TBD Checklist C - Method 1623_1623.1 Technical Review_Sample

Checklist C – Method 1623/1623.1 Technical Review – Sample Processing and Microscopy
Laboratory Name

Name and Affiliation of Evaluator

Date of Evaluation

Good Laboratory Practice (GLP) is generally defined as a system of management controls for the laboratories to ensure the consistency and reliability of results. Adapted
from other federal programs for the purposes of the Cryptosporidium Laboratory QA Evaluation Program, GLP includes personnel, equipment, and standard operating
procedures appropriate for the program.

Item to be evaluated

1
1.1

1.2

Reference*

Classification

1623

1623.1

Cert

Does laboratory appear to have
established appropriate safety and health
practices prior to use of this method?

5.0

5.0

4.1

Critical

Do all laboratory personnel wear gloves
when handling biohazard and toxic
compounds, and change gloves before
touching other surfaces and equipment?

5.3
5.4

5.3

4.1.6

Critical
GLP

Yes

Laboratory Facilities

1.3

Does the laboratory disinfect bench
surfaces before and after analyses?

-

-

4.1.3

Critical
GLP

1.4

Does the laboratory have adequate bench
space to perform the method?

-

-

2.0

Critical
GLP

1.5

Other than the issues noted in items 1.1
through 1.4 (if any), no other facility issues
were observed?

7.3

7.3

4.3.1

Requirement

-

-

4.2.2

Critical
GLP

2

Reagents

2.1

Is reagent water used to prepare all
reagents?

2.2

Are all reagents clearly labeled with
identity of reagent, date of preparation,
technician initials, and expiration date?

Satisfactory

G-24

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

2.3

3
3.1

3.2

Cert

Are SOPs available in the work area, and
does laboratory practice reflect written
procedures?

Was spike suspension vial vortexed for 30
seconds or per manufacturer’s
instructions?
Is the carboy used for method blank
randomly selected from carboy stock to
check efficacy of cleaning system?
Was the suspension vial adequately
rinsed?

3.4

Are SOPs for sample spiking available in
the work area, and does laboratory
practice reflect written procedures?

3.5

Other than issues noted for items 3.1
through 3.4 (if any) was sample spiking
demonstrated successfully?

Technician:
11.4.3.1.2

11.2.3.2

-

Method Procedure

-

-

7.1.5.3

Critical
GLP

11.4.3.1

11.2.3

-

Method Procedure
Critical
GLP

Filtration/Elution
Technician:

4.1 Envirochek ® HV filtration
Are all components required for
sample filtration present and in
good condition?

6.1
6.2.1-6.2.2
6.3

6.1 6.2.8

6.1.7

Requirement
GLP

4.1.2

Is the filter assembly set up
correctly?

Figure 3a

Figure 1

-

Method Procedure
GLP

4.1.3

Is the pump adequate for needs?

6.3.3

6.2.4

-

Requirement
GLP

4.1.4

Is the appropriate flow rate
maintained (approximately 2
L/min)?

12.2.1.2

12.2.1.2

-

Method Procedure

Is the volume filtered measured
using a flow totalizer or calibrated
carboy?

12.2.4.2

12.2.4.2

-

Requirement

4.1.1

4.1.5

Satisfactory
Yes

Critical
GLP

Sample Spiking

3.3

4

1623.1

Classification

G-25

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*

Classification

1623

1623.1

Cert

Is the system well maintained
and cleaned appropriately
following use?

4.5

4.5

6.1.7

Critical
GLP

4.1.7

Is the system able to maintain
seal during use with no leaks?

-

-

6.1.7

Requirement
GLP

4.1.8

Are SOPs for Envirochek® HV
filtration available in the work
area, and does laboratory
practice reflect written
procedures?

4.1.6

4.1.9

Yes

Critical
GLP

Other than issues noted for items
4.1.1 through 4.1.8 (if any) was
Envirochek® HV filtration
demonstrated successfully?

4.2 Envirochek® HV capsule filter elution

Technician:

4.2.1

Is the elution buffer prepared as
per Method?

