deparray™ forensic sample prep kit - arinahayat.com · deparray™ forensic sample prep kit 4 2....

For research use only. Not for use in diagnostic procedures. For in vitro use only.

DEPArray™ Forensic Sample Prep Kit

Resolve Heterogeneity in Forensic Biological Mixtures

USER MANUAL Version 2.0

Content version: July 2017 REF- -FORSR: DEPArray™ Forensic Sample Prep Kit – Reagents (Box 1 REF FORSR1 and

Box 2 REF FORSR2)

-FORSE: DEPArray™ Forensic Sample Prep Kit – Epithelial Cells

-FORSS: DEPArray™ Forensic Sample Prep Kit – Sperm Cells

-FORSW: DEPArray™ Forensic Sample Prep Kit – White Blood Cells

Store FORSR1, FORSE, FORSS, FORSW at +2…+8°C 20 reactions

Store FORSR2 at -25…-15°C


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1. Kit Contents ......................................................................................................................... 3 2. Storage and Handling ............................................................................................................ 4 3. Intended Use & Product Use Limitation .................................................................................. 4 4. Safety Information ............................................................................................................... 4 5. Technical Assistance ............................................................................................................. 5 6. Additional Required Reagents ............................................................................................... 5 7. Additional Required Materials and Equipment ....................................................................... 5 8. DEPArray™ Forensic Sample Prep Kit Description .................................................................... 6 9. How to Analyze DEPArray™ Forensic Sample Prep Kit Products................................................ 7 10. DEPArray™ Forensic Sample Prep Kit Downstream Applications .............................................. 7 11. Sample Specifications ........................................................................................................... 7 12. What to Do Before Starting ................................................................................................... 8 13. DEPArray™ Forensic Sample Prep Kit Procedure ..................................................................... 9 14. Patent & Trademark Information ......................................................................................... 33 15. Warranty ........................................................................................................................... 33 16. Annex 1: Chemicals Classifications ....................................................................................... 34 17. Appendix A: CellBrowser™ Cell Images ................................................................................. 36


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1. Kit Contents ▪ DEPArray™ Forensic Sample Prep Kit – Reagents (REF FORSR):

- Box 1 (REF FORSR1) - Box 2 (REF FORSR2)

▪ DEPArray™ Forensic Sample Prep Kit – Epithelial Cells (REF FORSE)

▪ DEPArray™ Forensic Sample Prep Kit – Sperm Cells (REF FORSS)

▪ DEPArray™ Forensic Sample Prep Kit – White Blood Cells (REF FORSW)

Box Storage Reagent Cap Color Vial Content

Nuclear marker cyan 1 vial/ 22ml

Water white 2 vials/ 1000ml

Sample prep buffer white 1 vial/ 900ml

Blocking buffer white 1 vial/ 900ml

Fix buffer white 20 vials/ 225ml

Perm buffer white 1 vial/ 1100ml

1 +2…+8°C

-25…-15°C2

Storage Reagent Cap Color Vial Content

+2…+8°C ECs marker green 1 vial/ 530ml


+2…+8°C SCs marker red 1 vial/ 900ml


+2…+8°C WBCs marker yellow 1 vial/ 170ml


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2. Storage and Handling Store DEPArray™ Forensic Sample Prep Kit at +2…+8°C upon arrival.

Attention: Store the DEPArray™ Forensic Sample Prep Kit – Reagents, Box 2 at -25…-15°C upon arrival. Thaw only before use.

IMPORTANT STORAGE INFORMATION:

Upon arrival, Perm Buffer must be thawed and aliquoted in 50 μl single-use vials; freeze the aliquots and thaw only before each use.

Prior to any use, thaw a single-use Fix Buffer vial. Avoid any further freeze-thaw cycle. Always keep Fix Buffer protected from light.

Nuclear marker, epithelial cells marker, sperm cells marker and white blood cells marker must be kept in the dark.

When stored and handled under these conditions the kit components are stable through the i n d i c a t e d expiration date. Handle and store reagents with the appropriate attention and care, and set up the reactions according to good laboratory practices.

Menarini Silicon Biosystems SpA recommends the users to follow the Guidelines for Research involving Recombinant DNA Molecules (NIH guidelines) Federal Register, July 5, 1994 (59 FR 34496) and any amendments thereto. Menarini Silicon Biosystems SpA disclaims any and all responsibility for any injury or damage which may be caused by any behavior not in compliance with the indicated guidelines.

3. Intended Use & Product Use Limitation The DEPArray™ Forensic Sample Prep Kit is intended for research use only. The DEPArray™ Forensic Sample Prep Kit is for in vitro use only. No claim or representation is made for any intended use to provide information for the diagnosis, prevention, or treatment of a disease.

4. Safety Information When working with chemicals always wear suitable personal protective equipment such as a lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDS). MSDS of each Menarini Silicon Biosystems kit and components are available online at http://www.siliconbiosystems.com/msds-documents.

http://www.siliconbiosystems.com/msds-documents


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5. Technical Assistance For technical assistance and additional information, please refer to Menarini Silicon Biosystems Customer Support :

e-mail: [email protected]

Phone : (+39 ) 051-9944100 (Mon-Fri, 9 am – 18 pm CET +01:00)

6. Additional Required Reagents Reagents listed below are not included in DEPArray™ Forensic Sample Prep Kit and shall be purchased separately by the user: PBS 1X CaCl2/MgCl2 free, pH 7,2 Recommended supplier: Thermo Fisher Scientific (Gibco), 500ml autoMACS Running Buffer – MACS Separation Buffer, 1,5l Recommended Supplier: Miltenyi Upon arrival, aliquot autoMACS Running Buffer – MACS Separation Buffer (25ml volume aliquots) and store at +2…+8°C protected from light. Use one aliquot for each sample preparation. Discard leftover buffer after use. Note: REF codes may differ within different countries. Please, verify the correct correspondent code for your country before ordering.

