流式质控方法课件

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Flow Cytometry Quality Assurance: A Different View Roger Scott Orlandi MT(ASCP) Flow Cytometry Supervisor Guthrie Clinic Laboratories Guthrie Square, Sayre PA 18840

Flow Cytometry QA In the following slides you will see Standardizations and Improvements that have been made to the Forms, Charts, and Documents used in Flow Cytometry and how they are part of the Quality Assurance Process.

02/01/09

Guthrie Clinic Laboratories

2

Flow Cytometry QA • The majority of Flow Samples here at Guthrie (Peripheral Blood and Bone Marrow) are held at Room Temperature. • Tissue Samples (Lymph Nodes, Liver and Spleens, etc.) and the storage of Samples for Absolute CD34 Count and CD34 Percentage are Refrigerated. • Laboratory Regulations state that Thermometers prior to being placed into service must be compared to a Certified Thermometer. • In Our Flow Department, all of the Thermometers used to document temperature are validated against a Certified Thermometer once a year.

02/01/09

Guthrie Clinic Laboratories

3

Flow Cytometry QA

Flow Cytometry Certified Thermometer Temperature Check Comparison Celcius=5/9(F-32)

Place the Certified Thermometer in the same area of the Flow thermometer for approximately 15 minutes. >>ACCEPTABLE RANGE = +1.0 degrees C <<

Thermometer Area

Date

Thermometer Reading

Certified Thermometer

Difference + 1.0 *C - OK

Sample Analysis Sample Preparation IEC Refrigerated Centrifuge

• This Chart is used to Document the Comparison of the Flow Thermometers to a Standard Certified Thermometer.

Refrigerator

• (One Time per Year)

Freezer

Comment:

__________________________________________________________________ __________________________________________________________________

Supervisor: __________________________________ Date: ________________________

02/01/09

Pathologist: __________________________________ Date: ________________________

Guthrie Clinic Laboratories

4

Flow Cytometry QA

Temperature Documentation Chart – Flow Cytometry

Month: __________________ Day Refrigerator Freezer (2 Š 8 *C)

Year: ______________

Sample Prep. Area (-5 to -25 *C) (18 Š 25 *C) (65 Š 75 *F)

Sample Analysis Hygrometer Area Analysis Area (18 Š 25 *C) (10 Š 90 %) (65 Š 75 *F)

IEC Centra Centrifuge (18 Š 25*C) (65 Š 75*F)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

02/01/09

• This Chart is used to Document the Flow Cytometry Temperatures: Room Temp (x2), Refrigerator, Freezer, and Centrifuge • (Daily)

Reviewed by: ____________________

Guthrie Clinic Laboratories

5

Flow Cytometry QA • The Staffing and Work Volume here at Guthrie allows for both Computer and Hand Logging Documentation of Samples. • The Sample Type and Billing Code Information are recorded in a Log Book. This book is used to aid in the Tracking of CPT Code Charges and Statistical Data Comparisons. 02/01/09

Guthrie Clinic Laboratories

6

Flow Cytometry QA Flow Sample Number

Date

PatientÕsName & Medical Record Number

 Flow  Sample Log Chart

Sample Send-Out (IP) = In-Patient Source Orders (Out) = Out-Patient Cancelled Flow Test Request

Flow Charge Codes Reports Back On File

­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

• This Chart is used to Log Flow Cytometry Samples • The Type of Sample, In-Pat/Out-Pat is Documented along with a running Total of How Many have been Tested and a listing of the Charge Codes for billing

• (Each Sample) 02/01/09

Guthrie Clinic Laboratories

7

Flow Cytometry QA • Before Flow Samples can be Prepared, the Department must assure that the equipment used in the process has been properly Quality Controlled. • The Delivery Volume of the MLA Pipetters is checked for accuracy using the OHAUS Digital Analytical Balance. • The OHAUS Digital Analytical Balance is validated by checking the readings against Certified Standard Weights. 02/01/09

Guthrie Clinic Laboratories

8

Flow Cytometry QA

Flow Cytometry OHAUS Analytical Balance Validation with Certified Standard Weights >>>ACCEPTABLE RANGE = +10% <<< Date: _________________