7.4.1

7.6.1

-

Method Procedure

4.2.2

Is the wrist-shaker assembly set
up correctly with arms fully
extended?

12.2.6.1.1

12.2.6.1

3.14.2

Method Procedure
GLP

-

12.2.7

-

1623
Recommendation
1623.1 Method
Procedure

Is volume of elution buffer
measured to ensure the use of
one 250 mL centrifuge tube?

12.2.6.2.2

12.2.8.2

-

Method Procedure

4.2.5

Are the samples shaken at an
appropriate speed?

12.2.6.2.3

12.2.8.3

3.14.3

Method Procedure

4.2.6

Are the samples shaken three
times for 5 minutes each time,
and each in a different
orientation?

12.2.6.2

12.2.8

-

Method Procedure

4.2.3

4.2.4

Is the dispersant addition
performed as per Method
1623.1?

Satisfactory

G-26

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

4.2.7

4.2.8

1623.1

Classification
Cert

Are SOPs for Envirochek® HV
capsule filter elution available in
the work area, and does
laboratory practice reflect written
procedures?

Yes

Critical
GLP

Other than issues noted for items
4.2.1 through 4.2.7 (if any) was
Envirochek® HV capsule filter
elution demonstrated
successfully?

4.3 Filta-Max®filtration

Technician:
®

4.3.1

Which filter is used – Filta-Max
(black end caps) or Filta-Max
xpress® (red end caps)?

4.3.2

Are all components required for
sample filtration present and in
good condition?

6.1
6.2.1
6.2.3
6.3

6.1
6.2.1-6.2.7
6.2.9

6.1.7

Requirement
GLP

4.3.3

Is the filter assembly set up
correctly?

Figure 3b

Figure 2

-

Method Procedure
GLP

4.3.4

Is appropriate flow rate
maintained of <4 L per minute for
Filta-Max®?

12.3.1.1.3

12.3.1.1.3

-

Method Procedure

Is the volume filtered measured
correctly using a flow meter or
calibrated carboy?

12.3.1.5.2

12.3.1.5.2

-

Requirement
GLP

Is system well maintained and
cleaned appropriately following
use?

12.3.4

12.3.4

6.1.7

Requirement
GLP

4.3.7

Is system able to maintain seal
during use with no leaks?

-

-

6.1.7

Requirement
GLP

4.3.8

Does the laboratory indicate on
the filter housing the correct
direction of flow?

12.3.1.3

12.3.1.3

-

Critical

4.3.5

4.3.6

Satisfactory

G-27

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

4.3.9

4.3.10

1623.1

Classification
Cert

Are SOPs for Filta-Max® filtration
available in the work area, and
does laboratory practice reflect
written procedures?

Yes

Critical
GLP

Other than issues noted for items
4.3.1 through 4.3.9 (if any) was
Filta-Max® filtration demonstrated
successfully?

4.4 Filta-Max® filter wash station elution

Technician:

4.4.1

Is an automatic or manual wash
station used?

4.4.2

Is the filter wash station set up
correctly?

12.3.2.1

12.3.2.1

-

Requirement
GLP

4.4.3

Is residual suspension rinsed
from all containers?

12.3.2.2.1d

12.3.2.2.1d

-

Critical

4.4.4

Is PBST used to elute the filter?

7.4.2.4

7.6.2.4

-

Method Procedure

4.4.5

Is an appropriate amount of
PBST used for each wash?
(approx. 600 mL)

12.3.2.2

12.3.2.2

-

Method Procedure

During the first wash, is the
plunger moved up and down 20
times?

12.3.2.2.1h

12.3.2.2.1h

-

Method Procedure

Is the plunger moved up and
down gently to avoid generating
excess foam?

12.3.2.2.1h

12.3.2.2.1h

-

Method Procedure

During the second wash, is the
plunger moved up and down 10
times?

12.3.2.2.2b

12.3.2.2.2b

-

Method Procedure

If the automatic washer is used,
is the machine operating
properly?

12.3.2.1

12.3.2.1

-

Requirement

Is the wash station cleaned
adequately between samples?

