concentration of BLOOD BORNE pathogens in whole blood for research use

INTRODUCTION

The InnovaPrep Concentrating Pipette (CP) is an automated, rapid micro-particle concentrator developed for general microbiology use. The system performs rapid ‘mechanical enrichment’ as a frontend to rapid microbial detection, replacing older, slower methods such as centrifugation or enrichment steps. The one-pass method provides sample volume reduction and removal of matrix associated inhibitors for modern direct detection.

The concentration process uses tangentially-loaded, dead-end filtration to capture particles onto the surface of a porous membrane filter within the InnovaPrep CP’s single-use p. Aer the sample has been processed and the particles have been trapped, InnovaPrep’s patented Wet Foam Elution™ process is employed to wash the particles off of the membrane surface into a very small liquid volume.

Concentrating clinical and blood product samples for research is one application of interest for this technology that holds promise by delivering concentrated samples of blood-borne bacteria to rapid detection methods such as real-me polymerase chain reaction systems. A simple protocol for preparing blood prior to concentration using the InnovaPrep CP is provided below. Many users have continued method development beyond this protocol with excellent success.

PROTOCOL OVERVIEW

The following preparation method is capable of rendering whole blood “filterable” through membrane filters that are used in the InnovaPrep CP. Because complex clinical samples can foul membrane filters, a means for removing or passing these components through the filter ps must be implemented. The lysis protocol uses a non-ionic surfactant to lyse red blood cells and then an enzyme to further break down the red blood cell components to the point that they will readily pass through the membrane filter used in the InnovaPrep Concentrating Pipette Tip (CPT). Note that Universal Precautions are required for working with blood and blood products.

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MATERIALS REQUIRED:

InnovaPrep Concentrating Pipette (CP-150) 

CP elution fluid (HC08000 or HC08001) 

CP Pipette filter p (CC08000, CC08018 or CC08022) 

BD sodium polyanethol sulfonate (SPS) Vacutainer  

Blood rocker

Lysis buffer (A solution of 0.01 M Na2PO4/1% Tween 20 buffer - adjusted to a pH of 9.0-11.5) 

≥500 LAPU/g Protease from Aspergillus oryzae (Sigma-Aldrich, Product Number: P6110)  

Shaking water bath

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STEP 1 - LYSIS

The following blood lysis method was demonstrated to make it possible to process volumes of 12 mL or more of human whole blood in the InnovaPrep CP to a final concentrate volume of approximately 200 µL for subsequent analysis.

1. Collect 5mL blood directly into pre-weighed, sterile BD SPS Vacutainer, containing sodium polyanethol sulfonate (SPS) and citrate dextrose additives for anticoagulation and preservation.
2. Place the filled vacutainers onto a blood rocker to reduce clotting.
3. Prepare a solution of 0.01 M Na2PO4/1% Tween 20 lysis buffer  
4. Adjust the pH of the lysis buffer to the range of 9.0-11.5.  Raising the pH to this range reduces the amount of me needed for an effective reaction to take place.
5. Using the stock 500 LAPU/g protease prepare a 50 LAPU/g solution.  
6. Add 24 mL of pH adjusted lysis buffer and 4 mL of 50 LAPU/g protease to a specimen cup.
7. Add the volume of either one or two vacutainers of blood to the specimen cup (up to 10 mL).  
8. Place the specimen cups into a shaking water bath.
9. Allow to incubate at 37° C for a period of 45 to 65 minutes. At the beginning of the incubation period the blood/buffer mixture will be an opaque blood-red fluid. Towards the end of the incubation period the sample quickly changes to a clear, dark reddish-brown color.  Once the color change occurs, allow incubation to continue for an additional 5 to 10 minutes to ensure that complete lysis has occurred.  Removing the sample too quickly may result in filter fouling and premature stoppage of the concentration run.

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STEP 2 - CONCENTRATION

1. Set up Concentrating Pipette as instructed in section 4 of the CP user guide.
2. Prime the fluid line as instructed in section 5.2.1 of CP user guide.
3. Insert a Concentrating Pipette Tip (CPT) and set menu parameters as instructed in section 6.2 of the CP user guide for the chosen CPT type.
4. Lower the CPT into the prepared blood sample.
5. Press – “run”on the user screen.
6. When the entire sample has been processed the CP will stop.  
7. Place a clean final sample container under the CPT.  The menu screen will prompt you to press “Extract”.
8. Press “Extract”.
9. The sample will dispense from the pipette p into the sample container and is ready for subsequent sample preparation and analysis step.

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HINTS & TROUBLESHOOTING GUIDE

A technical report entitled “Blood Methods Development Research” is available by request.  

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CONTACT INFORMATION

Technical Support and ordering information:

Phone:  InnovaPrep 1-816-619-3375  

Email: info@innovaprep.com

Mail: InnovaPrep 132 East Main Street, Drexel, MO 64742

PRODUCTS RECOMMENDED FOR YOU

The below items can be purchased directly from the InnovaPrep website, 

http://innovaprep.com/ordersupplies/concentrating-pipette-supplies/.

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