Weight

Balance Reading

(+ 10% Range)

10 g (#1)

(9.000-11.000g)

10 g (#2)

(9.000-11.000g)

5g

(4.500-5.500g)

2 g (#1)

(1.800-2.200g)

2 g (#2)

(1.800-2.200g)

1g

(0.990-1.010g)

500 mg

(0.450-0.550g)

200 mg (#1)

(0.180-0.220g)

200 mg (#2)

(0.180-0.220g)

100 mg

(0.090-0.110g)

50 mg

(0.045-0.055g)

20 mg (#1)

(0.018-0.022g)

20 mg (#2)

(0.018-0.022g)

Comment:

Results (Acceptable)

• This Chart is used to check the Flow Cytometry OHAUS Digital Analytical Balance against a Standard Set of Weights. • (One Time a Year)

__________________________________________________________________ __________________________________________________________________

Supervisor: __________________________________ Date: ________________________ Pathologist: __________________________________ Date: ________________________

02/01/09

Guthrie Clinic Laboratories

9

Flow Cytometry QA

• The MLA Pipetters that are used in Flow are checked and/or adjusted for Calibration two times per year and as needed if inconsistencies are observed.

02/01/09

Guthrie Clinic Laboratories

10

Flow Cytometry QA

Flow Cytometry Pipetter Calibration and Calibration Check

Warm up the OHAUS electronic balance for about one (1) to two (2) minutes before use. Take a weighing boat and TARE the balance. Using Reagent Grade Distilled Water, pipette the water into the boat five (5) times using a new pipette tip each time and wiping any excess water from the tip with a Kimwipe. Record the total weight on the documentation sheet. Divide the total by five (5). The value must be within the stated range or the pipetter must be adjusted and rechecked for calibration using the above stated procedure. Date:

Date:

Pipette Volume:

Pipette Volume:

(5X) Total:

(5X) Total:

(5X/5) Volume:

(5X/5) Volume:

Range:

Range:

Circle: Acceptable / Adjust

Circle: Acceptable / Adjust

Laboratorian Initials:

Laboratorian Initials:

Date:

Date:

Pipette Volume:

Pipette Volume:

(5X) Total:

(5X) Total:

(5X/5) Volume:

(5X/5) Volume:

Range:

Range:

Circle: Acceptable / Adjust

Circle: Acceptable / Adjust

Laboratorian Initials:

02/01/09

• (Two Times a Year)

Laboratorian Initials:

5 uL (+5%) Range = (0.0047-0.0053),

10 uL (+5%) Range = (0.0095-0.0105)

20 uL (+3%) Range = (0.0194-0.0206),

25 uL (+3%) Range = (0.0243-0.0257)

30 uL (+3%) Range = (0.0291-0.0309),

50 uL (+2%) Range = (0.0490-0.0510)

100 uL(+1%) Range = (0.0990-0.1010),

200 uL (+1%) Range = (0.1980-0.2020)

250 uL(+1%) Range = (0.2475-0.2525),

450 uL (+1%) Range = (0.4455-0.4545)

500 uL(+1%) Range = (0.4950-0.5050),

1800 uL(+1%) Range = (1.7820-1.8180)

1900 uL(+1%) Range = (1.8810-1.9190)

• This Chart is used when the Flow Cytometry MLA Pipetters are Checked and/or Adjusted for Calibration

Guthrie Clinic Laboratories

11

Flow Cytometry QA • The use of Timers in Flow is an important part of the Sample Preparation Process. • Time is used for Antibody Incubation, Sample Lysing, Centrifugation and Instrument Cleaning.

