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Duke Human Vaccine Institute

OUTLINE

Immunology Quality Assessment Purpose Grading Criteria

Proficiency Testing (PT) Cryopreservation Thawing

Troubleshooting Challenges Technique/Tips

Immunology Quality Assessment

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

IQA Global Laboratory Participants in the

Cryopreservation Proficiency Testing Program(94 participating laboratories as of June 2011)

65 in North America

13 in Central and South America

11 in Africa

5 in Asia

Purpose

What is the purpose of the IQA Cryopreservation Proficiency Testing (PT) Program?

1. Assess a laboratory’s ability to consistently process, cryopreserve

and ship viable peripheral blood mononuclear cells (PBMCs)

2. Troubleshoot and work with individual labs via conference calls, site visits or wet lab sessions performed at the IQA

3. Communicate with the networks to verify each lab’s status and collaborate to determine necessary corrective action

Quarterly Reminders Yearly Calendar (IQA website)

Participation Reminder

Collection Notification

Shipment Notification

Results Report Notification

Investigational Report (IR) Notification

Continuous communication via email/phone from IQA and network personnel

Grading Criteria

Grading Criteria Performance Evaluation

established by the IQA PBMC Cryopreservation PT Advisory Group (ICAG)

Scoring System for One Sample

% Viability % Viable Recovery Score/sample:

80-100% 70-120% 2

65-79% 50-69% 1

- 121-150% 1

< 65% < 50 % 0

- > 150% 0

No Submission 0

Grading Criteria

Combined Scoring System for Both Samples

Combined% Viability Score (both samples)

% Viability STATUS

Combined % Viable Recovery Score

(both samples)

% Viable Recovery STATUS

OVERALL STATUS (both viability and

viable recovery)

3-4 A 3-4 A Overall status is determined by the

lower of the %viability/ %viable recovery statuses.

2 PA 2 PA

0-1 OP 0-1 OP

Example: % Viability Status: A

% Viable Recovery Status: PA

Overall Status: PA

A: Approved

PA: Provisionally Approved

OP: On Probation

Grading CriteriaSite Vial ID Tech Viability (%)

Viability

Score

Viability

Status

Viable

Recovery

(%)

Viable

Recovery

Score

Viable

Recovery

Status

Combined

status

XXX 213V110000X BL 100 2A

95 2PA PA

XXX 213V110000X BL 97.5 2 68 1

An Investigation Report must be submitted to the IQA within 5 working days of receiving results report:

•If your site received a viability or percent viable recovery scores of less than 2 on one or both samples you are required

to complete and submit an IR and are advised to work within your lab and with the IQA.

Your lab must submit samples within 4 weeks of on probation status notification:

•If your site received a viability or percent viable recovery status of “On Probation” (OP), you are required to not only

complete and submit an IR but you must also process another set of samples to be tested by the IQAC

https://iqa.center.duke.edu/wysiwyg/downloads/IQA_Investigation_and_Review_Request_Form_v3.doc

Grading Criteria APPROVED – A Status

If a score of 3 is received, an Investigational Report (IR) must be completed (even though A status earned)

Eligible to perform PBMC cryopreservation for network protocols

Eligible to start new network protocols

Eligible to serve as an IQA back-up lab

Not exempt from future proficiency testing

Grading Criteria PROVISIONALLY APPROVED – PA Status

Required to complete an IR


Eligible to start new network protocols

NOT eligible to serve as an IQA back-up lab

Grading Criteria ON PROBATION – OP Status

Required to complete an IR and submit the equivalent of one round of IQA PT within 4 weeks (to improve score)


NOT eligible to start new network protocols OR serve as an IQA back-up lab

If lab remains OP for 2 consecutive rounds, re-certification* and network involvement must take place

(*Fast Track: submitting the equivalent of two rounds of samples;

with satisfactory results, lab becomes PA)

