Duke Human Vaccine Institute

OUTLINE• IQA Proficiency Testing Program

– Purpose– Workflow– Reminders

• PBMC Processing Methods– Isolation – Cell Counts– Freezing– Thawing

• Troubleshooting– Processing Errors– Common Clerical Errors– Poor Technique For Thawing PBMC

How Does The Cryopreservation Proficiency Program Work?

• To assess a laboratory’s ability to process, cryopreserve and ship viable PBMC specimens.

• Troubleshooting efforts would include conference calls, site visits or wet lab sessions performed at the IQA laboratory.

Website: www.iqa.center.duke.edu

Currently there are 65 domestic and 28 international laboratories enrolled in the program.

IQA Global Laboratory Participants in the Cryopreservation Proficiency Testing Program

93 participating laboratories

65 in North America

11 in Central and South America

11 in Africa

6 in Pacific Rim

IQA Sample Workflow

• Laboratories obtain and process samples quarterly.

• Frozen PBMC samples are shipped overnight to the IQA and tracking number is provided to IQA.

• IQA receives shipments, cryo samples are unpacked and inspected.

• LDMS information is uploaded.

IQA Sample Workflow

• Vials are stored in a vapor phase liquid nitrogen tank.

• Empty boxes are returned to labs if prepaid return label was provided to IQA.

• Samples are removed from vapor phase liquid nitrogen storage for evaluation.

• Vials that do not meet network criteria are confirmed by testing the second vial.

IQA Sample Workflow• Create/distribute summary spreadsheet.

• Laboratories are emailed to retrieve performance report on the IQA website (iqa.center.duke.edu).

• One week later the IQA begins troubleshooting unsatisfactory laboratory performances.

How Does The IQA Remind Labs To Participate In The Program?

• Laboratories remindersLaboratories reminders– Yearly Calendar (IQA website)– Email Participation Reminder (10 weeks prior to

shipping )– Email Collection Notification (1 week prior to

sample collection)– Email Shipment Notification (1-2 weeks prior to

due date)

Safety• Treat all human-derived specimens as infectious

using universal safety precautions.

• Wear a full-face shield and cryo-gloves duringhandling of frozen samples.

• Wear appropriate personal protective equipment.

• Process in Class II biological safety cabinet.

Peripheral Blood Peripheral Blood Mononuclear Cells (PBMC) Mononuclear Cells (PBMC)

IsolationIsolation

Plasma Isolation

Centrifuge EDTA , Heparin or ACD blood at 400 x g for 10 minutes.

Buffy Coat

Plasma

Basic PBMC Isolation Procedure

Diluted anticoagulated whole blood

Ficoll®

PBMC can be isolated from whole blood samples using different density gradient centrifugation procedures such as:

• Manual Ficoll®• Cell Separation Tube with Frit Barrier (CSTFB) • CPT tubes

Anticoagulated whole blood is layered over the separating medium:

plasma/platelets

separating medium granulocytes

erythrocytes

buffy coat (PBMC)

At the end of the centrifugation step, the following layers are visually observed from top to bottom.

The PBMC layer is then removed and washed twice to get rid of contaminants before cell type and cell viability can be confirmed.

Manual Ficoll® Methods

Overlay Underlay

Cell Separation Tube With Frit Barrier (CSTFB)

1. Load the Ficoll into a polypropylene tube with a frit (porous polyethylene barrier).

2. Centrifuge to settle the Ficoll below the frit.

3. Layer whole blood on top of the frit.

4. Centrifuge the tube.

Frit Barrier

BD Vacutainer® CPT™ Cell PreparationTube With Sodium Citrate

8 mL Draw Capacity(16 x 125mm silicone glass

coated tube )1.0 mL of 0.1 Molar

Sodium Citrate Solution (Top Fluid Layer)

Polyester Gel Barrier

FICOLL™ HYPAQUE™ density medium

Buffy Coat Isolation

Comparison of Manual Ficoll® Methods

Underlay Method Mononuclear layer

Overlay Method Mononuclear layer

Cell Separation Tube With Frit Barrier (CSTFB)

Plasma Layer

Ficoll®

Mononuclear Layer

Frit Barrier

Cell Preparation Tube (CPT)

Mononuclear Layer

Plasma Layer

Erythrocytes

Manual Cell CountsManual Cell Counts

What to Count on the Hemacytometer?