7. Additional Required Materials and Equipment epT.I.P.S. LoRetention Dualfilter pipette tips (0,1–10μl, 0,5–20μl, 10-100μl, 20–300μl, 50–1.000μl, 0,5-5ml) Current Supplier: Eppendorf.

Eppendorf Research® plus Pipette set, 1 each (0,1–10μl, 0,5–20μl, 10-100μl, 20–300μl, 50–1.000μl, 0,5-5ml) Current Supplier: Eppendorf. Polypropylene Conical Tubes 15 ml Current Supplier: BD Falcon™ Protein Lobind Tubes 1,5ml Current Supplier: Eppendorf Sterile Polystyrene Disposable Serological Pipets (10ml) Pipette Fillers Metal Tweezers Metal Scissors Parafilm® 1,5ml, 15ml and 50ml Tube Racks DNA IQ™ Spin Baskets for 1,5ml tube Recommended Supplier: Promega Burker Chamber A thermomixer suitable for 1,5ml and 15ml tubes

mailto:[email protected]


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Note: If the thermomixer is not suitable for 15ml tubes, use an adequate support on the thermomixer plate and fix it with adhesive tape. Swinging Bucket Rotor Centrifuge suitable for 1,5ml and 15ml tubes Refrigerated microcentrifuge Vortex -25…-15°C Storage Freezer +2…+8°C Storage Fridge Water Bath Sonicator with de-gas function Biohazard Hood Note: REF codes may differ within different countries. Please, verify the correct correspondent code for your country before ordering.

8. DEPArray™ Forensic Sample Prep Kit Description

DEPArray™ Forensic Sample Prep Kit is a sample preparation kit, specifically developed to allow the detection, identification and isolation of human sperm, epithelial and white blood cells using the DEPArray™ system starting from semen/saliva/blood forensic mixtures collected on swabs. Cell and nuclear specific immunofluorescent staining allow simultaneous, precise identification of sperm cells (APC channel), epithelial cells (FITC channel) and white blood cells (PE channel) with the DEPArray™ system. The procedure is suitable for preparation and analysis of mixed biological samples using both DEPArray™ V2 and DEPArray™ NxT platforms. The kit is composed of four modules that can be combined according to the user’s experimental needs. DEPArray™ Forensic Sample Prep Kit – Reagents (REF FORSR) is the mandatory module, containing common reagents for all staining combinations. DEPArray™ Forensic Sample Prep Kit – Epithelial Cells (REF FORSE), DEPArray™ Forensic Sample Prep Kit – Sperm Cells (REF FORSS) and DEPArray™ Forensic Sample Prep Kit – White Blood Cells (REF FORSW) can be used jointly or in combinations of two, to respectively detect epithelial cells, sperm cells and white blood cells, depending on the user’s needs. The kit content is intended for 20 preparations. The output of the DEPArray™ Forensic Sample Prep Kit procedure is a cell suspension in which cells are distinguishable by means of the linked antibody. See Appendix A – CellBrowser™ Cell Images for an example of images resulting from the staining for each cell type singularly (Fig. 1) or in heterogeneous clusters (Fig. 2). The analysis at the DEPArray™ system of such samples allows the recovery of 100% pure cells, single or in groups, from each cell type present in the mix.

The main features of the DEPArray™ Forensic Sample Prep Kit are: • Cell-specific staining of human epithelial, sperm and white blood cells; • Identification and recovery of different cell populations from complex

biological mixtures with the DEPArray™ system; • Possibility to obtain clear-cut genetic profiles from each contributor even

from minimal amounts of biological evidence by routine DNA identification methods.


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9. How to Analyze DEPArray™ Forensic Sample Prep Kit Products Biological samples prepared according to DEPArray™ Forensic Sample Prep Kit procedure are intended for DEPArray™ analysis using the DEPArray™ System and cartridges. See DEPArray™ User’s Manual V2 or DEPArray™ NxT User’s Manual for further instructions. To obtain genetic analysis of isolated cells, it is strongly advisable to use a single-tube cell lysis method, in order to avoid template loss; for this purpose, it is recommended to use DEPArray™ LysePrep Kit (REF DALYS), whose lysis reagents have to be added in the same tube where cells have been collected.

10. DEPArray™ Forensic Sample Prep Kit Downstream Applications Cells prepared according to DEPArray™ Forensic Sample Prep Kit procedure and isolated with the DEPArray™ system are compatible with standard DNA-based human identification kits, instrumentations, and software from a variety of suppliers. Internally validated and suggested downstream procedure includes: STR multiplex PCR amplification to be performed directly in the same tube in

which cells have been recovered and lysed. The following methods have been tested and validated: AmpFlSTR® NGM SElect™ PCR Amplification Kit and PowerPlex® Fusion 6C System, followed by Capillary electrophoresis (Applied Biosystems 3500/3500xL Genetic Analyzers). For data collection and analysis we recommend GeneMapper® ID-X Software.

NGS Analysis (e.g. Ion PGM™ System, Illumina®MiSeq FGx).

11. Sample Specifications

The DEPArray™ Forensic Sample Prep Kit has been validated for the identification and recovery of pure homogeneous pools of cells with the DEPArray™ starting from forensic biological mixtures [1].

The procedure is optimized for forensic biological evidences adsorbed on swabs; the analysis of evidence on different supports may require specific adaptations of the procedure.

When possible, collect the evidence using swabs. COPAN 4N6FLOQSwabs™ Genetics are recommended for a more efficient cell extraction yield. Store samples at +2…+8°C for a better cell preservation.

The use of PBS instead of water to wet the swab for evidence collection is mandatory to avoid cytolysis.

Mock samples processing is recommended to obtain expertise with DEPArray™ Forensic Sample Prep Kit procedure and DEPArray™ analysis.

Please contact Menarini Silicon Biosystems Customer Support to request assistance for the analysis of evidence not collected with the validated swabs.


