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Standard Operating Procedures

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Home Standard Operating Procedures Clinical Protocol I
** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Arterial or mixed venous blood will be collected from patients at the indicated time points following randomization.

The collection of blood should be obtained from an existing arterial or venous line, or by venipuncture (with a needle 20 gauge or larger), and should be performed by someone experienced in the technique and familiar with infectious precautions.

The use of pneumatic tube system for transfer of the collected blood to the laboratory is not recommended since it can activate white blood cells and promote unwanted release of mediators.

Processing of the blood should be done as soon as possible after collection in order to prevent the unwanted released of mediators caused by dying cells.

Specimen Collection & Handling

  1. Label all citrated plasma (CPT) tubes, with matching a small sterile polypropylene tube and 6 cryogenic freezing tubes per patient/ sample collection.
  2. A minimum of 4mL of whole blood is collected into a CPT tube. Inverted several times to mix the blood with the contained anticoagulant.
  3. Place the tube immediately on ice.
  4. In laboratory, invert the CPT tube once to mix. Centrifuge at 1700 x g (calculate the speed in rpm taking into account the size of your rotor) for 10 minutes at 4 C with brake off, in a horizontal, swinging bucket centrifuge.
  5. Carefully collect the plasma using a disposable plastic pipette or sterile glass pipette from the CPT tube and transfer into a new labeled 15mL conical tube being sure not to withdraw any of the white interfacial layer.
  6. Discard the pellet.
  7. Aliquot plasma into 2mL cryogenic freezing tubes by placing 250μL of plasma per tube. Store at ≤ –70°C until processing.

Special Note 1.1: only collect the plasma fraction to within 0.2mL of the interface layer. Note that after centrifugation you should have approximately 2mL of supernatant per tube so transfer only approximately 1.5mL of plasma.

Special Note 1.2: Processing and handling of Blood for analysis – please remember endotoxin is ubiquitous and can change expression of all mediators being assayed. Endotoxin free precautions should be taken for handling of all procedures (sterile precautions and using endotoxin free solutions should suffice)

Supplies

  • CPT tubes containing sodium citrate (Becton-Dickinson, #362760) – will accommodate 4.0mL.
  • 15 ml sterile polypropylene conical tubes (Falcon/Becton-Dickinson, #35-2097)
  • 2ml cryogenic freezing tubes (Corning, #430289) Internal thread
  • Sterile, endotoxin-free 10ml serological pipets (VWR, #53283- 740)
  • Sterile glass pasteur pipets (VWR, #14672-410)
  • Rubber bulbs for pasteur pipets (VWR, #56311-062)
  • P1000 pre-sterilized, pipet tips (VWR, # 53508-830)
  • Insulated Styrofoam box (VWR, #15713-5~9)
  • Ice bucket

Equipment

  • Clinical Centrifuge with swinging-bucket rotor.
  • Sterile tissue-culture hood, BSL-2 or equivalent
  • Laboratory pipetter capable of delivering 1.0mL of liquid (eg., Rainin P-I 000)
  • Ultra low temperature -70°C freezer
  • Access to a sterilizing autoclave

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Arterial or mixed venous blood will be collected from patients at the indicated time points following randomization.

The collection of blood should be obtained from an existing arterial or venous line, or by venipuncture (with a 20 gauge needle or larger), and should be performed by someone experienced in the technique and familiar with infectious precautions.

The use of pneumatic tube system is not recommended since it can activate white blood cells and promote unwanted release of mediators.

Processing of the blood should be done as soon as possible after collection in order to prevent the unwanted released of mediators caused by dying cells.

