B2- BLOOD PROCESSING
REDI-NET Consortium
BLOOD PROCESSING
WHOLE BLOOD AND BUFFY COAT LYSIS
CELL-FREE FLUID LYSIS (SERUM/PLASMA)
FTA CARD LYSIS
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093 and HT9425-23-C-0059. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This protocol details blood processing.
Before start
BEFORE START
- Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program.
- Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
- Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 2) to Thermo Scientific 1.5 ml Screw Cap Micro Tubes.
- Buffer ATL may form precipitates upon storage. If necessary, warm to
56°Cuntil the precipitates have fully dissolved. Prepare buffer ATL-DX: add 100 μl Reagent DX to 15 ml Buffer ATL. If smaller amounts are needed, transfer1.5mLof Buffer ATL into a sterile 2 ml vial and add10µLReagent DX. Mix well, after addition of Reagent DX. After preparation, the mixture is stable for 6 months atRoom temperature(15-25°C). - For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% isopropanol to ACB as indicated on the bottles.
- MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for
0h 3m 0s(before first use) or 1 minute (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended. - Aliquot nuclease-free water in big bottle into a few 15 ml tubes for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.
Steps
1. WHOLE BLOOD AND BUFFY COAT LYSIS
Add 200µL sterile 1x PBS to a 200µL whole blood or buffy coat sample.
Transfer 400µL blood and 1x PBS mixture to the bead tubes prepared in step 3 of Before Start section under the Guidelines & Warnings tab. Add 100µL of ATL-DX buffer.
Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material, 100µL EBV, and 100µL ZIKV standard into a tube from step 3 of Before Start section. Add 100µL ATL-DX buffer and 162.5µL sterile 1x PBS.
Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 100µL ATL-DX buffer and 400µL sterile 1x PBS.
Load all samples into the Bullet Blender (refill dry ice if necessary).
Set the speed at 12 and the time at 3. Press Start.
Let the samples settle for 0h 1m 0s in the Bullet Blender and then repeat step 7.
2. CELL-FREE FLUID LYSIS (SERUM/PLASMA)
Add up to 1.5mL fluid sample to a 2 ml microcentrifuge tube (depending on available volume, no less than 0.5 mL) and centrifuge the tube for 0h 5m 0s at maximum speed (12000- 14000 xg).
Use a pipet to remove the supernatant.
Add 500µL ATL-DX buffer and resuspend the pellet in by vortexing.
Transfer the entire pellet resuspension to the bead tubes prepared in step 3 of Before Start section.
Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material, 100µL EBV, and 100µL ZIKV standard into a tube from step 3 of Before Start section. Add 250µL ATL-DX buffer.
Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 500µL ATL-DX buffer.
Load all samples into the Bullet Blender (refill dry ice if necessary).
Set the speed at 12 and the time at 3. Press Start.
Let the samples settle for 0h 1m 0s in the Bullet Blender and then repeat step 16.
3. FTA CARD LYSIS
Punch FTA card with dried blood spots with a 3.0mm single-hole puncher.
Use a forceps to place three card punches of the same sample into a bead tube prepared in step 3 of Before Start section.
Add 20µL Proteinase K and 200µL QIAcard FTA Wash Buffer and 200µL ATL-DX buffer to the sample. Mix immediately by vortexing for 0h 0m 15s.
Include a positive control for each batch of samples: transfer 37.5µL ZymoBIOMICS Microbial Community Standard Material, 100µL EBV, and 100µL ZIKV standard into a tube from step 3 of Before Start section. Add 125µL QIAcard FTA Wash Buffer and 125µL ATL-DX buffer and 20µL proteinase K.
Include a negative control for each batch of samples: a bead tube from step 3 of Before Start section with 200µL QIAcard FTA Wash Buffer and 200µL ATL-DX buffer and 20µL proteinase K.
Incubate in a thermomixer at 1400rpm.
Refill the dry ice compartment of the Bullet Blender if necessary. After incubation, load all samples into the Bullet Blender.
Set the speed at 12 and the time at 3. Press Start.
Let the samples settle for 1 minute in the Bullet Blender and then repeat step 25.
4. INSTRUMENT SET UP
Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.