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12. What to Do Before Starting

1. Working Area Organization

The DEPArray™ Forensic Sample Prep Kit has been optimized to enable the identification and separation of distinct cell types in forensic biological mixtures. In order to prevent any contamination (from the lab environment or the operators), it is strongly recommended to: Wear nitrile/powder-free gloves for all protocol steps, and use only newly

opened packages of plastic ware (e.g. reaction tubes and pipet tips). Do not use autoclaved plastic ware.

Set up a separate, dedicated laboratory or working space for sample preparation and organize it with dedicated materials such as a biohazard hood, a thermomixer, a swinging-bucket rotor, a vortex, lab coats, a +2…+8°C fridge, a -25…-15°C freezer, etc.

Use only barrier tips: Eppendorf epT.I.P.S. LoRetention Dualfilter tips are strongly suggested to avoid cell loss; the use of 1000μl and 20μl or 5000μl and 20μl epT.I.P.S. LoRetention Dualfilter tips is also mandatory for the loading of DEPArray™ cartridges v2.0 and NxT respectively.

Perform downstream analysis (e.g., genotyping, NGS, etc) in a separate laboratory space using dedicated equipment and materials: this separation of spaces is crucial in order to avoid carryover of amplified DNA.

2. Pipetting Tips All pipetting must be carried out under a dedicated hood. The use of a

biohazard hood is recommended when handling cellular samples. After centrifugation steps, the DEPArray™ Forensic Sample Prep Kit procedure

requires the removal of the supernatant down to very small volumes: to avoid cell loss, always be sure to aspirate the supernatant following the buffer meniscus with the tip to avoid cell pellet resuspension and inadvertent removal.

3. Recommendation and suggestion

In order to ensure proper handling time and efficient execution of the procedure, it is advisable to prepare in parallel up to two biological samples, according to the instruction provided in this manual. The preparation of more than two samples is at the user's discretion and should be appropriately validated.


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13. DEPArray™ Forensic Sample Prep Kit Procedure

DEPArray™ Forensic Sample Prep Kit procedure overview DEPArray™ Forensic Sample Prep Kit is suitable for different procedures depending on the nature of the sample intended to process. Follow 13.a Epithelial Cells/ Sperm Cells Mix Sample Preparation if processing a Sperm Cells/Epithelial Cells mixed sample; otherwise, follow 13.b Epithelial Cells/Sperm Cells/White Blood Cells Mix Sample Preparation if presence of blood is expected or suspected in the sample (Fig. 1).

Fig. 1 DEPArray™ Forensic Sample Preparation procedure decision tree

DEPArray™ Forensic Sample Prep Kit procedure

Before starting, make sure that all the samples meet the requirements described in section 11 “Sample Specifications”, and that the working area is properly sterilized and equipped as described in section 12 “What to do before starting”.


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13.a Epithelial Cells/Sperm Cells Mix Sample Preparation

The DEPArray™ Forensic Sample Prep Kit procedure for Sperm/Epithelial Cells Mix is divided in five macro-steps, as shown in Fig. 2.

Fig. 2 DEPArray™ Forensic Sample Prep Kit procedure for Sperm Cells/Epithelial Cells Mix


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Before Starting

a. Allow an aliquot of Running Buffer to equilibrate at room temperature (10-15 minutes).

b. Clean and sterilize scissors and tweezers (UV radiation, 10’ is recommended) before starting sample preparation. Keep the sterilized instrument under the biological hood during the entire sample preparation to avoid contamination.

Scissors cleaning and sterilization is mandatory prior to each sample preparation.

Note: When processing more than one sample at the same time, remember to use distinct instruments; as an alternative if one single pair of scissors is available, remember to clean it before using with each different sample at each step to avoid cross-contamination.


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Step 1:

Sample Incubation

1.1 Label a 15ml clean polypropylene conical tube on both the side and the cap. 1.2 Add 8ml of Running Buffer to the pre-labelled tube. 1.3 Open the swab tube and insert the swab into the 15ml tube prepared in Step 1.2. Seal the cap with a Parafilm® strip. Discard the original swab tube. Do not throw the 15ml tube cap away.

If using a type of swab with shafts that do not fit the 15ml tube in length, cut the shaft and fix it to the top of the 15ml tube using Parafilm®. Always be sure the swab head is completely immersed into the buffer volume prior to fixing the shaft; if necessary, add additional Running Buffer into the tube.

1.4 Place the sample tube in the middle of the thermomixer plate; incubate the sample at 300 rpm for 30 minutes at room temperature.


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Step 2:

Cells Collection

2.1 Label a clean 1,5ml microcentrifuge tube and insert a spin basket inside it.

2.2 Remove the sample tube from the thermomixer plate. Remove the Parafilm® strip around the tube cap, open the cap and extract the swab.

Do not throw the 15ml tube away. 2.3 Place the swab head into the spin basket inserted into the 1,5ml tube from Step 2.1 and break the stem taking advantage of the breakpoint on the shaft (breakpoints are designed to be compatible with standard 1,5ml tubes).

When using swabs lacking of a breakpoint, cut the shaft close to the swab head using a pair of sterilized scissors, and let the swab head fall into the spin basket.

2.4 Set the pipet to 200μl of volume. Lift up the swab head using sterilized tweezers and add 200μl of Running Buffer directly onto the swab head, taking care to keep it right above the tube.

Do not place down the swab head into the spin basket: using the same pipette tip, set the pipet to 100μl of volume and collect the buffer from the bottom of the spin basket; wash the sample by pipetting ten times the buffer onto the evidence, keeping it lifted up with the tweezers.

2.5 Close the 1,5ml sample tube. Fill the 15ml sample tube with Running Buffer, and close it using its own cap kept from Step 1.3. 2.6 Centrifuge both sample tubes at 200g for 30 minutes at 22°C in a swinging-bucket rotor.