Grading Criteria ON HOLD/Not Approved – Hold/NA Status

Lab is enrolled in the IQA PT program, but currently not participating

NOT eligible to perform PBMC cryopreservation, take on new PBMC cryopreservation protocols, or serve as an IQA back-up lab

Lab must re-certify* when they wish to start the IQA PT program again

(*Fast Track: submitting the equivalent of two rounds of samples;

with satisfactory results, lab becomes PA)

March 2011 Results Data

Status:Overall %

Viability

Overall %

Recovery

Combined

Status

# of labs with an

Approved

(A) Status91 84 81

# of labs with a

Provisionally Approved

(PA) Status0 7 9

# of labs with an

On Probation

(OP) Status0 0 0

# of labs with an

on Hold

(Hold) StatusNA NA 4

94 Labs Enrolled

91 Participated March 2011

-2 Labs participated in the Fast Track

(received satisfactory results (A), but

are PA for the next round of testing)

-1 Lab was placed on Hold status

(by network)

-0 Labs are currently OP

Proficiency Testing

Quarterly IQA PT

Preparation

PBMC Processing

Shipment to IQA

Preparation

Obtain IRB Consent Form

Select/schedule two Donors (HIV +/-)

Collect blood from each donor

(ACD, EDTA or NaHep )

PBMC Processing

Dilute Whole blood/Ficoll

Isolate buffy coat/PBMC

Wash

Count cells

Adjust/re-suspend cells in Cryopreservation Solution

PBMC Processing

Ficoll®

Dilute Whole-blood and Layer over Density Gradient

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

Manual Ficoll® Cell Separation Tube with Frit Barrier (CSTFB) Cell Preparation Tubes (CPT)

Anti-coagulated whole blood is layered over the separating medium

Diluted anti-coagulated whole blood (manual)

Ficoll®

PBMC ProcessingIsolate Buffy Coat/PBMCs – POST Centrifugation

plasma/platelets

separating medium granulocytes

erythrocytes

Buffy Coat (PBMC)

PBMC ProcessingIsolate the Buffy Coat (PBMC)

Remove plasma layer Isolate and harvest buffy coat (PBMC layer)

Manual Ficoll® Methods

Overlay Underlay

Overlay: diluted blood is layered OVER room temperature Ficoll® Underlay: room temperature Ficoll® is added UNDER diluted blood

Centrifuge: 400 x g for 30 minutes, brake off

Manual Ficoll® Methods

Buffy Coat (PBMC)

(Underlay)

Buffy Coat (PBMC)

(Overlay)

Cell Separation Tube With Frit Barrier (CSTFB*)

Load room temperature Ficoll® into a polypropylene tube with a frit barrier (if necessary)

Centrifuge to settle the Ficoll® below the frit barrier(800 x g for 30 seconds)

Layer whole blood on top of the frit barrier

Centrifuge:(800 x g for 15 minutes, brake off)

*Accuspin/Sigma-Aldrich

Frit Barrier

Cell Separation Tube With Frit Barrier (CSTFB)

Plasma Layer

Ficoll®

Buffy Coat (PBMC)

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

Centrifuge: 1,500 to 1,800 RCF for a minimum of 20 minutes, brake off

*Becton, Dickinson and Co.

BD Vacutainer® CPT™ Cell PreparationTube With Sodium Citrate

Buffy Coat (PBMC)

Plasma Layer

Erythrocytes

Comparison of 3 Methods

Factors:Manual Ficoll

(overlay/underlay)Cell Separation Tube

w/ Frit BarrierCell Preparation Tube

(CPT)

Cost/Sample*$3.55/

20mL whole blood$13.10/

20mL whole blood$8.25/

8mL whole blood

Advantages Most cost effective Less time consumingConvenient

(can ship after spin),very time effective

Disadvantages Time consuming Cost Cost, volume limitation

*Prices are for reagents only, are approximate and may vary

This information is provided to give you a general idea of what method might be best for your lab

Review:

Ficoll® carefully (clean interface)

Precise buffy coat/PBMC isolation/pipetting

Attention to detail during set up

(centrifuge settings, reagents, volumes)