Specimen Collection & Handling

  1. For each patient, label two 15mL polystyrene collection tubes containing the following:
    • Tube #1 contains 360μL of sterile trisodium citrate (= “CIT” tube)
    • Tube #2 contains 360μL of sterile trisodium citrate + 80μl of 1M sterile benzamidine (=“CIT/BZ” tube)

      ** Please note that the sterile trisodium citrate can be obtained from the blue-topped Becton Dickinson vacutainer tubes (BD cat. No.366415). Each vacutainer tube should contain about 500μl of trisodium citrate, but the actual amount that can be removed is closer to 450μL.
  2. Draw 2 tubes of 4mL each of blood. IMMEDIATELY transfer the content of one tube into the “CIT” tube and the content of the other tube into the “CIT/BZ” tube.
  3. In laboratory, invert the tubes once to mix. Centrifuge at 1700 x g (calculate the speed in rpm taking into account the size of your rotor) for 10 minutes at 4 C with brake off, in a horizontal, swinging bucket centrifuge.
  4. Using a plastic transfer pipette, transfer each plasma into 8 cryogenic freezing tubes (ie. 16 cryogenic freezing tubes in total for each patient).
  5. Write either “CIT” or “CIT/BZ” on the tops of the cryogenic freezing tubes, and also write the patient ID number and the “day” (for example, “J1, Day 1").
  6. Store the plasma samples in a -80oC freezer in cardboard boxes or in Ziploc baggies (use one Ziploc bag per patient day).

Special Note 1.1: only collect the plasma fraction to within 0.2mL of the interface layer. Note that after centrifugation you should have approximately 2mL of supernatant per tube so transfer only approximately 1.5mL of plasma.

Special Note 1.2: Processing and handling of Blood for analysis – please remember endotoxin is ubiquitous and can change expression of all mediators being assayed.

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Arterial or mixed venous blood will be collected from patients at the indicated time points following randomization.

The collection of blood should be obtained from an existing arterial or venous line, or by venipuncture (with a 20 gauge needle or bigger), and should be performed by someone experienced in the technique and familiar with infectious precautions.

The use of pneumatic tube system is not recommended since it can activate white blood cells and promote unwanted release of mediators.

Processing of the blood should be done as soon as possible after collection in order to prevent the unwanted released of mediators caused by dying cells.

Specimen Collection & Handling

  1. Label all 1 BD serum tube (no. 367812), 1x15mL conical tube and 6 cryogenic freezing tubes per patient.
  2. Fill the tube with whole blood in the identified collection tube. Inverted gently several times to mix the blood with the contained pro-coagulant.
  3. Allow the blood to clot by standing tube vertically at room temperature (22 oC) for 60 minutes.
  4. Place the tube in wet ice for no longer than 2 hours before centrifuging.
  5. Centrifuge at 1200 x g (calculate the speed in rpm taking into account the size of your rotor) for 10 minutes at 4 C with brake off, in a horizontal, swinging bucket centrifuge.
  6. Carefully collect the serum using a disposable plastic pipette or sterile glass pipette from the vacutainer and transfer it into a new labeled 15mL conical tube making sure not to withdraw any of the white interfacial layer.
  7. Discard the pellet.
  8. Aliquot serum into 2mL cryogenic freezing tubes by placing 250μL of serum per tube. Store at ≤ –70°C until processing.

Special Note 1.1: only collect the serum fraction to within 0.2mL of the interface layer. Note that after centrifugation you should have approximately half the volume of blood as supernatant per tube.

Special Note 1.2: Processing and handling of Blood for analysis – please remember endotoxin is ubiquitous and can change expression of all mediators being assayed. Endotoxin free precautions should be taken for handling of all procedures (sterile precautions and using endotoxin free solutions should suffice).

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Arterial or mixed venous blood will be collected from patients at the indicated time points following randomization.

The collection of blood should be obtained from an existing arterial or venous line, or by venipuncture (with a needle 20 gauge or larger), and should be performed by someone experienced in the technique and familiar with infectious precautions.

The use of pneumatic tube system for transfer of the collected blood to the laboratory is not recommended since it can activate white blood cells and promote unwanted release of mediators.

Processing of the blood should be done as soon as possible after collection in order to prevent the unwanted released of mediators caused by dying cells.