Formulas For Cell Counts

• Percent viable cells :

# live cells _______ /# total cells (live+dead) _________x 100 = _______ %

• Total Viable Cell Count (Concentration):Total number of live cells counted X Dilution factor x resuspension volume x 104= __ # cellsNumber of squares counted

• Calculate cell yield per mL of whole blood:

(QC check)= (Cell Concentration/ Whole Blood Volume)

Cryopreservation

Why do we use DMSO and FBS as Why do we use DMSO and FBS as

Cryoprotective Agents?Cryoprotective Agents?

• DMSO helps dehydrate the cells prior to freezing therefore preventing the formation of ice crystals that will cause cells to lyse during thawing.

• During the freezing and thawing processes cells are deprived of nutrients and therefore, FBS contains an abundance of proteins.

Effects of Freezing Rates on Mononuclear Cells

PBMCIce Crystals

Slow cooling rate.•Cells dehydrate and shrink but they survive.•Less intracellular ice.•More osmotic imbalance.

Fast cooling rate.•Intracellular ice crystals form. •Less osmotic imbalance.• Cell death.

Cell Freezing ContainersCell Freezing Containers

• Cells are gradually cooled at a rate of approximately 1°C per minute using a rate-controlled freezer, or they are placed in a “Mr. Frosty”, “StrataCooler”, “Cool-Cell” in a -80°C freezer overnight.

• Latent heat released from the cooling cells is absorbed by the freezing chamber preventing the heat from damaging cells.

Thawing of Cryopreserved Thawing of Cryopreserved CellsCells

Thawing of Cryopreserved PBMCThawing of Cryopreserved PBMC

• If PBMC are not thawed properly, viability and cell recovery can be compromised; cells may not perform optimally in functional assays.

• Cells should be thawed quickly but diluted

slowly to remove DMSO. Cells that have DMSO permeated into their membranes are very fragile and must be pelleted and handled gently.

Why Are The Cryopreserved Cells Why Are The Cryopreserved Cells Diluted?Diluted?

• The gradual dilution of DMSO avoids the osmotic shock, and the warm temperature from the media assures that the cells can actively compensate the decreasing osmotic pressure.

Why use Benzonase® and RPMI Why use Benzonase® and RPMI during the thawing procedure?during the thawing procedure?

• Benzonase will remove the contaminated cellular DNA/RNA and RPMI (rich nutrient) is used to compensate from the stress that cells undergo during thawing.

• Use Benzonase when thawing cells for assays.

TroubleshootingTroubleshooting

Processing Errors Processing Errors Red blood cell contamination

• Density gradient media or centrifuge not (15-30°C) room temperature.

• Centrifugation time is too short.

Excess of platelets• Not removing most of the plasma above the interface.

No defined or distinct mononuclear layer – Centrifugation time is too short.

– Centrifuge not calibrated.

– The brake was left on.

– The centrifugation speed was too high.

– The centrifuge arms and buckets were not properly greased and oiled.

– Hyperlipemic sample.

Processing ErrorsProcessing Errors

Processing Errors Processing Errors Granulocyte contamination

– Removing excess amounts of the separation media with the PBMC band.

– Centrifuge was not set up at the appropriate speed.

– Density gradient media or centrifuge not (15-30°C) room temperature.

LDMS Computational ErrorLDMS Computational ErrorExample 1.Example 1.

Shipping Manifest Processing Report

Cell concentrations do not match.

How does a Clerical Error affectHow does a Clerical Error affectthe Results?the Results?

Total viable cell count performed by

the IQA (x10^6 cells per vial)

Sample cell concentration

reported by processing site

(x 10^6 cells per vial)

Total percent viable recovery Final performance

5.66 9.7 58% Unsatisfactory

5.66 5.4 104.8% Satisfactory

Formula Percent Viable Recovery:

Number of cells counted by IQA divided by number of cells reported by site.

Results multiplied by 100.

LDMS Computational ErrorLDMS Computational ErrorExample 2.Example 2.

Shipping Manifest Processing Report

Low cell yield:0.58 x106 cells/ml of

blood

Lab froze 3 vials at 10 million cells each, possibly

3.5 x106 cell/vial

Cell ViabilityCell Viability

Viability is too low

Processing Report Unexpected Viability

Freshly isolated PBMCviability should be >95%.

Yields outside the expected ranges may indicate:

•Long processing time•Poor technique

IQA PersonnelIQA Personnel

Daniella Livnat Project Officer for the IQAContract# HHSN27220070054C

Thomas DennyPrincipal Investigatorthomas.denny@duke.edu

IQA PersonnelIQA Personnel

Raul LouzaoIQA Program Managerraul.louzao@duke.edu

Tania GarreltsTania.garrelts@duke.edu

Special Thanks To:

Questions?Questions?