PBMC Processing



Wash (200-400 x g for 10 minutes)

Count cells

Adjust/Re-suspend cells in Cryopreservation Solution

Cell Counting Gently re-suspend cells

Thoroughly mix staining agent (0.4% trypan blue or 0.05% crystal violet) and cells together

Load hemacytometer (10uL) using correct cover slip

Let mixture stain; 8-12 seconds

Count viable and nonviable cells from outer 4 quadrants

Cell Counting (manually on a hemacytometer)

Cell Counting

Live/

Viable Cells

Dead/

Non-Viable Cells

Cell Counting - Problems

“Clean” Sample-mostly PBMCs (viable/non-viable)-easy to read/decipher

“Dirty” Sample-platelets/debris-more difficult to read

Cell Counting - Problems

Dead Cells/Poor Viability

Formulas For Cell Counting

Dilution Factor (DF):

final volume / aliquot volume

(final volume= aliquot + diluent)

Ex: (1mL cells + 9mL PBS) / 1mL cells = 10 DF

Ex: (20uL cells + 20uL trypan blue) / 20uL cells = 2 DF


Percent viable cells:

# live cells /# total cells (viable + non-viable) x 100

Ex: total viable cells: 226 total non-viable cells: 11total cells: 237

(226/237) x 100 = 95.4% viable Cells


Total Viable Cell Count (Concentration):

total viable cells X dilution factor x re-suspension volume (mL) x 104

squares (4)

Ex: total viable cells: 226

dilution factor: 10

volume: 2mL

104 : hemacytometer factor/chamber volume

(226/4) (10) (2mL) (10⁴) = 11.3x106 cells

Review:

Keep counts consistent

Outer four quadrants, top/left borders

Mix sample well for accurate representation

Check hemacytometer and cover slip

Don’t over fill chamber (only 10uL)

Double check calculations

Check yourself with automated cell counter

optional

PBMC Processing



Wash

Count cells

Adjust/Re-suspend cells in Cryopreservation Solution

Cryopreservation

Why do we use DMSO and FBS as Cryoprotective Agents?

DMSO helps dehydrate the cells prior to freezing preventingthe formation of ice crystals that could cause cells to lyseduring thawing.

FBS contains an abundance of proteins, which helps nourishthe cells during the freezing and thawing processes whencells are deprived of nutrients.

Cryopreservation Solution (CPS) – Freezing Media

Fetal Bovine Serum (FBS) Dimethyl sulfoxide (DMSO)

90% 10%

Formulas for Freezing

Determining final CPS re-suspension volume:

Total Cell Concentration/Freeze down concentration

= actual volume of CPS need

Ex: Total Cell Concentration: 11.3 X 10⁶ cells

Freeze down concentration: 1mL/5x10⁶cells

11.3 X 10⁶ cells / (1mL/5x10⁶cells) = 2.26mL

(round down to nearest whole mL)

Formulas for Freezing# of cells/vial:

(Total cell concentration/Final CPS volume) x Freezing volume*

= # cells (10⁶)/vial (mL)

*Freezing Volume: usually 1mL

Ex: Total cell concentration: 11.3 x 10⁶ cells

Final CPS volume: 2mL

Freezing volume: 1mL

(11.3 x 10⁶ cells/ 2mL) x 1mL = 5.65 x 10⁶ cells/mL

Freezing Containers

Once cells have been aliquoted, they 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, or CoolCell in a -80°C freezer overnight.