Specimen Collection & Handling

  1. Label all Stabilyte evacuated tubes (Biopool tubes), a 15mL conical tube and 6 cryogenic freezing tubes per patient/ sample collection.
  2. A minimum of 4mL of whole blood is collected into the Stabilyte tube. Gently inverted several times to mix the blood with the contained anticoagulant.
  3. In laboratory, invert the Stabilyte tube once to mix. Centrifuge at 3000 x g (calculate the speed in rpm taking into account the size of your rotor) for 15 minutes at 4 C with brake off, in a horizontal, swinging bucket centrifuge.
  4. Carefully collect the plasma using a disposable plastic transfer pipette from the Stabilyte tube and transfer into a new labeled 15mL conical tube being sure not to withdraw any of the white interfacial layer.
  5. Discard the pellet.
  6. Aliquot plasma into 2mL cryogenic freezing tubes by placing 250μL of plasma per tube. Store at ≤ –70°C until processing.

Special Note 1.1: only collect the plasma fraction to within 0.2mL of the interface layer. Note that after centrifugation you should have approximately 2mL of supernatant per tube so transfer only approximately 1.75mL of plasma.

Special Note 1.2: Processing and handling of Blood for analysis – please remember endotoxin is ubiquitous and can change expression of all mediators being assayed. Endotoxin free precautions should be taken for handling of all procedures (sterile precautions and using endotoxin free solutions should suffice)

Supplies

  • Stabilyte evacuated tubes (Biopool, cat nb 102080) – will accommodate 4.0ml.
  • 15mL sterile polypropylene conical tubes (Falcon/Becton-Dickinson, #35-2097)
  • 2ml cryogenic freezing tubes (Corning, #430289) Internal thread
  • Sterile, endotoxin-free 10ml serological pipets (VWR, #53283- 740)
  • Disposable Transfer Pipets, Sterile, Samco* Scientific (VWR, #14670-003)
  • P1000 pre-sterilized, pipet tips (VWR, # 53508-830)
  • Insulated Styrofoam box (VWR, #15713-5~9)
  • ice bucket

Equipment

  • Clinical Centrifuge with swinging-bucket rotor.
  • Sterile tissue-culture hood, BSL-2 or equivalent
  • Laboratory pipetter capable of delivering 1.0mL of liquid ( eg., Rainin P-I 000)
  • Ultra low temperature -70°C freezer
  • Access to a sterilizing autoclave

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Cerebrospinal fluid (CSF) will be collected from patients at the indicated time points following randomization.

The collection of CSF should be obtained from an existing line, under sterile conditions, and should be performed by someone experienced in the technique and familiar with infectious precautions.

The use of pneumatic tube system for transfer of the collected CSF to the laboratory is not recommended since it can activate white blood cells and promote unwanted release of mediators.

Processing of the CSF should be done as soon as possible after collection in order to prevent the unwanted released of mediators caused by dying cells.

Specimen Collection & Handling

  1. Label all Stabilyte evacuated tubes (Biopool tubes), a 15mL conical tube and 6 cryogenic freezing tubes per patient/ sample collection.
  2. A minimum of 4mL of CSF is collected, drop by drop, directly into the Stabilyte tube. Gently inverted several times to mix the CSF with the contained anticoagulant.
  3. In laboratory, invert the Stabilyte tube once to mix. Centrifuge at 3000 x g (calculate the speed in rpm taking into account the size of your rotor) for 15 minutes at 4 C with brake off, in a horizontal, swinging bucket centrifuge.
  4. Carefully collect the CSF using a disposable plastic transfer pipette from the Stabilyte tube and transfer into a new labeled 15mL conical tube being sure not to withdraw any of the red and white blood cells interfacial layers.
  5. Discard the pellet.
  6. Aliquot CSF into 2mL cryogenic freezing tubes by placing 250μL of plasma per tube. Store at ≤ –70°C until processing.