During this time: - Thaw a single-use 50μl aliquot of Perm Buffer - Allow water to equilibrate at room temperature; - Label one 1,5ml microcentrifuge tube for Perm Buffer dil. 1:10; - Label one 1,5ml microcentrifuge tube for Sample Prep Buffer/Perm Buffer Mix.

2.7 Discard the swab head and the spin basket from the 1,5ml sample tube. 2.8 Gently aspirate and remove the supernatant from the 15ml sample tube without disturbing the cell pellet: use a 10ml serological pipette to aspirate the liquid down to the 2ml marked line; then use 1000μl and 200μl pipette tips to progressively remove the liquid volume. Leave approximately 100μl of liquid in the tube.

Always be careful to gently aspirate the supernatant following the buffer


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meniscus with the tip, to avoid pellet resuspension.

2.9 Using a 200μl pipette tip, gently aspirate and remove the supernatant from the 1,5ml sample tube without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.

Always be careful to gently aspirate the supernatant following the buffer meniscus with the tip, to avoid pellet resuspension.

2.10 In order to combine the two pellets, resuspend the pellet in the 15ml sample tube with

a 200μl pipette tip and transfer the sample into the 1,5ml sample tube.

Note: to prevent cell loss, keep the pipette tip to use it in the next step.

2.11 To minimize cell loss, add 50μl of Running Buffer to the bottom of the 15ml sample tube using a clean tip, without touching the tube walls. Discard the tip. Wash the bottom of the tube by gently resuspending 5 times the buffer with the same tip used in the previous step, then transfer the volume into the 1,5ml sample tube.


2.12 Repeat the washing Step 2.11, and then discard the 15ml tube. 2.13 Close the 1,5ml sample tube and discard the tip.

2.14 Centrifuge the sample at 300g for 5 minutes at 22°C in a swinging-bucket rotor.

During this time, prepare Sample Prep Buffer/Perm Buffer Mix, needed in Step 3.2: - Mix well the thawed Perm Buffer vial content by vortexing, briefly spinning down and pipette the entire volume until complete resuspension: the solution must be clear, with no visible floating particles or sedimentation; - Dilute Perm Buffer 1:10 in water: dispense 90μl of water into the pre-labelled Perm Buffer dil. 1:10 tube, add 10μl of Perm Buffer and mix well the entire volume. - Dispense 40μl of Sample Prep Buffer into the pre-labelled Sample Prep Buffer/Perm Buffer Mix tube, add 1μl of diluted Perm Buffer and mix well the entire volume ten times. - Store Sample Prep Buffer/Perm Buffer Mix at room temperature for Step 3.2.

2.15 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately up to 20μl of volume in the tube.


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Step 3:

Immuno-fluorescent staining- epithelial and sperm cells

3.1 Adjust the sample volume to 20μl with PBS 1X: add the complementary volume with a clean tip, dispensing it onto the meniscus of the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire volume ten times using the same tip previously used to resuspend the pellet. Discard the tip. 3.2 Add 41μl of Sample Preparation Buffer/Perm Buffer Mix to the sample, set the pipette to 60μl and mix well the cell suspension by gently pipetting ten times.


Note: from this moment on, protect the sample from light

3.3 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering with aluminum foil. 3.4 Using clean pipette tips, add 40μl of Blocking Buffer and 1μl of Nuclear Marker to the sample by dispensing them onto the meniscus of the liquid; discard the tips. Set the pipette to 100μl of volume and mix well the cell suspension using the same tip used in the previous step by gently pipetting ten times.


3.5 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering it with aluminum foil.

During this time, thaw one aliquot of Fix Buffer needed in Step 4.3. Place it at room temperature in the dark, covering it with aluminum foil.

During this time, if interested in ECs staining, centrifuge ECs Marker at +4°C for 5 minutes at maximum speed in a refrigerated microcentrifuge for Step 3.6.

3.6

NOTE: if not interested in epithelial cells staining do not add Epithelial Cells marker; if not interested in sperm cells staining do not add Sperm Cells marker. Complement the missing volume with an equal volume of Running Buffer (24 or 40μl respectively)

Using a clean pipette tip, add 40μl of Sperm Cells Marker and 24μl of Epithelial Cells Marker to the sample by dispensing them onto the meniscus of the liquid; discard the tip. Set the pipette to 160μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times, then discard the tip.

Aspirate the Epithelial Cells Marker from the meniscus of the liquid.


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3.7 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering with aluminum foil. 3.8 Stop the reaction by adding 1ml of Running Buffer to the sample tube without touching the liquid or resuspending. 3.9 Centrifuge the sample at 300g for 15 minutes at 22°C in a swinging-bucket rotor.

If proceeding with DEPArray™ analysis immediately at the end of sample preparation, during this time thaw one vial of SB115 buffer.

3.10 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately 20μl of volume in the tube.

Always be careful to gently aspirate the supernatant following the liquid meniscus with the tip to avoid cell resuspension.


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Step 4:

Fixation

4.1 Add 550μl of Running Buffer with a clean tip. Resuspend well the cell suspension by gently pipetting the entire volume at least ten times and measure the exact sample volume.

Note: to prevent cell loss, keep the pipette tip to use it in the next step. 4.2 Adjust the measured sample volume to 600μl with Running Buffer: add the complementary volume with a clean tip, dispensing it onto the tube wall close to the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire volume ten times with the same tip used in the previous step to resuspend the pellet.


4.3 Using a clean pipette tip, add 200μl of Fix Buffer to the sample tube, dispensing it onto the tube wall close to the liquid without touching it; discard the tip. Set the pipette to 800μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times. Discard the tip. 4.4 Incubate the sample for 10 minutes at room temperature in the dark, covering with aluminum foil. 4.5 Stop the reaction by adding 700µl of PBS 1X to the sample tube without touching the liquid or resuspending. 4.6 Centrifuge the sample at 350g for 10 minutes at 22°C in a swinging-bucket rotor. 4.7 Gently aspirate and remove the supernatant as possible without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.