02/01/09

• Timers are validated against a Certified Calibrated Stopwatch two times per year. Guthrie Clinic Laboratories

12

Flow Cytometry QA

Flow Cytometry Timer Maintenance Validation Records

The timers in Flow Cytometry are checked semi-annually by a Flow Cytometry Laboratorian using a Calibrated Checked Stopwatch. The Stopwatch is checked against the USNO Master Clock web site visual clock. The interval is 60 sec. If the web site is not available, the Stopwatch is checked against the USNO Master Clock Voice Announcer at (202)762-1401 or (202)762-1069. The interval is 50 sec. The time is recorded on the chart below. The timers and the IEC CENTRO-7R CENTRIFUGE (IC7R) are checked at a five (5) minute time setting. The allowable variance on any of the timers or centrifuges is 10% (4'30"-5'30"). The Clay Adams SERO-FUGE II CENTRIFUGE (CASF) is checked at the two (2) minute time setting. The allowable variance on this centrifuge is also 10% (1'48"-2'12"). Times will be recorded to the nearest second ("). If any timer or centrifuge timer is out of range, the process will be repeated. Should the timer continue to be out of range following two (2) repeats, the timer will be discarded and replaced. A centrifuge timer will have BioMed repair or replace the timer so that the timer will meet the above stated 10% range. The Flow Cytometry Laboratorian testing the timers, the Cytometry Supervisor, and the Cytometry Director will all initial the report when the timer Preventive Maintenance check has been completed. ACCEPTABLE RANGE AT FIVE (5) MINUTES +10% - (4'30"-5'30") ACCEPTABLE RANGE AT TWO (2) MINUTES +10% - (1'48"-2'12") The Control Company Digital Timer = CCDT Date

Calibrated Stop Watch 60 seconds

CCDT CCDT CCDT Elect Ct-UP #1 #2 #1

Elect #2

Elect Timex IC7R #3 Ironman Fuge

• This Chart is used to check the Flow Cytometry Timers against a Standard Calibrated Stopwatch.

CASF Tech Supv Directors Fuge Init Init Init

• (Two Times a Year)

02/01/09

Guthrie Clinic Laboratories

13

Flow Cytometry QA • On an Average, there is about 3 pieces of Glassware Washed in Flow Weekly. This works out to about 156 pieces a Year. • A review of the CAP General Check List gives this presenter the impression that if each batch of Glassware cannot be check for Soap Residue, then an adequate percentage of the Glassware should be checked for Soap Residue. • Here in the Guthrie Flow Department a Random piece of Glassware is checked for Soap Residue Monthly by the Supervisor or Designee. This works out to be about 8% of the total Glassware Washed. 02/01/09

Guthrie Clinic Laboratories

14

Flow Cytometry QA Flow Cytometry Glass Cleaning Soap Residue Validation Chart

Date

Glassware Description

Result Of Test

Indicator Validation

• This Chart is used to Document the Checking of Flow Glassware for Soap Residue.

• (Once a Month)

02/01/09

Guthrie Clinic Laboratories

15

Flow Cytometry QA • The Flow Cytometry Reagent List is Updated Daily and Every Time there is a New Preparation of Sample Reagents (Super PBS, PBS, or Lyse Reagent), New Antibody Lot Numbers, and Instrument Calibration Beads. All are Documented on the Reagent List. • A Flow Cytometry Reagent List is Attached to Every Patient Sample Report. 02/01/09

Guthrie Clinic Laboratories

16

Flow Cytometry QA in 2002-2003 Guthrie Clinic Laboratories Flow Cytometry Reagent List Date: ____________November 12, 2002_____ _ Room Temp: _____21.0 degrees C______________ Sheath Fluid Lot#: _______1470_________________ Exp. Date: _____October 31, 2005 __________ Phos. Buff. Saline Lot#: _ 1127891 __________ Exp. Date: _____February 28, 2004 _________ 1:10 Working Dil. Prep: _____November 11, 2002 Exp. Date: _____November 18, 2002__________ Lysing Reagent Lot#: _____82091 ____ Exp. Date: _____March 31, 2004___ _______ 1:10 Working Dil. Prep: ___October 25, 2002______ Exp. Date: _____November 25, 2002 ____ _ 2% Paraformaldehyde Lot#: _ 516159 ___ Prep: October 15, 2002 __ Exp. Date: __November 15, 2002__ _ _ _ 1% Working Paraform Lot#: __ 516159 __________ Prep: November 12, 2002__ Exp. Date: __November 12, 2002__________ CaliBRITE Beads Lot#: _______45175 _______ Exp. Date: _January 31, 2003 ______________ Monoclonal Antibody Inventory as of Date: _______ _November 05, 2002___________