Special Note 1.1: only collect the CSF fraction to within 0.2mL of the interface layer. Note that after centrifugation you should have approximately 2mL of supernatant per tube so transfer only approximately 1.75mL of CSF.

Special Note 1.2: Processing and handling of CSF for analysis – please remember endotoxin is ubiquitous and can change expression of all mediators being assayed. Endotoxin free precautions should be taken for handling of all procedures (sterile precautions and using endotoxin free solutions should suffice)

Supplies

  • Stabilyte evacuated tubes (Biopool, cat nb 102080) – will accommodate 4.0mL.
  • 15mL sterile polypropylene conical tubes (Falcon/Becton-Dickinson, #35-2097)
  • 2ml cryogenic freezing tubes (VWR, #89092-262) Internal thread
  • Sterile, endotoxin-free 10mL serological pipets (VWR, #53283- 740)
  • Disposable Transfer Pipets, Sterile, Samco* Scientific (VWR, #14670-003)
  • P1000 pre-sterilized, pipet tips (VWR, # 53508-830)
  • Insulated Styrofoam box (VWR, #15713-5~9)
  • ice bucket

Equipment

  • Clinical Centrifuge with swinging-bucket rotor.
  • Sterile tissue-culture hood, BSL-2 or equivalent
  • Laboratory pipetter capable of delivering 1.0mL of liquid (eg., Rainin P-I 000)
  • Ultra low temperature -70°C freezer
  • Access to a sterilizing autoclave

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

The purpose of this procedure is to obtain total cellular RNA from human whole blood for the subsequent analysis of mRNA populations, either by the polymerase chain reaction (following reverse transcription) or by microarray analyses. This procedure does not contain an initial separation of leukocytes, but rather relies on the denaturing of proteins and precipitation of RNA/DNA from whole blood directly. Following lysis, total cellular RNA is removed from DNA and proteins by precipitation and chromatographic separation. A more detailed description of the principle and protocols can be found in the accompanying documentation from the PAXgene Blood RNA Tube Circular and the PAXgene Blood RNA Kit Handbook.

It is essential that prior to the use of the PAXgene system for collecting blood, the operator has fully read the PAXgene Blood RNA Handbook and understands the procedures and their potential risks to the operator and the patient.

METHOD

N.B. All centrifugations are done at room temperature.

  1. Arterial or venous whole blood is collected into a room temperature (18-25o C) PAXgene Blood RNA Tube (PAXgene, PreAnalytiX Inc., Hombrehtikon, Switzerland, distributed by QIAGEN, cat# 762115). Allow at least 10 seconds for a complete blood draw to take place.