4.8 If proceeding with DEPArray™ analysis, continue from step 5.1. For sample storage, add 50µl of Running Buffer to the sample, resuspend ten times then store the sample at +2…+8°C protected from light until processing. We suggest to process samples within few days from preparation in order to avoid cell clumping. When processing, continue from Step 5.1 to end.


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Step 5:

Washing

before DEPArray™ Analysis

5.1 Add 1ml of SB115 buffer to the sample tube: energetically dispense the buffer towards the side of the tube avoiding direct contact with the cell pellet. 5.2 Centrifuge the sample at 350g for 10 minutes at 22°C in a swinging-bucket rotor. 5.3 Gently aspirate and remove the supernatant as possible without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.


5.4 Repeat Steps 5.1 and 5.2

During this time:

FOR DEPARRAY™ NxT analysis - Label three 1,5ml microcentrifuge tubes for SB115 buffer - Aliquot 1ml of SB115 buffer into each tube - Close the tubes and wrap them with Parafilm® - Sonicate the SB115 aliquots for 10 minutes in the water bath sonicator - Aspirate the sonicated SB115 buffer aliquots from each tube and collect them into a single 50ml tube. Be careful to avoid the formation of bubbles

FOR DEPARRAY™ v2.0 analysis - Label one 1,5ml microcentrifuge tube for SB115 buffer - Aliquot 1ml of SB115 buffer into the tube - Close the tube and wrap it with Parafilm® - Sonicate the SB115 aliquot for 10 minutes in the water bath sonicator

5.5 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately 10μl of volume in the tube (use the round mark at the bottom of the 1,5ml tube as a reference for 10μl volume).

Always be careful to gently aspirate the surnatant following the fluid meniscus with the tip to avoid cell resuspension.

5.6 Resuspend well the pellet by gently pipetting the cell suspension several times and measure the exact sample volume.

Note: to prevent cell loss, keep the pipette tip to use it in the next step. 5.7 Adjust the sample volume with SB115 buffer for the DEPArray™ cartridge loading according to the instructions sheet of the DEPArray™ cartridge: add the complementary volume plus 1 microliter extra for cell count with a clean tip, dispensing it onto the meniscus of the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire


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volume ten times with the same tip used in the previous step to resuspend the pellet. 5.8 Count cells to assess the sample concentration prior DEPArray™ analysis using Forensic Application (e.g.: dilute 1μl of sample into 9μl of SB115 and count the cells using a Burker Chamber). Load up to maximum 6000 cells into the DEPArray™ NxT cartridge; load up to 6000 cells into the DEPArray™ A300K DS V2.0 cartridge.

Note: Avoid the formation of bubbles when pipetting to ensure further efficient sample loading into the DEPArray™ cartridge.


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13.b White Blood Cells/Epithelial Cells/Sperm Cells Mix Sample Preparation

The DEPArray™ Forensic Sample Prep Kit procedure for White Blood Cells/Epithelial Cells/Sperm Cells Mix is divided in six macro-steps, as shown in Fig. 3.

Fig. 3 DEPArray™ Forensic Sample Prep Kit procedure for White Blood Cells/Epithelial Cells/Sperm Cells Mix


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Before starting

a. Allow an aliquot of Running Buffer to equilibrate at room temperature (10-15 minutes).

b. Clean and sterilize scissors and tweezers (UV radiation, 10’ is recommended) before starting sample preparation. Keep the sterilized instrument under the biological hood during the entire sample preparation to avoid contamination.

Scissors cleaning and sterilization is mandatory prior to each sample preparation.

Note: When processing more than one sample at the same time, remember to use distinct instruments; as an alternative if one single pair of scissors is available, remember to clean it before using with each different sample at each step to avoid cross-contamination.


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Step 1:

Sample Incubation

1.1Label a 15ml clean polypropylene conical tube on both the side and the cap. 1.2 Add 8ml of Running Buffer to the pre-labelled tube. 1.3 Open the swab tube and insert the swab into the 15ml tube prepared in Step 1.2. Seal the cap with a Parafilm® strip. Discard the original swab tube. Do not throw the 15ml tube cap away.

If using a type of swab with shafts that do not fit the 15ml tube in length, cut the shaft and fix it to the top of the 15ml tube using Parafilm®. Always be sure the swab head is completely immersed into the buffer volume prior to fixing the shaft; if necessary, add additional Running Buffer into the tube.

1.4 Place the sample tube in the middle of the thermomixer plate; incubate the sample at 300 rpm for 30 minutes at room temperature.

During this time, centrifuge the WBCs Marker at maximum speed for 5 minutes at 4°C in a refrigerated microcentrifuge for Step 4.3. Store at +2…+8° afterwards.


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Step 2:

Cells Collection

2.1 Label a clean 1,5ml microcentrifuge tube and insert a spin basket inside it.

2.2 Remove the sample tube from the thermomixer plate. Remove the Parafilm® strip around the tube cap, open the cap and extract the swab.

Do not throw the 15ml tube away. 2.3 Place the swab head into the spin basket inserted into the 1,5ml tube from Step 2.1 and break the stem taking advantage of the breakpoint on the shaft (breakpoints are designed to be compatible with standard 1,5ml tubes).

When using swabs lacking of a breakpoint, cut the shaft close to the swab head using a pair of sterilized scissors, and let the swab head fall into the spin basket.

2.4 Set the pipet to 200μl of volume. Lift up the swab head using sterilized tweezers and add 200μl of Running Buffer directly onto the swab head, taking care to keep it right above the tube.

Do not place down the swab head into the spin basket: using the same pipette tip, set the pipet to 100μl of volume and collect the buffer from the bottom of the spin basket; wash the sample by pipetting ten times the buffer onto the evidence, keeping it lifted up with the tweezers.