02/01/09

Reagent Vol. Vial Date Exp. Extra Vial Date Exp. Comp Lot# Opened Date Vial Lot# Opened Date Testing ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG2a/IgG1 (#340752) 1/3 40856A 09/03/02 03/31/2004 Yes 40856B 11/12/02 03/31/2004 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG1-PerCP (#340762) Full 38100 09/03/02 11/30/2003 09/03/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG1-APC (#340754) 1/3 29411 03/12/02 12/31/2002 Yes 40570 11/12/02 08/31/2003 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD3/CD8/CD45/CD4 (#340499) 2/3 43635 09/20/02 10/31/2003 Yes 46368 10/24/02 10/31/2003 10/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD3/CD16+56/CD45/CD19 (#340500) 7/8 41790A 08/02/02 11/30/2003 Yes 41790B 10/22/02 11/30/2003 10/22/02 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD14/CD64 (#341125) (1:2) 1/5 25677 09/19/01 11/30/2002 Yes 44804 10/22/02 02/28/2004 10/22/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD33-APC (#340680) 1/20 24797 09/19/01 09/30/2002 Yes 39366 07/23/02 04/30/2004 07/23/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------HLA-DR/CD13 (341145) (1:2) 1/2 35462 06/05/02 04/03/2003 Yes 48018 11/12/02 11/30/2004 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD34 ŠAPC (#340667) (1:12) Full 26828 11/12/01 03/31/2003 11/12/01 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD5/CD10/CD19 (#341139) Full 49222 10/30/02 05/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KAPPA/LAMBDA/CD19 (#341151) 7/8 40583 08/20/02 08/31/2003 08/20/02 KAPPA/LAMBDA (DAKO)-poly 1/3 091(101) 09/24/02 09/30/2004 09/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD20-APC (#340940) 1/20 40313 08/05/02 04/30/2003 Yes 47327 10/22/02 07/31/2003 10/22/02 CD19-PerCP-Cy5.5 (#340950) 2/3 46362 10/15/02 01/31/2004 10/15/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD2/CD7/CD3 (#341131) 2/3 39977 08/05/02 02/29/2004 08/05/02 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD25- APC (#340938) 1/5 35957 05/01/02 01/31/2003 Yes 44322 10/30/02 05/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------FMC7/CD23/CD19 (#341149) 7/8 42383 08/05/02 04/30/2003 08/05/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD5- APC (#340658) 1/5 32658 05/01/02 03/31/03 Yes 46856 10/30/02 10/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD103/CD22/CD20 (#341123) Full 44802 10/22/02 01/31/2004 10/22/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD11c Š APC (#340714) 1/3 41411 07/23/02 09/30/2003 Yes 41411 10/30/02 09/30/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD7/CD33 (#341141) (1:2) 1/2 26129 10/09/01 01/31/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD38/CD56/CD19 (#341133) (1:2) 7/8 40652 09/24/02 11/30/2003 09/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD38 Š APC (#340677) 7/8 43830 10/15/02 08/31/2004 10/15/02 CD45 Š APC (#340942) 2/3 33887 04/09/02 08/31/2003 Yes 48054 11/12/02 11/30/2003 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD15/CD117 (#341127) (1:2) 1/10 25960 09/19/01 11/30/2002 Yes 44562 10/30/02 09/30/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD45/Glycophorin-A/CD41a (1:4) Full 25812 09/19/01 01/31/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD10 Š APC (#340922) 1/20 24512 09/19/01 05/31/2003 Yes 39970 09/03/02 08/31/2003 09/03/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD4/CD25/CD3 (#341135) 7/8 25333 11/08/01 12/31/2002 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD45 - PerCP-Cy5.5 (#340952) 1/3 37532 06/28/02 09/30/2003 06/28/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD14 Š APC (#340684) 1/20 26285 01/02/02 02/28/2003 Yes 32976 06/05/02 06/30/2003 06/05/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PerCP-Cy5.5 Beads (#345036) Full 40513P 10/09/02 02/28/2003 Yes 40513P 02/28/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------APC Beads (#340487) Full 38159K 10/0902 04/30/2004 Yes 43179H 07/31/2004 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TdT Š FITC (SUPERTECHS) 2/3 01250 06/18/02 12/04/2003 06/18/02