12.3.4.2

12.3.4.2

-

Requirement
GLP

4.4.6

4.4.7

4.4.8

4.4.9

4.4.10

Satisfactory

G-28

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

4.4.11

4.4.12

5

1623.1

Classification
Cert

Are SOPs for Filta-Max® filter
wash station elution available in
the work area, and does
laboratory practice reflect written
procedures?

Yes

Critical
GLP

Other than issues noted for items
4.4.1 through 4.4.11 (if any) was
Filta-Max® filter wash station
elution demonstrated
successfully?

Concentration

5.1 Filta-Max® filter sample concentration

Technician:
12.3.3.2.1b

12.3.3.2.1a

-

Requirement
GLP

NOTE
pg 43

NOTE
pg 34

-

Method Procedure

Is concentration performed after
each of the washes?

12.3.2.2.1j

12.3.2.2.1j

-

Method Procedure

Is the sample concentrated so
that some liquid remains above
the filter (enough to cover the stir
bar about half-way)?

12.3.3.2.1c

12.3.3.2.1b

-

Method Procedure

Are the stir bar and concentration
tube rinsed after each
concentration and the liquid
added to the concentrate?

12.3.3.2.1c

12.3.3.2.1b

-

Requirement

5.1.6

Was the filter membrane washed
twice with 5 mL of PBST?

12.3.3.2.3

12.3.3.2.3

-

Method Procedure

5.1.7

Are SOPs for Filta-Max® filter
sample concentration available in
the work area, and does
laboratory practice reflect written
procedures?

5.1.1

Is concentrator set up correctly?

5.1.2

Is the force of the vacuum
maintained below 30 cm Hg?

5.1.3
5.1.4

5.1.5

Satisfactory

Critical
GLP

G-29

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

5.1.8

1623.1

Classification
Cert

Yes

Other than issues noted for items
5.1.1 through 5.1.7 (if any) was
Filta-Max® filter sample
concentration demonstrated
successfully?

5.2 Envirochek® HV and Filta-Max® filter sample centrifugation

Technician:

Is the sample centrifuged at 1500
x G (maximum 2000 x G) using a
swinging bucket rotor?

13.2.1
and
NOTE
pg 46

13.2.1
and
NOTE
pg 37

-

Method Procedure
GLP

5.2.2

Are the centrifuge tubes properly
balanced prior to centrifugation?

-

13.2.1

3.15.4

Critical

5.2.3

Does lab have easily accessible
method for determining relative
centrifugal force of centrifuges?

-

-

3.15.1

Critical
GLP

Is the sample centrifuged for 15
minutes, with time beginning
when centrifuge reaches desired
speed?

13.2.1

13.2.1

-

Method Procedure

Is the centrifuge slowly
decelerated at the end without
the brake?

13.2.1

13.2.1

-

Method Procedure

5.2.6

Is the pellet volume determined?

13.2.1

13.2.1

5.2.3

Requirement

5.2.7

Is there a set of standards for
comparison of pellet size?

-

-

5.2.3

Recommendation
GLP

5.2.8

Are SOPs for Envirochek® and
Filta-Max® filter sample
centrifugation available in the
work area, and does laboratory
practice reflect written
procedures?

5.2.1

5.2.4

5.2.5

5.2.9

Satisfactory

Critical
GLP

Other than issues noted for items
5.2.1 through 5.2.8 (if any) was
Envirochek® HV or Filta-Max®
filter sample centrifugation
demonstrated successfully?

G-30

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*
1623

6

1623.1

Classification
Cert

Purification and Slide Preparation

6.1

Is an approved IMS kit/manufacturer
used?

6.2

Is the supernatant from the centrifuged
sample aspirated no lower than 5 mL of
supernatant above every 0.5 mL pellet or
portion of 0.5 mL pellet?
6.2.1

6.2.2

Yes
Technician:

7.5

7.7.1

-

Method Procedure
GLP

13.2.2

13.2.2
13.2.3

5.2.2
5.2.3

Requirement

Are the samples aspirated using
the pipette, with the documented
internal diameter, as specified in
the SOP?