2.5 Close the 1,5ml sample tube. Fill the 15ml sample tube with Running Buffer, and close it using its own cap kept from Step 1.3. 2.6 Centrifuge both sample tubes at 200g for 30 minutes at 22°C in a swinging-bucket rotor.

Note: during this time, thaw one Fix Buffer vial. Place it at room temperature in the dark, covering it with aluminum foil.

2.7 Discard the swab head and the spin basket from the 1,5ml sample tube. 2.8 Gently aspirate and remove the supernatant from the 15ml sample tube without disturbing the cell pellet: use a 10ml serological pipette to aspirate the liquid down to the 2ml marked line; then use 1000μl and 200μl pipette tips to progressively reduce the liquid volume. Leave approximately 100μl of liquid in the tube.


2.9 Using a 200μl pipette tip, gently aspirate and remove the supernatant from the 1,5ml sample


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tube without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.


2.10 In order to combine the two pellets, resuspend the pellet in the 15ml sample tube with

a 200μl pipette tip and transfer the sample into the 1,5ml sample tube.


2.11 To minimize cell loss, add 50μl of Running Buffer to the bottom of the 15ml sample tube using a clean tip, without touching the tube walls. Discard the tip. Wash the bottom of the tube by gently resuspending 5 times the buffer with the same tip used in the previous step, then transfer the volume into the 1,5ml sample tube.


2.12 Repeat the washing Step 2.11, and then discard the 15ml tube. 2.13 Close the 1,5ml sample tube and discard the tip.

2.14 Centrifuge the sample at 300g for 5 minutes at 22°C in a swinging-bucket rotor. 2.15 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately 20μl of volume in the tube.


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Step 3:

Pre-staining Fixation




3.3 Using a clean pipette tip, add 60μl of Fix Buffer to the sample tube, dispensing it onto the tube wall close to the liquid without touching it; discard the tip. Set the pipette to 800μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times. Discard the tip.

Do not throw Fix Buffer vial. Keep it protected from light until Step 5.3

NOTE: from this moment on, protect the sample from light 3.4 Incubate the sample for 10 minutes at room temperature in the dark, covering with aluminum foil. 3.5 Stop the reaction by adding 700µl of PBS 1X to the sample tube without touching the liquid or resuspending. 3.6 Centrifuge the sample at 350g for 10 minutes at 22°C in a swinging-bucket rotor. 3.7 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately 20μl of volume in the tube.



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Step 4A:

Immuno-fluorescent staining– white blood cells

4.1 Add 30μl of Running Buffer to the sample, resuspend well the cell pellet by gently pipetting and measure the sample volume.

Note: to prevent cell loss, keep the pipette tip to use it in the next step. 4.2 Adjust the sample volume to 100μl with Running Buffer: add the complementary volume with a clean tip, dispensing it onto the meniscus of the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire volume ten times with the same tip used in the previous step.

Note: to prevent cell loss, keep the pipette tip to use it in the next step. 4.3 Using a clean pipette tip, add 7,5μl of White Blood Cells Marker to the sample by dispensing it onto the meniscus of the liquid; discard the tip. Set the pipette to 105μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times. Discard the tip.

Aspirate the White Blood Cells Marker from the meniscus of the liquid

4.4 Incubate the sample on the thermomixer at 300 rpm for 10 minutes in the dark at room temperature, covering with aluminum foil.

During this time: - Thaw a single-use 50μl aliquot of Perm Buffer - Allow water to equilibrate at room temperature; - Label one 1,5ml microcentrifuge tube for Perm Buffer dil. 1:10; - Label one 1,5ml microcentrifuge tube for Sample Prep Buffer/Perm Buffer Mix.

4.5 Stop the reaction by adding 1ml of Running Buffer to the sample tube without touching the liquid or resuspending. 4.6 Centrifuge the cell suspension at 350 g for 10 minutes at 22°C in a swinging-bucket rotor.

During this time, prepare Sample Prep Buffer/Perm Buffer Mix, needed in Step 4.10:


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- Mix well the thawed Perm Buffer vial content by vortexing, briefly spinning down and pipette the entire volume until complete resuspension: the solution must be clear, with no visible floating particles or sedimentation; - Dilute Perm Buffer 1:10 in water: dispense 90μl of water into the pre-labelled Perm Buffer dil. 1:10 tube, add 10μl of Perm Buffer and mix well the entire volume. - Dispense 40μl of Sample Prep Buffer into the pre-labelled Sample Prep Buffer/Perm Buffer Mix tube, add 1μl of diluted Perm Buffer and mix well the entire volume ten times. - Store Sample Prep Buffer/Perm Buffer Mix at room temperature for Step 4.10.



4.8 Resuspend well the cell pellet by gently pipetting and measure the sample volume with a 20μl tip.



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Step 4B:

Immuno-fluorescent staining-

epithelial and sperm cells

4.9 Adjust the sample volume to 20μl with PBS 1X: add the complementary volume with a clean tip, dispensing it onto the meniscus of the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire volume ten times using the same tip previously used to resuspend the pellet. Discard the tip. 4.10 Add 41μl of Sample Preparation Buffer/Perm Buffer Mix to the sample, set the pipette to 60μl and mix well the cell suspension by gently pipetting ten times.


4.11 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering with aluminum foil. 4.12 Using clean pipette tips, add 40μl of Blocking Buffer and 1μl of Nuclear Marker to the sample by dispensing them onto the meniscus of the liquid; discard the tips. Set the pipette to 100μl of volume and mix well the cell suspension using the same tip used in the previous step by gently pipetting ten times.


4.13 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering it with aluminum foil.

During this time, if interested in ECs staining, centrifuge ECs Marker at +4°C for 5 minutes at maximum speed in a refrigerated microcentrifuge for Step 4.14.