• This Chart is the Flow Reagent List. • A Copy of the Reagent List is attached to all Flow Sample Reports. • The Reagent List is updated Daily and whenever a new lot number of Reagent is received. •

(Each Sample Preparation)

Guthrie Clinic Laboratories

17

Flow Cytometry QA • All New Lot#s of Flow Cytometry Antibodies are tested against the present (Old or In-Use) Antibodies before using the New Lot# of Antibody for Patient Testing. • The Percentage Difference between Old and New Lot#s of Reagents MUST be within the Laboratory’s Stated Acceptable Range (GCL = +5%) before the New Lot# is used for Patient Sample Testing.

02/01/09

Guthrie Clinic Laboratories

18

Flow Cytometry QA Guthrie Clinic Laboratories Flow Cytometry New Lot# Comparison Testing Studies Immunophenotyping Worksheet Date: ____________November 12, 2002_____ _ Room Temp: _____21.0 degrees C______________ Sheath Fluid Lot#: _______1470_________________ Exp. Date: _____October 31, 2005 __________ Phos. Buff. Saline Lot#: _ 1127891 __________ Exp. Date: _____February 28, 2004 _________ 1:10 Working Dil. Prep: _____November 11, 2002 Exp. Date: _____November 18, 2002__________ Lysing Reagent Lot#: _____82091 ____ Exp. Date: _____March 31, 2004___ _______ 1:10 Working Dil. Prep: ___October 25, 2002______ Exp. Date: _____November 25, 2002 ____ _ 2% Paraformaldehyde Lot#: _ 516159 ___ Prep: October 15, 2002 __ Exp. Date: __November 15, 2002__ _ _ _ 1% Working Paraform Lot#: __ 516159 __________ Prep: November 12, 2002__ Exp. Date: __November 12, 2002__________ CaliBRITE Beads Lot#: _______45175 _______ Exp. Date: _January 31, 2003 ______________ Monoclonal Antibody Inventory as of Date: _______ _November 05, 2002___________

Control ID#: ___________________________ MRN#: ___________________________ Reagent

Old Lot# Outdate

CD Marker

Old Lot# %-(+) Cells

New Lot# Outdate

CD Marker

• This Chart is an example of the Flow Cytometry Antibody Comparison Testing Worksheet.

New Lot# %-(+) Cells

(+5 to PASS)

(+5 to PASS)

(+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS)

• All New Lot#s of Flow Cytometry Antibodies are tested against the present (Old or InUse) Antibodies before using the new Lot# of Antibody for Patient Testing.

(+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS)

02/01/09

Guthrie Clinic Laboratories

19

Flow Cytometry QA Guthrie Clinic Laboratories Flow Cytometry Reagent List Date: ____________November 12, 2002_____ _ Room Temp: _____21.0 degrees C______________ Sheath Fluid Lot#: _______1470_________________ Exp. Date: _____October 31, 2005 __________ Phos. Buff. Saline Lot#: _ 1127891 __________ Exp. Date: _____February 28, 2004 _________ 1:10 Working Dil. Prep: _____November 11, 2002 Exp. Date: _____November 18, 2002__________ Lysing Reagent Lot#: _____82091 ____ Exp. Date: _____March 31, 2004___ _______ 1:10 Working Dil. Prep: ___October 25, 2002______ Exp. Date: _____November 25, 2002 ____ _ 2% Paraformaldehyde Lot#: _ 516159 ___ Prep: October 15, 2002 __ Exp. Date: __November 15, 2002__ _ _ _ 1% Working Paraform Lot#: __ 516159 __________ Prep: November 12, 2002__ Exp. Date: __November 12, 2002__________ CaliBRITE Beads Lot#: _______45175 _______ Exp. Date: _January 31, 2003 ______________ Monoclonal Antibody Inventory as of Date: _______ _November 05, 2002___________

