SYSMEX ogt Cytocell Multiprobe OctoChrome Instructions For Use Manual

Instructions For Use
REF: PMP 802 / PMP 804 / PMP 803
Chromoprobe Multiprobe
FOR PROFESSIONAL USE ONLY
ENGLISH/FRANÇAIS/ITALIANO/DEUTSCH/ESPAÑOL
Further information available at www.cytocell.com
Fluorescence In Situ Hybridisation (FISH) is a technique that allows DNA
sequences to be detected on metaphase chromosomes or in interphase nuclei
from fixed cytogenetic samples. The technique uses DNA probes that hybridise
to entire chromosomes or single unique sequences, and serves as a powerful
adjunct to classic cytogenetics. Recent developments have meant that this
valuable technique can now be applied as an essential diagnostic tool in prenatal,
haematological and pathological chromosomal analysis. Target DNA, after
fixation and denaturation, is available for annealing to a similarly denatured,
fluorescently labelled DNA probe, which has a complementary sequence.
Following hybridisation, unbound and non-specifically bound DNA probe is
removed and the DNA is counterstained for visualisation. Fluorescence
microscopy then allows the visualisation of the hybridised probe on the target
material.
Probe Information
Cytocell's Chromoprobe Multiprobe
square Multiprobe device and the whole chromosome painting probe (labelled in
3 different colours), to allow all 24 chromosomes to be identified on a single slide.
The OctoChrome™ device allows the simultaneous analysis of the whole
genome on one slide in one hybridisation.
Probe Specification
Each square of the OctoChrome™ device carries whole chromosome painting
probes for three different chromosomes in three different colour fluorophores,
red, green and blue (Texas Red, FITC and Aqua/DEAC spectra respectively)
which are visible simultaneously with a DAPI/FITC/Texas Red triple filter or
specific single filters.
The arrangement of chromosome combinations on the OctoChrome™ device
has been designed to facilitate the identification of the non-random chromosome
rearrangements found in the most common leukaemias (Figure 1).
The OctoChrome™ is intended for FISH on metaphase chromosomes from fixed
cultured peripheral blood cells.
Figure 1. Location of the probes on the Chromoprobe Multiprobe
OctoChrome™.
Materials Provided
Each kit contains the following reagents, which are sufficient for either 2 (PMP
802), 5 (PMP 804) or 10 (PMP 803) patient samples:
1. 2, 5 or 10 Chromoprobe Multiprobe
directly labelled probes:
Amount of red painting probe: 12-16ng per square
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OctoChrome™
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OctoChrome™ combines the utility of an 8
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OctoChrome™ devices coated with
Amount of green painting probe: 36-51ng per square
Amount of blue painting probe: 112-122ng per square
2. 4, 7 or 12 glass slides printed with a special template
3. 500µl Hybridisation Solution (Formamide, Dextran Sulphate, SSC)
4. 1 Cytocell Slide Surface Thermometer
5. 1 Cytocell Chromoprobe Multiprobe
Warnings and Precautions
1. For in vitro diagnostic use. For professional use only.
2. Wear gloves when handling Hybridisation solution, DNA probes and DAPI
counterstain.
3. Hybridisation Solution contains formamide, which is a teratogen; do not
breathe fumes or allow skin contact. Wear gloves, a lab coat, and handle in
a fume hood. Upon disposal, flush with a large volume of water.
4. DAPI is a potential carcinogen. Handle with care; wear gloves and a lab
coat. Upon disposal, flush with a large volume of water.
Dispose of all hazardous materials according to your institution's guidelines
5.
for hazardous waste disposal.
6. Operators must be capable of visually distinguishing between red, blue and
green.
7. Failure to adhere to the protocol may affect the performance and lead to
false positive/negative results.
Storage and Handling
The Chromoprobe Multiprobe
indicated on the kit label. Do not freeze.
Equipment and Materials Necessary but not Supplied
1. 500µl Counterstain (DAPI antifade (ES: 0.125µg/ml DAPI (4,6-diamidino-2-
phenylindole)).
2. Hotplate (with a solid plate and accurate temperature control up to 80ºC).
3. Variable volume micropipettes and tips range 1µl - 200µl.
4. Water bath with accurate temperature control at 72ºC.
5. Microcentrifuge tubes (0.5ml).
6. Fluorescence
Recommendation section).
7. Plastic or glass coplin jars.
8. Forceps.
9. Fluorescence grade microscope lens immersion oil.
10. Bench top centrifuge.
11. Microscope slides.
12. Timer.
13. 37ºC water bath without stirrer.
Fluorescence Microscope Recommendation
Use a 100-watt mercury lamp and plan apochromat objectives x63 or x100 for
optimal visualisation. Use a triple bandpass filter DAPI/FITC/Texas Red for
optimal visualisation of the green and red fluorophores and DAPI simultaneously.
The aqua fluorophore has specificity to the Aqua and DEAC spectrum (single
bandpass Aqua or DEAC filter is required).
Check the fluorescence microscope before use to ensure it is operating correctly.
Use immersion oil that is suitable for fluorescence microscopy and formulated for
low autofluorescence. Avoid mixing DAPI Antifade with microscope immersion
oil as this will obscure signals. Follow manufacturers' recommendations in
regards to the life of the lamp and the age of the filters.
Sample Preparation
The Chromoprobe Multiprobe
peripheral blood cells fixed in Carnoy's fixative that should be prepared according
to the laboratory or institution guidelines.
Prepare air dried samples on Cytocell Chromoprobe Multiprobe
according to Cytocell's protocol below. Baking or otherwise ageing slides is not
recommended as it may reduce signal fluorescence.
Chromoprobe Multiprobe
Please note: The probes used on the Chromoprobe Multiprobe
directly labelled with fluorophores, which are light sensitive. Ensure that
exposure of the probes to laboratory lights is limited at all times (it is not
necessary to work in the dark).
1. Slide preparation
a) Clean a template slide. Soak the template slide for 2 minutes in 100%
methanol and polish dry with a clean soft tissue.
b) Establish the correct mitotic index. It is important that the intended sample
has a sufficiently high mitotic index to allow detection of chromosome
abnormalities. To check the density of the sample, using a micropipette (e.g.
a Gilson P10 or P20) pipette 4µl of the cell suspension onto one of the areas
of the spare template slide and allow to air dry. The small volume used
means that you usually have to gently touch the slide with the pipette tip to
transfer the suspension. Examine by phase contrast microscopy. If the cell
density is too high, dilute the suspension with fresh fixative. If the mitotic
index is too low, spin down the fixed cell suspension at 160xg for 10 minutes.
Note the volume of supernatant, remove, and re-suspend the cell pellet in a
smaller volume of fresh fixative. If cell sample density has been altered, spot
4µl of the concentrated sample onto another square of your test slide and
re-examine by phase contrast microscopy.
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Please Note: 50µl is the minimum volume required for the protocol.
c) Quality control of samples. Samples should be examined for cytoplasm
since this will interfere with the in situ protocol. If the chromosomes appear
to be enclosed by a granular material when examined under phase contrast
microscopy, then this will compromise results. One method for reducing
cytoplasm is to spot 4µl of your sample onto the template slide and watch
the fixative as it spreads out: in the normal situation, the fixative will spread
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Hybridisation Chamber
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kit should be stored at 2-8ºC until the expiry date
microscope (Please see Fluorescence
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OctoChrome™ is designed for use on cultured
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PI008/CE v011.00/2019-04-30
Microscope
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template slides
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device are
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