-

NOTE
pg 37

-

Critical

Is the proper rate (mL/min) or
pressure (psi) maintained
throughout aspiration?

-

13.2.2
13.2.3

-

Method Procedure

6.3

Is the pellet vortexed a sufficient time for
resuspension?

13.2.3
13.2.4.1.3
13.2.4.2

13.2.2.1
13.2.3.1.2
13.2.3.2

-

Method Procedure

6.4

Is the resuspended pellet volume
quantitatively transferred to the flat-sided
tube (2 rinses)?

13.3.2.1

13.3.2.1

-

Method Procedure

Are the IMS beads thoroughly
resuspended prior to addition to the flatsided tube?

13.3.2.2
13.3.2.4

13.3.2.2
13.3.2.4

-

Method Procedure

6.6

Is the flat-sided tube rotated at 18 rpm for
1 hour at room temperature?

13.3.2.6

13.3.2.6

-

Method Procedure

6.7

Is the rotating mixer calibrated annually?

-

-

3.17.4

Critical
GLP

6.8

Is flat-sided tube correctly placed in
magnet and rocked through 90 degrees
about once per second?

13.3.2.713.3.2.9

13.3.2.713.3.2.9

-

Method Procedure

Is all the liquid removed when decanting is
performed with the magnet up?

13.3.2.11

13.3.2.11

-

Method Procedure

13.3.2.13

13.3.2.14

-

Method Procedure

6.5

6.9

6.10 Is the sample quantitatively transferred
from the flat-sided tube to the
microcentrifuge tube (2 rinses)?

Satisfactory

G-31

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

6.11 Are the beads rinsed with PBS while
inside the microcentrifuge tube?

Reference*

Classification

1623

1623.1

Cert

13.3.4

13.3.2.17

-

Yes
1623
Recommendation
1623.1 Requirement

6.12 Is standard NaOH (5 µL, 1N) and standard
HCl (50 µL, 0.1N) used?

NOTE
pg 49 & 50

NOTE
pg 42

3.17.5

Requirement
GLP

6.13 Is sample vortexed vigorously for 50
seconds immediately after the addition of
acid and 30 seconds after the sample has
set for 10 minutes at room temperature?

13.3.3.213.3.3.4

13.3.3.213.3.3.4

-

Method Procedure

6.14 Is a second dissociation performed?

13.3.3.10
NOTE
pg 49

13.3.3.10
NOTE
pg 41

5.2.4

Requirement

13.3.3.10

13.3.3.10
13.4.5

-

13.3.3.7

13.3.3.7

-

6.15 When the second dissociation is
performed, does the laboratory:
(A) use a second slide
(B) add the additional volume to the
original slide?
6.16 Are the slides clearly labeled so they can
be associated with the correct sample?

Circle one:
A

B

Requirement

6.17 What type of slides is used?

GLP

6.18 Is slide dried at: (A) room temperature, (B)
35º to 42ºC, or (C) in the refrigerator?

13.3.3.12

13.3.3.12

-

6.19 If the slide is warmed, is incubator or slide
warmer calibrated and labeled?

-

-

3.4

6.20 Are SOPs available in the work area for
sample purification and slide preparation,
and does laboratory practice reflect written
procedures?

Circle one:
A

B
Critical
GLP
Critical
GLP

6.21 Other than issues noted for items 6.1
through 6.20 (if any) was purification and
slide preparation demonstrated
successfully?

7
7.1

Sample Staining
What staining kit/manufacturer is used?

Technician:
14.2

14.2

Satisfactory

3.18.1

GLP

G-32

C

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

Reference*

Classification

1623

1623.1

Cert

Yes

7.2

Is FITC stain applied according to
manufacturer’s directions?

14.2

14.2

5.3.2

Method Procedure

7.3

Are positive and negative staining controls
performed?

14.1

14.1

5.3.5

Requirement

7.4

Are the slides incubated in a humid
chamber in the dark at room temperature
for approximately 30 minutes or per
manufacturer’s directions?