    The collection of blood should be obtained from an existing arterial or venous line, or venipuncture should be performed by someone experienced in the technique and familiar with infectious precautions. The PAXgene Blood RNA tube is held vertically, below the donor’s arm, during blood collection. If the PAXgene Blood RNA tube is the only tube to be collected, draw into a “discard tube” prior to using the PAXgene Blood RNA tube. Otherwise, the PAXgene Blood RNA tube should be the last tube drawn.
  2. After blood collection, gently invert the PAXgene Blood RNA tube 8-10 times. Store the PAXgene Blood RNA tube at room temperature until the sample is processed.
  3. After collection of the blood sample, incubate the PAXgene Blood RNA tube for at least 2 hours at room temperature to ensure complete lysis of the blood cellular constituents.
  4. Centrifuge the PAXgene Blood RNA tube for 10 minutes at 3,300 x g in a swinging-bucket centrifuge (in a Beckman Model GPR centrifuge, or equivalent). All centrifugation steps in this protocol are carried out at room temperature, unless otherwise specified.
  5. Remove and decant/discard the supernatant. Dry the rim of the tube with a clean Kimwipe. Add 5mL RNase-free water (provided in the PAXgene kit) to the pellet and close the tube using a fresh secondary Hemogard closure (provided in the PAXgene kit).
  6. Vortex thoroughly to resuspend the pellet, then centrifuge for 10 min at 3300 x g in a swinging bucket centrifuge. Decant and discard the entire supernatant.
  7. Thoroughly resuspend the pellet in 360μL Buffer BR1 (PAXgene kit) by vortexing.
  8. Using a micropipette, transfer the sample (usually 500-1200μL) into a 1.5mL microcentrifuge tube (USA Scientific Cat# 1415-2600). Add 300μL Buffer BR2 (PAXgene) and 40μL Proteinase K solution (PAXgene).
  9. Mix by vortexing, and incubate for 20 minutes at 55oC using a shaker-incubator, heating block, or water bath. If using a heating block or water bath, vortex each sample once during the incubation. Do not allow the temperature of the sample to decrease during vortexing.
  10. Centrifuge for 20 minutes at maximum speed in a microcentrifuge (Eppendorf model 5415C, maximum speed 14,000 rpm or 16,000 x g). Transfer the supernatant to a new 1.5mL microcentrifuge tube.
  11. Add 350 μl of 100% ethanol. Mix by vortexing and centrifuge briefly (1-2 seconds, 1,000 x g) to remove drops from the inside of the tube lid. Do not centrifuge for longer than 1-2 seconds as this may result in pelleting of the nucleic acids and reduced RNA yield.
  12. Apply 700 μl of sample to the PAXgene column sitting in a 2mL processing tube (all supplied in the PAXgene kit). Centrifuge for 1 minute at $8,000 x g. Place the PAXgene column in a new 2mL processing tube and discard the old processing tube containing the flow-through.
  13. Apply the remaining sample to the PAXgene column and centrifuge for 1 minute at $8,000 x g. Again, place the PAXgene column in a new 2mL processing tube and discard the old processing tube containing flow-through.
  14. Pipet 350μL Buffer BR3 to the PAXgene column and centrifuge for 1 minute at $8,000 x g. Place the PAXgene column in a new 2mL processing tube and discard the old processing tube containing flow-through.
  15. Prepare DNase I stock by dissolving solid DNase I (1500 Kunitz units; Qiagen, cat#79254) in 550μL of RNase free water and mix by inversion (1500 Kuntz Units/0.55mL). Pipet 10μL DNase I stock solution into 70μL of Buffer RDD. Mix by gently flicking the tube (do not vortex) and centrifuge briefly.
  16. Pipet DNase I incubation mix (80μL) directly onto PAXgene column and place upright at room temperature for 15 minutes.
  17. Pipet 350μL Buffer BR3 to the PAXgene column and centrifuge for 1 minute at $8,000 x g. Place the PAXgene column in a new 2mL processing tube and discard the old processing tube containing flow-through.
  18. Apply 500μL Buffer BR4 to the PAXgene column and centrifuge for 1 minute at $8,000 x g. Place the PAXgene column in a new 2mL processing tube and discard the old processing tube containing flow-through. Note that Buffer BR4 is supplied as a concentrate. Ensure that the ethanol is added to Buffer BR4 prior to use.
  19. Add another 500μL Buffer BR4 to the PAXgene column. Centrifuge for 3 minutes at maximum speed to dry the PAXgene column membrane.
  20. To eliminate residual Buffer BR4, discard the tube containing the flow-through, place the PAXgene column in a 2mL processing tube (USA Scientific Cat# 1620-2700), and centrifuge for 1 minute at full speed.
  21. Discard the tube containing the flow-through and transfer the PAXgene column to a 1.5mL elution tube (supplied as part of the PAXgene kit). Pipet 40μL Buffer BR5 directly on to the PAXgene column membrane (without touching the membrane with the pipet tip) and centrifuge for 1 minute at 8,000 x g.
  22. Repeat the elution step (step 17) as described, using 40μL Buffer BR5.
  23. Incubate the eluate for 5 minutes at 65oC in a heating block or water bath. Following incubation, chill immediately on ice.
  24. Add 0.1 volumes of 3 M sodium acetate pH 5.2 to the eluate and then add 2.2 volumes of ice-cold absolute ethanol. Vortex and store overnight at -20 C.
  25. Centrifuge at 16,000 x g (maximum) at 4 C for 30 minutes. Decant the ethanol, and Speed-Vac on low heat the remaining ethanol to dryness (approximately 5 minutes).
  26. Resuspend the RNA pellet in 20μl of RNase-free water. Combine the two samples into a single Eppendorf tube with an Eppendorf pipette.
  27. Label and store sample at -80o C until RNA analysis.

