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Standard operating procedures (SOP)

+Why have an SOP?

  Important to set up validated methodology which can be quality controlled and applied in a repeatable and reliable fashion on a continuing basis. All testing and results are open to legal verification for the purposes of test correctness and for billing purposes. An SOP is just one part of the requirements of an accredited testing laboratory.

  The contents of an SOP will vary from lab to lab and from organisation to organisation, but there are basic requirements which also aid in the teaching needs of new technologists

+SOP Cover Page   Institution Name

  Name of the test being outlined

  Accession number of the SOP

  Date formulated and by whom

  Name of persons checking and approving the SOP

  Review Dates (usually annually)

  The SOP will be document controlled. No one may write on the official document or copy the original for their own use. Archived copies will be kept and changes tracked.

  No tippex allowed under any circumstances. Black pens only for work sheets and other lab logs. No pencil notes.

+ SOP Purpose and Principle   These will explain the reason for the test and will include some detail behind

why certain reagents are used.

  In our example the following are mentioned colcemid, “closed culture” system, pH indicators, FCS (fetal calf serum), and a mitogen called phytoheamagglutinin (PHA), hypotonic potassium chloride.

  Colcemid disrupts the spindle and halts the cell in metaphase so that the chromosomes may be visualised

  A “closed culture” system is where the there is no supply of gases to aid in the control of the pH as the cells respirate. The buffer in the culture media must be able to control the pH between 7.2 and 7.4. Some culture media come with an added pH indicator. This can show infection (yellow – alkaline) or overgrowth by the sample and the buffer is exhausted. If the culture media is pink it indicates that there is too little CO2 and therefore acid conditions. Both scenarios are not conducive to good cell growth.

  Fetal Calf serum provides growth factors

  Mitogen called PHA is derived from kidney beans and acts like an antigen stimulating the T-cells to divide in culture (stimulated as they are during an infection). This will increase the cell yield for chromosome analysis.

  Hypotonic KCl to swell the cells before harvest

+   Responsibility: who is going to do the test?

  Health and Safety: MSDS – material data safety sheet supplied by the manufacturer.   Storage – delivery logs and usage logs must be kept   Protective clothing, coats, gloves, goggles, masks   Disposal SOP’s

  Materials and Reagents: Names the requirements for the method along with catalogue and purchasing information, expiry dates logged. How to store your chemicals. How do you prepare a 70 % solution of ethanol?

  Equipment: All equipment has to be noted on an acquisition register and have either a clear serial number or institution reference number for traceability. The equipment book of life will tell you when the piece was bought and how much it cost, when it will need to be replaced, when it was last serviced and when it is due for its next service. It will also contain the decontamination certificates that were done before each service.

+Biosafety Laminar Flow Cabinets

The Biosafety Cabinet chosen for the Cytogenetics laboratory provides protection for the technologist from diseases carried by the sample and is also important in providing a sterile area in which the sample can be prepared for culture. Infection through poor culture technique will result in loss of the sample. It is not always possible to get repeat samples and it could be costly, and dangerous as in the case of amniocentesis samples, chorionic villus samples, cord blood, and bone marrow samples.

+ Biosafety Laminar Flow Cabinets 1.  Placing of the cabinet – should be in area in a

separate room (preferably a culture room – dedicated purpose) with good ventilation and an external exhaust.

2.  HEPA filter (High Energy Particulate Arrestant). Excludes particles smaller than 0.3 um

3.  Clean air blown from top down through the filters and over the working area.

4.  HEPA filter also used to clean air that has been through the cabinet and may carry infectious material.

5.  Air drawn in at the front where the technologist is sitting, is sucked down before it reaches the work area, and is filtered.

6.  “Smoke test” done to check airflow. 7.  Daily cleaning before and after working in the

cabinet – can use 70% ethanol 8.  Decontamination essential before servicing

+Sterile (Aseptic) Technique   Protective clothing

  Wash hands before and after handling specimens and reagents

  Wash all surfaces with alcohol, including the necks of reagent bottles and the surfaces of pipettors

  DO not touch the edge of containers with the pipette

  Do not take lids off and set them “upwards”

  Only open containers for the time needed to add reagents or sample.

  Do not block the airflow of the cabinet with reagent bottles.

  Unwise to use an open flame in the hood as it will disturb airflow

+Equipment for lymphocyte culture

Cuture media - RPMI

Automatic pipettor plus single wrap disposable 10 ml pipettes

Universal Tubes

PHA

Heparinised blood tubes

+Equipment for lymphocyte culture (Cont)

Plastic pasteur pipette

Incubator

+Sampling

  Heparinised Blood (1ml minimum for adults)

  Unacceptable samples: wrong anticoagulant, clotted, haemolysed, lipaemic, more than 48 hours old, frozen, samples taken from patients on antibiotics.

  Urgent samples:   Cordocentesis (PUBS – perumbilical blood sample)

  Blood from parents of a fetus with abnormalities seen on ultrasound

  Blood from newborns with congenital abnormalities.

+Why are some samples considered more urgent than others?

  Cordocentesis – often done when the amniotic fluid indicates a problem that needs further testing or because the pregnancy is quite advanced

  Abnormality detected on ultrasound

  Amniocentesis chromosome results show – for example – a marker chromosome making it difficult to give a prognosis. Parents of the fetus need to be checked to see if they are carrying the marker. (The same goes for a translocation, possibly balanced being found in the fetus – the parents need to be checked)

  Blood from newborns who clearly show clinical signs of a congenital abnormality

+Cultures set up in duplicate as part of quality control

  Work done separately by two technologists to reduce the risk of losing cultures due to infection which will outgrow the lymphocytes.(that can be microbial or fungal growth).