02/01/09

Reagent Vol. Vial Date Exp. Extra Vial Date Exp. Comp Lot# Opened Date Vial Lot# Opened Date Testing ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG2a/IgG1 (#340752) 1/3 40856A 09/03/02 03/31/2004 Yes 40856B 11/12/02 03/31/2004 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG1-PerCP (#340762) Full 38100 09/03/02 11/30/2003 09/03/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IgG1-APC (#340754) 1/3 29411 03/12/02 12/31/2002 Yes 40570 11/12/02 08/31/2003 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD3/CD8/CD45/CD4 (#340499) 2/3 43635 09/20/02 10/31/2003 Yes 46368 10/24/02 10/31/2003 10/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD3/CD16+56/CD45/CD19 (#340500) 7/8 41790A 08/02/02 11/30/2003 Yes 41790B 10/22/02 11/30/2003 10/22/02 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD14/CD64 (#341125) (1:2) 1/5 25677 09/19/01 11/30/2002 Yes 44804 10/22/02 02/28/2004 10/22/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD33-APC (#340680) 1/20 24797 09/19/01 09/30/2002 Yes 39366 07/23/02 04/30/2004 07/23/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------HLA-DR/CD13 (341145) (1:2) 1/2 35462 06/05/02 04/03/2003 Yes 48018 11/12/02 11/30/2004 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD34 ŠAPC (#340667) (1:12) Full 26828 11/12/01 03/31/2003 11/12/01 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD5/CD10/CD19 (#341139) Full 49222 10/30/02 05/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KAPPA/LAMBDA/CD19 (#341151) 7/8 40583 08/20/02 08/31/2003 08/20/02 KAPPA/LAMBDA (DAKO)-poly 1/3 091(101) 09/24/02 09/30/2004 09/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD20-APC (#340940) 1/20 40313 08/05/02 04/30/2003 Yes 47327 10/22/02 07/31/2003 10/22/02 CD19-PerCP-Cy5.5 (#340950) 2/3 46362 10/15/02 01/31/2004 10/15/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD2/CD7/CD3 (#341131) 2/3 39977 08/05/02 02/29/2004 08/05/02 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD25- APC (#340938) 1/5 35957 05/01/02 01/31/2003 Yes 44322 10/30/02 05/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------FMC7/CD23/CD19 (#341149) 7/8 42383 08/05/02 04/30/2003 08/05/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD5- APC (#340658) 1/5 32658 05/01/02 03/31/03 Yes 46856 10/30/02 10/31/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD103/CD22/CD20 (#341123) Full 44802 10/22/02 01/31/2004 10/22/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD11c Š APC (#340714) 1/3 41411 07/23/02 09/30/2003 Yes 41411 10/30/02 09/30/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD7/CD33 (#341141) (1:2) 1/2 26129 10/09/01 01/31/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD38/CD56/CD19 (#341133) (1:2) 7/8 40652 09/24/02 11/30/2003 09/24/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD38 Š APC (#340677) 7/8 43830 10/15/02 08/31/2004 10/15/02 CD45 Š APC (#340942) 2/3 33887 04/09/02 08/31/2003 Yes 48054 11/12/02 11/30/2003 11/12/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD15/CD117 (#341127) (1:2) 1/10 25960 09/19/01 11/30/2002 Yes 44562 10/30/02 09/30/2003 10/30/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD45/Glycophorin-A/CD41a (1:4) Full 25812 09/19/01 01/31/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD10 Š APC (#340922) 1/20 24512 09/19/01 05/31/2003 Yes 39970 09/03/02 08/31/2003 09/03/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD4/CD25/CD3 (#341135) 7/8 25333 11/08/01 12/31/2002 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD45 - PerCP-Cy5.5 (#340952) 1/3 37532 06/28/02 09/30/2003 06/28/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CD14 Š APC (#340684) 1/20 26285 01/02/02 02/28/2003 Yes 32976 06/05/02 06/30/2003 06/05/02 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PerCP-Cy5.5 Beads (#345036) Full 40513P 10/09/02 02/28/2003 Yes 40513P 02/28/2003 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------APC Beads (#340487) Full 38159K 10/0902 04/30/2004 Yes 43179H 07/31/2004 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TdT Š FITC (SUPERTECHS) 2/3 01250 06/18/02 12/04/2003 06/18/02

