat No: G-7528)
0.66 g of tri-Sodium citrate (Merck Cat No: 10242 )
0.26 g of Citric acid monohydrate (Merck Cat No: 10081)
1.8 g of Sodium chloride (Merck Cat No: 10241)
Dissolve in 150 mL of fresh deionised water, and make up to 200 mL.
Filter under vacuum through an FP Vericel 0.45 mm filter.
Store in 20 mL aliquots at -20oC (freezer 4, tandem room). Mark each bottle with the date of preparation, initials of person making up solution, and an expiry date equal to 2 years later. Thaw out a bottle as required and then store at 2-8 oC (fridge 16, metabolic lab) until used up. Each bottle contains enough for at least four preparations.
Filtered 0.9% Sodium Chloride.
Dissolve 4.50 g of sodium chloride (Merck Cat No:102415K) in 500 mL of fresh deionised water.
Filter under vacuum through an FP Vericel 0.45 mm filter.
Store in the appropriate 500 mL plastic bottle at 2-8oC (fridge 16, metabolic lab).
Mark the bottle with the date of preparation, initials of person making up solution, and an expiry date equal to 3 months later.
Filtered 3.6% Sodium Chloride.
Dissolve 18.0 g of sodium chloride (Merck Cat No: 10241) in 500 mL of fresh deionised water.
Filter under vacuum through an FP Vericel 0.45 mm filter
Store in the appropriate millimatic dispenser at 2-8oC (fridge 16, metabolic lab).
Mark the dispenser with the date of preparation, initials of person making up solution, and an expiry date equal to 3 months later.
Filtered Deionised Water (for hypotonic lysis and pre-sonicator steps).
Use fresh deionised water.
Filter under vacuum through an FP Vericel 0.45 mm filter.
Store in the millimatic dispenser at 2-8oC (fridge 16, metabolic lab).
Mark the dispenser with the date of preparation, initials of person making up solution, and an expiry date equal to 3 months later.
5% Decon 90 for cleaning MSE Soniprep 150.
Use a plain 25ml plastic universal. Add 1mL of concentrated Decon 90 solution.
Make up to the 20mL line with unfiltered deionised water. Mark the universal with the date of preparation, initials of person making up solution, and an expiry date equal to 3 months later.
Deionised Water for cleaning MSE Soniprep 150.
Use 3 plain 25 mL plastic universals and Label 1, 2 and 3. Add fresh deionised water straight from the deioniser. Mark each universal with the date of ‘preparation’, initials of person preparing, and an expiry date equal to 3 months later.
12% 5-Sulphosalicylic Acid.
Dissolve 12 g of 5-sulphosalicylic acid (Sigma Cat no: S-2130) in 100 mL of water. Do not filter!
Store at 2-8C (fridge 16, metabolic lab). Mark the bottle with the date of preparation, initials of person making up solution, and an expiry date equal to 5 years later.
8. Calibration
Not applicable
9. quality control
As with all laboratories performing white cell analysis, there is no external quality assessment scheme that covers this preparation stage of the assay. A small pool of carefully trained staff perform this procedure to minimise variability.
10. computer / telepath codes
Specimen type: Blood
Set name: WCYS1J
11. procedure or methodology
There are 3 stages to this procedure:
1a: Separating white cells from most of the red cells
1b: Hypotonic Lysis and Removal of the Remaining Red Cells
1c: Ultrasonic Disintegration of White Cells and Precipitation of Protein.
Stage 1a: Separating white cells from most of the red cells.
Remove the ACD-Dextran from the fridge and check that there is enough for all the preparations.
Remove another aliquot from the freezer if necessary.
Crush a full tray of ice using the ice crusher.
Add a little tap water to crushed ice in a 250 mL beaker or similar container.
Label two 10 mL conical bottomed tubes with the batch number and patient name.
Add the blood and an equal volume of ACD-Dextran to one of the tubes (note if any clots are present).
Cap, mix by inversion and place in the wet ice for 45 minutes. This allows red cells to precipitate.
Cool the Mistral 3000i centrifuge by running program #2 10 minutes before the 45 minute period ends.
Take the sample from the ice and transfer the supernatent into the other 10ml conical bottomed tube.
Centrifuge the supernatant using program #2 (450g, 4oC, 10 minutes)
CHECK THAT THE SETTINGS ARE CORRECT (especially 450g and not 450rpm!).
Discard the supernatant and proceed to the next step IMMEDIATELY.
Stage 1b: Hypotonic Lysis and Removal of the Remaining Red Cells.
Add 0.8 mlLof 0.9% sodium chloride, and resuspend the cells by gentle vortexing for about 10 seconds.
Add 2.4 mL of filtered deionised water
Vortex for 45 seconds (during this time many of the unwanted red cells are haemolysed).
Add 0.8 mL of 3.6% sodium chloride without delay (rescues the white cells).
Vortex for 5 seconds.
Centrifuge using program #2 (450g, 4oC) but for 5 minutes (again, check the settings)
Using a pastette draw up supernatant, and very gently blow the red cells off the surface of the white cells and discard the unwanted red cell containing supernatant. DO NOT DISTURB THE WHITE CELLS.
If a large proportion of red cells cannot be aspirated off, another hypotonic lysis step may be repeated but the white cell yield will be significantly lower.
Add 0.75 mL of 0.9% sodium chloride.
Resuspend the white cells by vortexing for 10 seconds.
Transfer to a 1.5 mL plastic centrifuge tube using a plastic pastette.
Add a further 0.75 mL of 0.9 % sodium chloride to the 10 mL tube, mix gently, and transfer to the 1.5 mL plastic centrifuge tube.
Centrifuge at 5,000 rpm for 4 minutes in the micro-centaur centrifuge.
Using an 250 mL pipette tip, remove any remaining red cells, and then remove all the supernatant.
Add 150 mL of filtered deionised water to the white cells and vortex for 20 seconds.
NOTE: If the white cell yield is very low a repeat sample may be needed - inform a senior member of staff if this occurs.
Stage 1c: Ultrasonic Disintegration of White Cells and Precipitation of Protein.
SONIPREP 150
Disintegrator
DANGER
Ultrasound Noise
CLOSE the instrument door, lock it, and apply your ear protectors before operating the probe!
Never operate with the door open.
Ask other people to leave the room (they do not have ear protection).
The disintegration probe is cleaned by operating for 5 seconds in each of the following:-
5% Decon