  Harvesting and analysis is also done in duplicate by two technologists. This increases the rate of chromosome abnormality detection, and reduces the transcription error rate

  Culture media will be tested at 370Cwithout sample to check that the pH remains constant and there is no infection.

  Validation: All methods should be tested to see if behave as they should in your laboratory. Historical data can be used to establish trends and acceptable yield of metaphase cells for analysis. Samples can be cultured simultaneously at your and another laboratory to see if the methodology works. The same tests can be used for training and competency records

+ Infection in the incubator!

  Decontamination of Cultures with Antibiotics

  When an irreplaceable culture becomes contaminated, determine if the contamination is bacteria, fungus, mycoplasma, or yeast.

  Isolate the contaminated culture from other cell lines

  Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters. Antibiotics and antimycotics at high concentrations can be toxic to some cell lines. Therefore, perform a dose response test to determine the level at which an antibiotic or antimycotic becomes toxic

  Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding

+Back to the SOP

  The Sign Sheet   Signed by all who read and understand the SOP.

  Indicates responsibility – if there is something you do not understand in the SOP – ask and make sure you know what to do if given this SOP to follow.

  Part of training and continuing competencies.

  Part of making sure everyone knows there has been a change to the method.

+Blood Lymphocyte Harvest

  Dividing cells in culture are halted in metaphase by the addition of colcemid. Ethidium bromide is added to lengthen the chromosomes, which it does by intercalating between the DNA. Hypotonic Potassium chloride (KCL) is added to swell the cells. Methanol/ acetic acid fixative is used to precipitate the nucleic acids thereby fixing the chromosomes and to precipitate the proteins, which can then be washed off. Slides are prepared by dropping the cells onto clean, cold slides. The cells break open on the slide to release the chromosomes. Heat is applied to encourage the chromosomes to spread which facilitates counting and analysis.

+Some Terminology

  Supernatant – fluid above the cells

  Spin at 500g (why not rpm? – it depends on the centrifuge and the distance of the base of the tube from the centre of the rotor)

+G-banding

  The trypsin is thought to increase the permeability of the chromosome structure. This process is stopped by the rinse in cold buffer. The stain then binds preferentially to different areas of the DNA forming the appearance of light and dark bands. Dark bands are rich in A + T and replicate late in the S phase. They do not seem to have many active genes. Depending on the stage of mitosis at which the chromosome is banded one can see from 400 up to 850 bands.

  400 bands are the minimum acceptable standard for certain clinical referrals.

  550 bands are the recommended standard.

  Method:

+Reason for referral Banding points

  Aneuploidies and known large structural rearrangements 400

  Expected small structural rearrangements 400

  Possible small unknown structural anomalies (e.g. clinically: recurrent abortion, dysmorphic features, delayed development) 550

  Microdeletion syndromes (FISH is the preferred method of analysis where available) 850

+ The Ananlysis:

  Count the chromosomes.   Analyze each set of chromosomes, comparing length and number

of bands. Note normal variances as well such qh+, large satellites, extra large Y.

  Note breaks and gaps and marker chromosomes. Use the clinical data to insure a thorough search through all the metaphases.

  Confirm that the patient gender matches the result that is found in the analysis.

  Always analyse a minimum of 10 metaphase spreads. The analysis is always split between 2 technologists to provide internal quality control.

  Space is provided on the vernier sheet for a technical drawing of a metaphase spread for analysis.

  Each technologist will select one metaphase for imaging and karyotyping.

  Routine stain can be done to investigate marker chromosomes and breaks and gaps.

  If necessary do further stains to solve uncertain findings

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The vernier sheet Note: Five metaphase cells can be analysed and their position recorded for image capture and karyotyping.

+Special Stains

  Quinacrine Stain (Q-Banding) – good for the Y chromosome

  Nor Stain (Silverstain for Satellites)

  C-Banding (Barium hydroxide stain for centromeres)

+Trouble-shooting What to check when things go wrong

Indication Little or no growth. Established cultures dying

Little or no growth. Established cultures dying

Medium deep pink

Little or no growth. Established cultures dying

Medium yellow and / or cloudy (acid conditions)

Little or no growth. Established cultures dying Medium pink indicates alkaline conditions and could be lack of CO2

Poor metaphase yield from blood cultures

Reason

Temperature out of range

Medium expired causing change in pH

• Infected medium

• Poor aseptic technique

Could be lack of CO2

PHA omitted from culture •FCS omitted from neonate culture •Patient may be on antibiotics that interfere with cell growth •Sample clotted •Incorrect anticoagulant •Incorrect transport conditions •Toxic collection equipment

Solution Check temperature of incubator and logs. Check equipment SOP for fault finding, and service agent details.

Check expiry date of culture medium in use. If expired take fresh medium. Check temperature of medium storage conditions are correct. Follow up with equipment manual and service conditions.

• Do a sterility check on culture medium in use: Check microscopically for contamination on stained and unstained samples to confirm infection.

NB Viral contamination will not be apparent. • Fungal contamination is easily seen by hyphae and

spores. • Review aseptic technique

Check gas cylinders for CO2 supply. • Use Fyrite to check correct concentration. • If equipment error is suspected then check

equipment manual and servicing details

Add PHA to the repeat culture • Add FCS to the repeat culture • Check with the referring doctor and request repeat

when patient is off antibiotics • Request Repeat sample • Request Heparin sample • Address as a non conformance with transport staff • Recommend the shortest possible contact time with

the rubber stopper of the syringe