Rate controlled freezing is important so that you avoid rapid intracellular freezing and excess extracellular ice formation, both lead to cell death

Freezing Containers

Stratagene StrataCooler® Cryo:

Holds up to 32 cryovials

~$159.00/unit

Freezing Containers

NALGENE ® Mr. Frosty

Must be stored at an ambient temperature (15-30°C)

Uses isopropanol, must be filled to correct level and replaced every fifth freeze/thaw cycle (with record log)


~$75.00-80.00/unit

(+isopropanol)

Freezing Containers

BioCision ® CoolCell

Allow inner-ring and foam to reach ambient temperature (15-30°C) after each use


~$100.00/unit

Freezing ContainersFactors: StataCooler® Mr. Frosty CoolCell

# Cryovials 32 18 12

Cost/unit* $159.00$75.00-80.00

(+isopropanol)$100.00

AdvantagesLarge sample-size,

No IsopropanolCost No Isopropanol

Disadvantages CostUp-keep, properwaste disposal

Small sample-size

Pre-chill each freezing container to 2-8°C prior to each use

(per HANC Cross-Network PBMC Processing SOP, draft)

*Costs are approximate and may vary. This information is provided to

give you a general idea of what method might be best for your lab

Review: Double check all calculations/dilutions

Perform the final centrifugation and re-suspension with CPS carefully

Keep cells chilled while adding CPS and freeze immediately after

Keep freezing container in a secure location within -80°C freezer, away from door/possible temperature fluctuation

Make sure freezers have been calibrated and are monitored

Shipment to IQAC

Prepare Shipment to Lab 213:

Enter specimen into LDMS/Label

Package Specimen

(Category B Biological Substance):

Include LDMS manifest, box report, batch file and completed return label

Ship 4 vials (2 from each donor) @ 5x10⁶cells/vial to the IQA during designated dates

Thawing

Thawing Process for IQA PT

Thaw/Dilute

Wash

Assess Viability/Viable Recovery

Report Results

Lab Communication

Thawing

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

Cells should be thawed quickly (to avoid the formation of ice crystals) but diluted slowly (drop-wise) to remove DMSO.

RPMI 1640 is nutrient rich and nourishes the cells as they undergo stress during the thawing process.

Troubleshooting

Challenges

Processing Challenges

Granulocyte Contamination

Centrifuge not set at correct speed/time

Ficoll® not room temperature

Collected from below the PBMC band (Ficoll®)

Fix/Prevention:

Count sample at higher magnification

Careful attention during PBMC isolation/set-up

Platelet Contamination

Could cause cell clumping/misleading counts

Sample quality

Collected from above the PBMC band (plasma)

Fix/Prevention:

Extra wash

Careful/accurate counting

Processing Challenges

No Distinct Buffy Coat/PBMC Layer

Could cause cellular contamination

Possible causes

Incorrect time, speed, temperature of centrifuge

Brake left ON

Layers disrupted/sample dropped or jarred

Poor Ficoll® technique, not room temperature

Fix/Prevention: Remix sample and repeat Ficoll® steps, careful handling and setup

Processing ChallengesCell concentration differ (after thawing procedure)

(inaccurate % Viable Recovery is the reason almost all labs fail!)

Contaminated sample = misleading counts

Calculation errors (fail to include DF, use incorrect DF, incorrect volume, etc)

Dilution errors Skip final centrifugation CPS made incorrectly Poor mixing (uneven distribution between vials)

VS…

Why use swinging buckets?As the rotor begins to rotate

the buckets swing out to a

horizontal position, allowing

the cells to pellet at the

bottom of the tubes.

A vertical rotor has no swing

and cells are forced to the

side of the tube, which is not

ideal for pellet applications.

This is why centrifugation

speed and time are important

factors (as it all affects the

way the cells are manipulated)

Technique/Tips

Technique/Tips

Read and follow the SOP

Check all reagents before processing (temperature, expiration dates, volumes)

Carefully inspect sample (before and throughout processing) to check for any irregularities (hemolysis, clumping, no pellet, poor layering, etc…)

Careful and precise pipetting/isolation

Handle sample gently, but make sure to mix well and re-suspend your pellet thoroughly

Technique/Tips

Mix sample well before each step

Work quickly, but not hastily

Count accurately

Double check all calculations/dilutions

Perform final re-suspension and aliquot samples into vials carefully

Freeze immediately

Remember to treat all human-derived specimen as infectious using universal safety precautions

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