Reagents

PAXgene Blood RNA Tube (PreAnalytiX, A Qiagen/BD Company) Cat# 762115

Absolute ethanol, USP grade (AAPER Alcohol and Chemical Corp, Shelbyville, Kentucky).

NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

The bronchoalveolar lavage (BAL) must be processed immediately after collection.

  1. Label all the tubes and slides needed for this procedure BEFORE the start of the protocol.
  2. ICU Doc’s will collect BALF in sterile traps (tri-trap assembly). Tubes should be documented for volume of normal saline infused, anatomical area lavaged (i.e. right middle lobe, and volume aspirated back into trap) for the bronchopist.
  3. Give one aliquot to the ICU nurse to be sent to Microbio/Virology/Cyto. (They will need approximately 20ml of fluid.)
  4. Place the two remaining traps on ice.
  5. After the procedure, wait for the bronchopist to fill out the paperwork, obtain a copy immediately and transport the specimens back to the lab for processing.
  6. Pool two traps of BALF and record the volume obtained.
  7. Filter the lavage sample through a 40μm nylon filter into a sterile 50mL falcon tube.
  8. Record the volume obtained after filtering (some fluid loss is expected).
  9. Transfer 2mL of filtered lavage sample to a clean and identified 15mL Falcon tube.
  10. Centrifuge the remaining fluid at 1200rpm (235 g) for 15 minutes with brakes at 4°C.
  11. In the mean time, determine the number of cells present in the sample:
    1. Mix 10µl of lavage fluid (from the 0.5 ml saved above) with 80l PBS + 10l Turks.
    2. Load 10μl of this mix into each chamber of a hemocytometer. Count four squares in each chamber. Maintain two counts, one viable, one non-viable.
    3. Calculate and record cell concentration for every chamber, as follows:
    4. (Total cells counted/4) x 10 x 4000 = number of cells per mL
    5. Do this for each chamber and average the numbers.
    6. Calculate and record total cell number by multiplying “cells per ml” by the volume of BAL after filtering (less 2mL fraction removed).
    7. Cell viability will be recorded as % of viable cells: (viable cells/total cells) x 100
  12. Pour the centrifuged lavage supernatant into a 50mL Falcon tube and place on ice.
  13. Put 1.5mL of supernatant into each of the ten cryovials and store the remaining supernatant in 10mL aliquots in the 15mL identified conical Falcon tubes.
  14. Place all aliquoted supernatant into a cryopreservation box into a -70C freezer.
  15. Set up the slides with the cytofunnels in the cytospin.
  16. Compute the volume needed for 50,000 cells per slide.
    1. (5x104cells/slide) / (Y x 104cells/mL) x 1000µL/mL = X μL/slide
    2. X = µL to give 50 000 cells/slide.
    3. Y = cell concentration from Step 6.
  17. Multiply X by 12.
  18. Remove this volume from the 2mL BAL aliquot.
  19. Bring total volume to 6mL with PBS in a separate tube. Mix well.
  20. Load 500µL into each cytofunnel.
  21. Centrifuge the slides in the cytospin at 600 rpm (Cytospin2 by Shandon) for 10 minutes
  22. Discard funnels and allow the slides to air dry at least two hours.
  23. Stain two slides with Hemacolor stains as follows:
    1. 30 seconds in fixative #1
    2. 15 seconds in eosin Y (red) #2
    3. 15 seconds in thiazine (blue) #3
    4. Rinse in distilled water
  24. All remaining slides are fixed. At least two slides should be fixed in each of the following ways: Methanol, acetone or formalin. Indicate on the slide with #2 pencil how slide was fixed.