14.3

14.3

5.3.3

Method Procedure

Are the labeling reagents rinsed away
properly after incubation, without
disturbing the sample?

14.5

14.5

-

Method Procedure

7.6

Was the working DAPI stain prepared the
day it was used?

7.7.2

7.9.2

3.19.2

Method Procedure

7.7

Is stock DAPI stored at 1 to 10oC in the
dark?

7.7.1

7.9.1

3.19.1

Method Procedure

7.8

Is the DAPI stain applied properly and
allowed to stand for a minimum of 1
minute?

14.6

14.6

-

Method Procedure

Is the DAPI stain rinsed away properly
without disturbing the sample?

14.7

14.7

-

Method Procedure

14.8

14.8

-

Method Procedure

7.5

7.9

7.10 Is the mounting media applied properly?
7.10.1

What type of mounting media is
used?

7.8

7.10

-

GLP

7.10.2

Are all the edges of the cover slip
sealed well with clear fingernail
polish, unless Elvanol® is used?

7.9
14.9

7.11
14.9

-

Method Procedure

14.10

14.10

5.3.6

Method Procedure

7.11 Are the finished slides stored in a humid
chamber in the dark at 1 to 10oC (humid
chamber not required for Elvanol®)?
7.12 Are SOPs for sample staining available in
the work area, and does laboratory
practice reflect written procedures?

Satisfactory

Critical
GLP

7.13 Other than issues noted for items 7.1
through 7.12 (if any) was sample staining
demonstrated successfully?

G-33

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

8
8.1

8.2

8.3

8.4

Reference*

Classification

1623

1623.1

Cert

Is microscope equipped with appropriate
excitation and band pass filters for
examining FITC labeled specimens as
demonstrated with lab, and auditor
provided, positive staining control?

6.9.2

6.7.2

3.22.3

Requirement
GLP

Is microscope equipped with appropriate
excitation and band pass filters for
examining DAPI labeled specimens as
demonstrated with lab, and auditor
provided, positive staining control?

6.9.3

6.7.3

3.22.3

Requirement
GLP

Does the microscope have appropriate
objectives and filters for DIC, which
change easily to and from
epifluorescence?

6.9.1

6.7.1

3.22.4

Requirement
GLP

Are all portions of the microscope, from
the light sources to the oculars, properly
adjusted?

10.3

10.0
Appendix B

3.22.6

Requirement

-

Figure 4

Visual
Guide

Requirement

Yes

Microscope and Examination

8.5

Is the DIC image appropriate for each
laboratory microscope?

8.6

Is microscope cleaned after every
session?

10.4

10.9.8

3.22.11

Requirement
GLP

8.7

Does the microscope have a 20X
scanning objective?

6.9.1

6.7.1

3.22.8

Requirement
GLP

8.8

Does the microscope have a 100X oil
immersion objective?

6.9.1

6.7.1

3.22.8

Requirement
GLP

8.9

Is the microscope equipped with an ocular
micrometer?

6.9.1

6.7.1

3.22.9

Requirement
GLP

6.9.1
10.3.5

6.7.1
App. B 3

3.22.9

Requirement

8.11 Is a calibration table for 100X objective
located close to the microscope(s)?

10.3.5.7

App. B 3.7

3.22.9

Requirement

8.12 Has the mercury bulb been used less than
the maximum hours recommended by the
manufacturer?

10.3.2.11

App.B 1.11

3.22.12

Requirement

8.10 Is a stage micrometer available to
laboratory?

Satisfactory

G-34

No

NA

UNK

Comments/
Response Requested

Item to be evaluated

8.13 Does the laboratory have a preventative
maintenance agreement in place to
service the microscope annually?
8.14 Are SOPs for sample examination
available in the work area, and does
laboratory practice reflect written
procedures?

Reference*

Classification

1623

1623.1

Cert

-

-

3.22.6

Satisfactory
Yes

Critical
GLP

Critical
GLP

8.15 Other than issues noted for items 8.1
through 8.13 (if any) was Microscope and
Examination demonstrated successfully?