Guthrie Clinic Laboratories Flow Cytometry New Lot# Comparison Testing Studies Immunophenotyping Worksheet Date: ____________November 12, 2002_____ _ Room Temp: _____21.0 degrees C______________ Sheath Fluid Lot#: _______1470_________________ Exp. Date: _____October 31, 2005 __________ Phos. Buff. Saline Lot#: _ 1127891 __________ Exp. Date: _____February 28, 2004 _________ 1:10 Working Dil. Prep: _____November 11, 2002 Exp. Date: _____November 18, 2002__________ Lysing Reagent Lot#: _____82091 ____ Exp. Date: _____March 31, 2004___ _______ 1:10 Working Dil. Prep: ___October 25, 2002______ Exp. Date: _____November 25, 2002 ____ _ 2% Paraformaldehyde Lot#: _ 516159 ___ Prep: October 15, 2002 __ Exp. Date: __November 15, 2002__ _ _ _ 1% Working Paraform Lot#: __ 516159 __________ Prep: November 12, 2002__ Exp. Date: __November 12, 2002__________ CaliBRITE Beads Lot#: _______45175 _______ Exp. Date: _January 31, 2003 ______________ Monoclonal Antibody Inventory as of Date: _______ _November 05, 2002___________

Control ID#: ___________________________ MRN#: ___________________________ Reagent

Old Lot# Outdate

CD Marker

Old Lot# %-(+) Cells

Guthrie Clinic Laboratories

New Lot# Outdate

CD Marker

New Lot# %-(+) Cells

(+5 to PASS)

(+5 to PASS)

(+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS) (+5 to PASS)

20

Flow Cytometry QA

• The Becton Dickinson FACSCalibur is the Flow Cytometer that is used at Guthrie for Sample Data Acquisition. • FACSCalibur Instrument Checks are Documented each time the instrument is used for Testing.

02/01/09

Guthrie Clinic Laboratories

21

Flow Cytometry QA _________=Total Laser Hours To Date  (Includes:  _____________)

Serial Number:  E 4225 

Reviewed by:  ____________________ ____________________

FACSCalibur Cytometer Daily Maintenance Log



Month:  _________________

Date

This Chart is used to Log all FACSCalibur Instrument Checks.

Time On Prime Fluidics Laser Curnt F­Comp Volts No Tube Tube (Low) Tube (Med) Tube (High) Bleach Soln 5 Minutes DH2O 5 Minutes Time Off



(Each Time Instrument Used)

Laser Hrs Daily Total Hours For Month Operators Initials

Monthly Maintenance Procedure BYPASS THE SHEATH FILTER Instrument Notes: 1.  Remove sheath reservoir and disconnect sheath filter. 2.  Install a spare reservoir with 1­2 L of 1:5 dilution of bleach. 3.  Connect sheath tubing, bypassing sheath filter.  Prime flow cell 3 times. 4.  Install 1:10 dilution of bleach on the SIP and run for 15 min. on HI flow rate. 5.  Repeat steps 2­4 using DI Water for 25 min. 6.  Reconnect the original sheath reservoir and sheath filter. 7. Replace the AIR Filter.  Clean dirty air filter.

Initials & Date:  _________________________________

02/01/09

_____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________

Guthrie Clinic Laboratories

22

Flow Cytometry QA • B/D CaliBRITE 3 and APC beads are tested using the FACSComp application on our B/D FACSCalibur cytometer instrument. • The beads check target settings and determine the PMT voltages for FL1, FL2, FL3, and FL4. • The application then makes compensation adjustments on the beads fluorescence.

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Guthrie Clinic Laboratories

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Flow Cytometry QA

02/01/09



B/D CaliBRITE 3 and APC Beads are tested using B/D FACSComp.



Run Daily.



PMT voltages and Fluorescence Compensation are checked for significant day to day variation.



Changes in PMT voltages and Compensation may be indicators to the Operator that an instrument problem exists.