Labeling Specimens

Labels are provided for you in the specimen Handling Kit (contact Handling Center). Please make sure labels are as follows:

  • Patient study ID number
  • Patient biospecimen number
  • Biospecimen type
  • Day post entry into the study
  • Date and time that the specimen was drawn
  • Please use blue or black ink when completing the label.
  • Make sure you use a pen that does not smudge with water or alcohol (Markette permanent marker).
  • Secure and cover the entire prepared label with Scotch tape.

Shipping Specimens

  • To ship the specimen on dry ice, please use a well-labeled, sealed plastic 12 x 75mm serum tube bagged and put on dry ice. Zip lock bag.
  • It is fundamental that you use “a lot” of dry ice, in case for some reason the transport takes longer than expected (to prevent thawing). A rule of thumb is 10x more volume of dry ice than volume of specimens.
  • Specimens should be sent via FedEx or Purolator. Your hospital’s shipping and receiving department should have the necessary packaging materials, forms and dry ice labels.
  • Then please courier the blood to the receiving centre.
  • Notify the receiving centre by telephone on day of shipping.

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

DNA isolation will be performed using the QIAamp DNA mini kit from QIAGEN (Cat no. 51304). This protocol is a summary of the one given with the kit. Please refer to vendor’s protocol for additional information.

Important points before starting:

  • All centrifugation steps are carried out at room temperature.
  • Use carrier DNA if the sample contains less than 10 000 genome equivalents.
  • 200μL of whole blood yields 3 to 12μg of DNA. Preparation of buffy coat is recommended if a higher yield is required.

Things to do before starting:

  • Equilibrate samples to room temperature.
  • Heat a water bath or heating block to 56oC for use in step 4.
  • Equilibrate Buffer AE or distilled water to room temperature for elution in step 11.
  • Ensure that buffer AW1, buffer AW2 and QIAGEN Protease have been prepared according to the instructions on page 17.
  • If a precipitate has formed in Buffer AL, dissolve by incubating at 56oC.

PROCEDURE:

  1. Pipet 20μL QIAGEN Protease (or proteinase K) into the bottom of a 1.5mL microcentrifuge tube.
  2. Add 200μL sample to the microcentrifuge tube. Use up to 200μL whole blood, plasma, serum, buffy coat or body fluids, or up to 5x106 lymphocytes in 200μL of PBS.
    1. If RNA-free genomic DNA is required, 4μL of an RNase A stock solution (100mg/ml) should be added to the sample before addition of buffer AL.
  3. Add 200μL of Buffer AL to the sample. Mix thoroughly by pulse-vortexing for 15 sec.
  4. Incubate at 56oC for 10 minutes.
  5. Briefly centrifuge the 1.5mL microcentrifuge tube to remove drops from the inside of the lid.
  6. Add 200μL ethanol (96-100%) to the sample, and mix again by pulse-vortexing for 15 seconds. After mixing, briefly centrifuge the 1.5mL microcentrifuge tube to remove drops from the inside of the lid.
  7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2mL collection tube) without wetting the rim.
  8. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 minute. If the lysate has not completely passed through the column after centrifugation, centrifuge again at higher speed until the QIAamp Mini spin column is empty. (N.B. When preparing DNA from buffy coat or lymphocytes, centrifugation at full speed is recommended to avoid clogging.)
  9. Place the QIAamp Mini spin column in a clean 2ml collection tube and discard the tube containing the filtrate.
  10. Carefully open the QIAamp Mini spin column and add 500μL Buffer AW1 without wetting the rim.
  11. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 minute.
  12. Place the QIAamp Mini spin column in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
  13. Carefully open the QIAamp Mini spin column and add 500μL of Buffer AW2 without wetting the rim.
  14. Close the cap and centrifuge at full speed (20 000 x g or 14 000 rpm) for 3 minutes.
  15. Place the QIAamp Mini spin column in a new 2mL collection tube and discard the old collection tube with the filtrate.
  16. Centrifuge at full speed for 1 minute.
  17. Place the QIAamp mini spin column in a clean, properly identified 1.5mL microcentrifuge tube and discard the collection tube containing the filtrate.
  18. Carefully open the QIAamp Mini spin column and add 200μL of Buffer AE or distilled water.
  19. Incubate at room temperature for 5 minutes.
  20. Centrifuge at 6000 x g for 1 minute.
  21. Discard the column and close the cap of microcentrifuge tube.
  22. Store the sample at -80oC until use.