4.14

NOTE: if not interested in epithelial cells staining do not add Epithelial Cells marker; if not interested in sperm cells staining do not add Sperm Cells marker. Complement the missing volume with an equal volume of Running Buffer (24 or 40μl respectively)

Using a clean pipette tip, add 40μl of Sperm Cells Marker and 24μl of Epithelial Cells Marker to the sample by dispensing them onto the meniscus of the liquid; discard the tip. Set the pipette to 160μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times, then discard the tip.

Aspirate the Epithelial Cells Marker from the meniscus of the liquid.


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4.15 Incubate the sample on the thermomixer at 300 rpm for 15 minutes at room temperature in the dark, covering with aluminum foil. 4.16 Stop the reaction by adding 1ml of Running Buffer to the sample tube without touching the liquid or resuspending. 4.17 Centrifuge the sample at 350g for 15 minutes at 22°C in a swinging-bucket rotor.

If proceeding with DEPArray™ analysis immediately at the end of sample preparation, during this time thaw one vial of SB115 buffer.




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Step 5:

Post- staining Fixation




5.3 Using a clean pipette tip, add 140μl of Fix Buffer to the sample tube, dispensing it onto the tube wall close to the liquid without touching it; discard the tip. Set the pipette to 800μl and mix well the cell suspension with the same tip used in the previous step by gently pipetting ten times. Discard the tip. 5.4 Incubate the sample for 10 minutes at room temperature in the dark, covering with aluminum foil. 5.5 Stop the reaction by adding 700µl of PBS 1X to the sample tube without touching the liquid or resuspending. 5.6 Centrifuge the sample at 350g for 10 minutes at 22°C in a swinging-bucket rotor. 5.7 Gently aspirate and remove the supernatant as possible without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.


5.8 If proceeding with DEPArray™ analysis, continue from step 6.1. For sample storage, add 50µl of Running Buffer to the sample, resuspend ten times then store the sample at +2…+8°C protected from light until processing. We suggest to process samples within few days from preparation in order to avoid cell clumping. When processing, continue from Step 6.1 to end.


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Step 6:

Washing

before DEPArray™ Analysis

6.1 Add 1ml of SB115 buffer to the sample tube: energetically dispense the buffer towards the side of the tube avoiding direct contact with the cell pellet. 6.2 Centrifuge the sample at 350g for 10 minutes at 22°C in a swinging-bucket rotor. 6.3 Gently aspirate and remove the supernatant as possible without disturbing the cell pellet. Leave approximately 50μl of volume in the tube.


6.4 Repeat Steps 6.1 and 6.2

During this time:

FOR DEPARRAY™ NxT analysis - Label three 1,5ml microcentrifuge tubes for SB115 buffer - Aliquot 1ml of SB115 buffer into each tube - Close the tubes and wrap them with Parafilm® - Sonicate the SB115 aliquots for 10 minutes in the sonicator water bath - Aspirate the sonicated SB115 buffer aliquots from each tube and collect them into a single 50ml tube. Be careful to avoid the formation of bubbles

FOR DEPARRAY™ v2.0 analysis - Label one 1,5ml microcentrifuge tube for SB115 buffer - Aliquot 1ml of SB115 buffer into the tube - Close the tube and wrap it with Parafilm® - Sonicate the SB115 aliquot for 10 minutes in the sonicator water bath

6.5 Gently aspirate and remove the supernatant without disturbing the cell pellet. Leave approximately 10μl of volume in the tube (use the round mark at the bottom of the 1,5 tube as a reference for 10μl volume).

Always be careful to gently aspirate the surnatant following the fluid meniscus with the tip to avoid cell resuspension.

6.6 Resuspend well the pellet by gently pipetting the cell suspension several times and measure the exact sample volume.

Note: to prevent cell loss, keep the pipette tip to use it in the next step. 6.7 Adjust the sample volume with SB115 Buffer for the DEPArray™ cartridge loading according to the instructions sheet of the DEPArray™ cartridge: add the complementary volume plus 1 microliter extra for cell count with a clean tip, dispensing it onto the meniscus of the liquid then discard the tip. Mix well the cell suspension by gently pipetting the entire


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volume ten times with the same tip used in the previous step to resuspend the pellet. 6.8 Count cells to assess the sample concentration prior DEPArray™ analysis using Forensic Application (e.g.: dilute 1μl of sample into 9μl of SB115 and count the cells using a Burker Chamber). Load up to maximum 6000 cells into the DEPArray™ NxT cartridge; load up to 6000 cells into the DEPArray™ A300K DS V2.0 cartridge.

Note: Avoid the formation of bubbles when pipetting to ensure further efficient sample loading into the DEPArray™ cartridge.


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14. Patent & Trademark Information

Menarini Silicon Biosystems SpA products may not be transferred to third parties, resold, modified for resale, used to manufacture commercial products without written approval of Menarini Silicon Biosystems SpA.

DEPArray™ is a trademark of Menarini Silicon Biosystems SpA, or its subsidiaries which may be registered in certain jurisdictions. Other brands and product names are trademarks of their respective holders.

15. Warranty

This product is warranted only to be free from defects in workmanship and material at the time of delivery to the customer. Menarini Silicon Biosystems SpA makes no warranty or representation, either expressed or implied, with respect to the fitness of a product for a particular purpose. There are no warranties, expressed or implied, which extend beyond the Technical Specifications of the products. Menarini Silicon Biosystems SpA’s liability is limited to either replacement of the products or refund of the purchase price. Menarini Silicon Biosystems SpA is not liable for any property damage, personal injury or economic loss caused by the product.

Notice to Buyer/User: Information presented herein is accurate and reliable to the best of our knowledge and belief, but is not guaranteed to be so. Nothing herein is to be construed as recommending any practice or any product in violation of any patent or in violation of any law or regulation. It is the user's responsibility to determine for himself or herself the suitability of any material and/or procedure for a specific purpose and to adopt such safety precautions as may be necessary.