G-35

No

NA

UNK

Comments/
Response Requested

9
9.1

9.2

9.3

Positive Staining Control and OPR Slides
Does the laboratory’s positive staining
control slide contain (oo)cysts at the
appropriate fluorescence intensity for
FITC?

15.2.1.3

15.2.1.3

5.4.8
5.4.9.2
5.4.10.2

Requirement

Does the laboratory’s positive staining
control slide contain (oo)cysts at the
appropriate fluorescence intensity for
DAPI?

15.2.1.3

15.2.1.3

5.4.8
5.4.9.3
5.4.10.3

Requirement

-

-

5.4.3

Recommendation

14.1.1
15.2.1.3

14.1.1
15.2.1.3

7.1.8.1

Requirement

-

Figure 4

Visual
Guide

Requirement

Does the laboratory’s OPR slide contain
(oo)cysts at the appropriate fluorescence
intensity for FITC?

15.2.2.1
15.2.3.1

15.2.2.2
15.2.3.2

5.4.9.2
5.4.10.2

Requirement

Does the laboratory’s OPR slide contain
(oo)cysts at the appropriate fluorescence
intensity for DAPI?

15.2.2.2
15.2.3.2

15.2.2.3
15.2.3.3

5.4.9.3
5.4.10.3

Recommendation

Does the laboratory’s OPR slide contain
an appropriate level of background
fluorescence?

-

-

5.4.3

Requirement

Does the laboratory’s OPR slide exhibit
appropriate contrast and organism
features by DIC?

9.7.1.1

9.8.1.1
Figure 4

Visual
Guide

Requirement

10.6.3.1

9.10.3.1

7.1.9.4

Requirement

Does the laboratory’s positive staining
control slide contain an appropriate level
of background fluorescence?

9.4

Is concentration of oocysts on the positive
staining control slide appropriate?

9.5

Does the laboratory’s positive staining
control exhibit appropriate contrast and
organism features by DIC?

9.6

9.7

9.8

9.9

9.10 Does the technical auditor’s count of
Cryptosporidium oocysts and Giardia
cysts on the OPR slide sent by the
laboratory agree within 10% of laboratory
count?

Comments:

G-36

10 Onsite Sample Processing
Method Step

Name

Position

Demonstrated Technique
Successfully
yes/no

Spiking – (filter type)
Filtration - (filter type)
Spiking flat-sided tube, and
processing IMS control
Aspiration and transfer from 250 mL
bottle

11 Onsite Blind Spike Results
Sample

Crypto Spike
Value

Crypto Count

Crypto
Recovery (%)

Giardia Spike
Value

Giardia Count

Giardia
Recovery (%)

12 Evaluation of Onsite Sample Processing and Blind Spike Results – Comments and Recommendations
Classification

Comments

G-37

Response Requested

13 Was analyst microscope operation acceptable? (yes/no)

Name

Position

Requirement

Requirement

Requirement

Method 1623: 10.3.4.1

Method 1623: 10.3.4.2-3

Method 1623: 10.3.6

Method 1623.1: 10.7.1

Method 1623.1: 10.7.2-3

Method 1623.1: 10.8

Adjust Interpupillary
Distance

Focus both eyepieces

Establish Kohler Illumination

14 Slide Count and Analyst Verification Results (yes/no)

Analyst

Requirement

Requirement

Method 1623:
10.6.3.1

Method 1623:
10.6.3.1

Method 1623.1:
9.10.3.1

Method 1623.1:
9.10.3.1

Crypto Count Within
10% of Target Count

Giardia Count
Within 10% of
Target Count

Requirement
Method 1623: 15.2
Method 1623.1: 15.2
Examine and Record
Characteristics

Requirement

Requirement

Method 1623: 15.2.2.3
15.2.3.3

Method 1623: 15.2.2.3
15.2.3.3

Method 1623.1: 15.2.2.4
15.2.3.4

Method 1623.1: 15.2.2.4
15.2.3.4

Measurement (100X)

Demonstrated Internal
Structures

15 Evaluation of Analyst Microscopy and Examination Skills – Comments and Recommendations
Classification

Comments

Response Requested

G-38