Guthrie Clinic Laboratories

24

Flow Cytometry QA • CD-Chex CD34 Whole Blood Control Manufactured by Streck Laboratories INC. is used as our System Process Assayed CD34 Control Material. • Streck offers a Quality Control Program for this Product. We fax our data to their Statistics Department. When the data has been compared to other users, Streck emails the Flow Supervisor that the information is available for Printing and/or Downloading. • The following Chart is used to track the Control Data and Document any Comments such as Antibody Lot Number Changes and Gating (Manual or Auto) Used. 02/01/09

Guthrie Clinic Laboratories

25

Flow Cytometry QA

CD­34 Positive Control Log Chart

Lot#: __________________

Expiration Date: ______________

Becton Dickinson FACSCalibur Flow Cytometer 

 

=  Out of Range Parameter

Date

Tech Abs.#CD34/uL CD34% Abs. WBC Cnt. Comments: Analysis Init. Gating Target: _______ (+7.0) _______(+0.10) _______ (+1.0) Used Range: ( ) ( ) ( ) ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

• This Chart is used to Document the Streck CD34 Positive Control Results.

• The Absolute CD34 and CD34 Percent is recorded along with the Control’s WBC Count Result.



(Each Day CD34 Counts are Performed)

Reviewed by: ____________________

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Guthrie Clinic Laboratories

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Flow Cytometry QA • The CD34 Control Whole Blood Statistics that come back from Streck include the Mean, SD, and CV, for the CD34%, CD34#, and WBC for our Laboratory and the Other Laboratories in the Program. • The Data is evaluated by the Supervisor and then reviewed by the Director of Flow Cytometry.

02/01/09

Guthrie Clinic Laboratories

27

Flow Cytometry QA • The next Chart is used to keep a running tabulation of the types of specimens and what days of the week Flow is processing samples. • The Data is used to trend Flow ordering patterns. It shows what days of the week Flow is the busiest. It is used as a tool to ensure optimal coverage at times of heaviest workload.

02/01/09

Guthrie Clinic Laboratories

28

Flow Cytometry QA

Flow Sample Statistics By Day Of The Week Green Ink = Rejected Sample (See Flow Log Book for Details)

Year: ______________ Sample Type ImmunoComp Samples

Monday

Red Ink

Tuesday

= Proficiency Testing Samples

Wednesday

Thursday



The Types of Flow Samples and what day of the week the Sample is Prepared is Documented.



This Data is Compiled and Used to Generate the Flow Sample Statistics.



(Sample Type by Day of the Week)

Date Range: _______________ Friday

Summary

(PB) Leukemia Lymphoma Samples

(BM) Leukemia Lymphoma Samples Lymph Node Tissue Leukemia Lymphoma (CD-34) Absolut Counts Fluids/BAL Leukemia Lymphoma ___________________________________________________________________________________________ ___________________________________________________________________________________________

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Guthrie Clinic Laboratories

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Flow Cytometry QA • State Reportable Results • The Guthrie Clinic Flow Laboratory is located in Northeastern Pennsylvania, three miles from the New York State Border. • We analyze samples from both Pennsylvania and New York State. • Both PA and NY have different CD4% and Absolute CD4 Count values that require reporting to the state’s Department of Health.

02/01/09

Guthrie Clinic Laboratories

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Flow Cytometry QA NYS DOH Flow Reportable Testing Audit Data Patient MRN

02/01/09



A random check of Sample Results that require reporting to the state is done at least twice a year to assure that the State Deportment’s of Health are getting the reportable information sent to them from our Laboratory.

• • • • •

Patient Information Date Sample was Collected Date Processed Date Reported to the Doctors Office Date Reported to the Dept. of Health

Date Date Requesting Date Date Sample Sample Office Office NYS DOH Collected Processed Location Notified Notified

Guthrie Clinic Laboratories

31

Flow Cytometry QA

• In Conclusion: • The Information Documented on the Charts used in Flow helps to assure accurate results and is a major part of the Guthrie Clinic Flow Cytometry Quality Assurance Process.

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Guthrie Clinic Laboratories

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