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16. Annex 1: Chemicals Classifications

EU Regulation 1272/08 (“CLP” Regulation) art. 28 sub. 1 and sub. 3, art. 29 sub. 2 and Annex I sub. 1.5.2.1 allow to report in the secondary label only the most important information about chemicals classification. The complete classification of the chemicals is listed below.

DEPArray™ Forensic Sample Prep Kit – Reagents, Nuclear Marker.

Contains bisBenzimide H 33342 trihydrochloride EC: 1272/2008; CAS: 23491-52-3.

Reagent (Vial)

CLP symbols

H and P Phrases Text

DEPArray™ Forensic Sample Prep Kit – Reagents, Nuclear Marker.

H302 Harmful if swallowed

H315 Causes skin irritation

H335 May cause respiratory irritation

H351 Suspected of causing cancer

P280 Wear protective gloves/ protective clothing/ eye protection/ face protection

P301 + P312 + P330

IF SWALLOWED: Call a POISON CENTER or doctor/ physician if you feel unwell. Rinse mouth

DEPArray™ Forensic Sample Prep Kit – Reagents, Water.

Classified as not dangerous under EU Regulation 1272/08.

DEPArray™ Forensic Sample Prep Kit – Reagents, Sample Prep Buffer.


DEPArray™ Forensic Sample Prep Kit – Reagents, Perm Buffer.

Contains (R*,R*)-1,4-Dimercaptobutane-2,3-diol. EC; CAS: 3483-12-3.

Reagent (Vial)

CLP symbols


DEPArray™ Forensic Sample Prep Kit – Reagents, Perm Buffer

H302 Harmful if swallowed

H315 Causes skin irritation

H319 Causes serious eye irritation

P261 Avoid breathing

P262 Do not get in eyes, on skin, or on clothing

P270 Do not eat, drink or smoke when using this product

P302 +P352 IF ON SKIN; wash with plenty of soap and water

P305+P351+P338 IF IN EYES; Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing


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DEPArray™ Forensic Sample Prep Kit – Reagents, Blocking Solution.


DEPArray™ Forensic Sample Prep Kit – Reagents, Fix Buffer.

Contains Paraformaldehyde EC: EC: 1272/2008; CAS: 30525-89-4.

Reagent (Vial)

CLP symbols


DEPArray™ Forensic Sample Prep Kit – Reagents, Fix Buffer

H302 H312

Harmful if swallowed or if in contact with skin

H315 Harmful in contact with skin

H317 May cause an allergic skin reaction

H318 Causes serious eye damage

H335 May cause respiratory irritation

H351 Suspected of causing cancer.

P210 Keep away from heat/sparks/open flames/hot surfaces. - No smoking

P261 Avoid breathing dust

P280 Wear protective gloves/ eye protection/ face protection

P305 + P351 + P338

IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing

DEPArray™ Forensic Sample Prep Kit – Epithelial Cells, ECs marker.


DEPArray™ Forensic Sample Prep Kit – Sperm Cells, SCs marker.


DEPArray™ Forensic Sample Prep Kit – White Blood Cells, WBCs marker.



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17. Appendix A: CellBrowser™ Cell Images

In the images below, examples of immunofluorescent staining are reported for each cell type: epithelial cells (ECs) are identifiable in FITC fluorescence channel (Fig. 1a, top); sperm cells (SCs) are identifiable in APC fluorescence channel (Fig. 1b, middle); white blood cells (WBCs) are identifiable in PE fluorescence channel (Fig. 1c,

bottom). In addition to the specific immunofluorescent staining, all cell types are detectable thanks to the nuclear staining (DAPI fluorescence channel) and distinguishable for their characteristic morphology.

Fig. 2 Image Gallery cells identified at the DEPArray™ System.

a) FITC+ Epithelial Cells. Epithelial cells morphology, typically tapered, can be variable but

always clearly distinguishable in BF (variable dimension, typically 20-60mm).

b) APC+ Sperm Cells. In addition to the immunofluorescent staining, the typical sperm

morphology can be observed in BF (diameter 5-7mm).

c) PE+ White Blood Cells. White blood cells normally appear translucent and rounded when

observed in BF (diameter 7-15mm).


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In order to prevent cell recovery contamination, it is very important to recognize heterogeneous clusters (i.e. cell aggregates composed by two or more different cell types). The specific immunofluorescent staining obtained by the DEPArray™ Forensic Sample Prep Kit, combined with cell morphological observation from the CellBrowser™ image gallery, enables the identification of heterogeneous clusters as shown in Fig. 2, which should be discarded.

Fig. 2 Image Gallery of heterogeneous cell clusters identified at the DEPArray™ System.

a) FITC+/APC+/PE- heterogeneous epithelial/sperm cell cluster.

b) FITC+/PE+/APC- heterogeneous epithelial/white blood cell cluster.

c) PE+/APC+/FITC- heterogeneous white blood cell/sperm cell cluster WBC/SC cluster.

Cytokeratin is reported to be an Epithelial Cell marker showing different levels of

expression among cells deriving from distinct Epithelial tissues (e.g. Epithelial Cells present in saliva rather than in vaginal fluid) as well as cells deriving from the same Epithelial tissue (e.g. Epithelial Cells coming from different areas of the oral mucosa present in saliva) [2]. Therefore, Cytokeratin epithelial staining efficacy may result highly variable among different samples. Nevertheless, Epithelial Cells remain still clearly detectable at the DEPArray™ System by means of bright nuclear staining and identifiable by their characteristic morphology.


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Reference [1] Fontana F et al. Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach. Forensic Sci Int Genet; 29:225-241 (2017) doi: 10.1016/j.fsigen.2017.04.023. [2] Oda B, Dale BA, Bourekis G. Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival cells. In Vitro Cell Dev Biol.; 26(6):596-603 (1990).

https://www.ncbi.nlm.nih.gov/pubmed/28511094

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Notes

deparray™ forensic sample prep kit - arinahayat.com · deparray™ forensic sample prep kit 4 2....

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