5. SET UP THE PROCESSING PLATES
Set up the Tip Comb, Wash, and Elution Plates outside the instrument according to the following table.
| A | B | C | D | E |
|---|---|---|---|---|
| Plate ID | Plate position | Plate type | Reagent | Volume per well |
| Tip comb | 7 | Place a 96 Deep-well Tip comb in a deep-well plate | ||
| Elution | 6 | Deep-Well | Nuclease-free water | 75 µL |
| Wash 4 | 5 | Deep-Well | 100% ethanol | 750 µL |
| Wash 3 | 4 | Deep-Well | 80% ethanol | 750 µL |
| Wash 2 | 3 | Deep-Well | Buffer AW2 | 700 µL |
| Wash 1 | 2 | Deep-Well | Buffer AW1 | 700 µL |
| Sample | 1 | Sample Lysate | Lysate and lysis buffer | 985 µL |
6. EXTRACTION
Centrifuge the bead tubes with lysate from the sample lysis step for 12000x g.
Add 20µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
Transfer 270µL supernatant (for FTA card samples of section 3, transfer 290µL supernatant) without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix; avoid foaming (it can be mixed on a Hula mixer for 0h 2m 0s). Add 695µL mixture to each sample. Each well including sample lysate should be 985µL.
Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
Start the run, then load the prepared plates into position when prompted by the instrument.
7. QUANTIFICATION AND STORAGE
After the running protocol is completed (~0h 35m 0s), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
In 0.6 mL microcentrifuge tubes, use 1µL total nucleic acid from whole blood/buffy coat samples, or 3µL total nucleic acid from FTA card/cell-free fluid samples, for DNA and RNA concentration measurements using Qubit 4 Fluorometer following manufacturer instructions.
Proceed with sample testing following the REDI-NET SOP B-4 Blood Testing or store at -20°C for less than 2 weeks.
8. INSTRUMENT SET UP
Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.
Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
9. SET UP THE SAMPLE PLATE AND ELUTION STRIP
Set up the Sample Plate according to the table below:
| A | B | C | D |
|---|---|---|---|
| Row ID | Plate Row | Reagent | Volume per well |
| Sample row | A | Lysate and lysis buffer | 985 µL |
| Wash 1 | B | Buffer AW1 | 700 µL |
| Wash 2 | C | Buffer AW2 | 700 µL |
| Wash 3 | D | 80% ethanol | 750 µL |
| Wash 4 | E | 100% ethanol | 750 µL |
| Tip Comb | F | Tip Comb | |
| **** | G | Empty | |
| **** | H |
Set up the Elution Strip according to the table below:
| A | B | C | D |
|---|---|---|---|
| Strip ID | Row | Reagent | Volume per well |
| Elution | A | Nuclease-free water | 75 µL |
10. EXTRACTION
Centrifuge the bead tubes with lysate from the sample lysis step for 12000x g.
Add 20µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
Transfer 270µL supernatant (for FTA card samples of section 3, transfer 290µL supernatant) without any particle carryover to the wells of the Deep-well Row A. This row becomes the Sample Row.
Add 135µL Buffer VXL, 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the Sample Plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix and avoid foaming. Add 695µL mixture to each sample. Each well including sample lysate should be 985 uL.
Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
Start the run, then load the prepared plate and Elution Strip into position when prompted by the instrument.
11. QUANTIFICATION AND STORAGE
After the running protocol is completed (~0h 35m 0s), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
In 0.6 mL microcentrifuge tubes, use 1µL total nucleic acid from whole blood/buffy coat samples, or 3µL total nucleic acid from FTA card/cell-free fluid samples, for DNA and RNA concentration measurements using Qubit 4 Fluorometer following manufacturer instructions.
Proceed with sample testing following the REDI-NET SOP B-4 Blood Testing or store at -20°C for less than 2 weeks.
APPENDIX 3. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
DNA quantification:
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200µL (i.e., if using 1µL of sample, add 199µL of 1x HS dsDNA Qubit Assay). Vortex for 5 - 10 seconds, then incubate for 0h 2m 0s at Room temperature before reading.
RNA Quantification:
In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:
| A | B | C |
|---|---|---|
| Reagents | Volume/sample | Volume for n+1 sample |
| Qubit RNA HS Assay buffer | 199 µL | …. µL |
| Qubit RNA HS Assay Dye | 1 µL | …. µL |
In a new 0.6 ml tube, mix 197µL of Qubit HS RNA Assay working solution and 3µL of the sample. Incubate for 0h 2m 0s at Room temperature before reading.