For complete information, refer to the protocol “QIAamp DNA Mini and Blood Mini Handbook”, version of November 2007, p.27-29.

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

  1. If an external ventricular drain (EVD) is placed as part of the routine care, fresh cerebrospinal fluid (CSF) samples will be taken at the time of placement of the EVD and at indicated time points. CSF obtained by lumbar puncture is also acceptable.
  2. 5mL of CSF per sample will be collected using standard sterile techniques in a 10mL red cap tube (BD cat. No. 366430)1. (CSF that has been sitting in the collection chamber has to be discarded and cannot be used for sampling).
  3. The sample has to be stored on ice immediately following collection and until processing, which should occur as soon as possible after collection.
  4. The sample will be centrifuged at 5000 rpm (750 x g) for 10 minutes at 4oC.
  5. CSF samples drawn from the top of the red blood cell plug after centrifuging will be divided into 5 x 1000L aliquots in properly identified tubes.
  6. The samples are stored frozen at ≤ –70 C.

Notes:

  1. If CSF flow through the EVD is sluggish or stops it may be impossible to obtain 5mL of CSF. After establishing the reason for the CSF flow problems as much CSF as is possible should be obtained (up to 5mL) over 5 - 10 minutes. Fluid should not be drawn out of the EVD using negative pressure since this may obstruct the tip of the catheter. CSF should be allowed to flow freely. Strict sterile technique should be used if a 3-way stopcock the closed EVD system is entered to obtain CSF samples.

** NOTE: The following procedure is to be performed wearing laboratory coat, gloves, eye protection, and mask.

PRINCIPLE

Sterile urine will be collected from patients at the indicated time points following randomization.

The collection of urine should be obtained from an indwelling urinary catheter.

Specimen Collection & Handling

  1. Label all 4 x 2mL cryogenic freezing tubes per sample per patient.
  2. Approximately 15mL of sterile urine is to be collected from the side port of each patient’s indwelling urinary catheter and placed into a sterile 50mL polypropylene tube.
  3. In the laboratory, the urine is centrifuged at 1500 x g for 5 minutes with brakes off, in a horizontal, swinging bucket centrifuge.
  4. Urine is then aliquoted into the labeled 2mL cryogenic freezing tubes using sterile techniques.
  5. Urine sediment is discarded.
  6. The aliquot are stored at ≤ –70°C until processing

Supplies

  • 2mL cryogenic freezing tubes
  • 15mL sterile polypropylene conical tubes (Falcon/Becton-Dickinson, #35-2097)
  • Sterile, endotoxin-free 10mL serological pipets (VWR, #53283- 740)
  • Rubber bulbs for pasteur pipets (VWR, #56311-062)
  • P1000 pre-sterilized, pipet tips (VWR, # 53508-830)

Equipment

  • Clinical Centrifuge with swinging-bucket rotor. Must accommodate 15mL conical tubes [Put some dry cotton or gauge at the bottom of the 15mL tube holders in order to avoid sticking of CPT (glass) tubes].
  • Ultra low temperature -70°C freezer