Auditing Cryptosporidium/

Giardia Laboratories

Debra Waller

NJDEP-Office of Quality Assurance

debra.waller@dep.state.nj.us

August 2013

Note: This presentation does not address all of the

requirements of the analytical methods (1623 or

1623.1)

For example the presentation does not address

microscopic calibration, the IMS portion and only

refers to some of the portions of the examination

phase (typical size, shape and magnification used).

Note: This presentation is also based on the use of an

*Envirocheck® HV filter for sample filtration, *Easy

Stain®, and that the laboratory has performed the

required Initial Demonstration of Capability (IDC). Also

all negative and positive staining controls were

acceptable and monthly analyst verifications are

performed as required and a digital photo library is

retained by the single Principal Analyst for the

purposes of this presentation. Control charts are

retained for OPR and MS data as required.

*Inclusion of these products are for example only and not as an endorsement.

Sample Volumes

• Must collect and filter at least 10L of sample (method verified for 10L and 50L samples only).

• Volume must be noted in the bench sheet to the nearest 0.25L.

Note: A laboratory can use other sample volumes provided that the laboratory demonstrates equivalent or superior performance through an approved initial demonstration of capability (IPR, MS/MSD for 1623.1, OPR and MB) prior to changes being made for the analysis of regulatory samples. For the purposes of this presentation the laboratory has not performed an IDC for alternative volumes.

Sample Collection and Filtration Records

• Must maintain a record of:

o Sample number or ID

o Sample turbidity (if not provided, must be tested)

o Sample type and date and time of collection

o Date and time of receipt in the laboratory

o Temperature at receipt (<20°C and not frozen)

o Sample filtration start date and time

o Name/initials of the person that filtered the sample (client or lab)

o Lot number and number of filters used for the sample filtration

Holding Times • 4 days (96 hours) between collection/filtration and

elution

• 1 working day between elution and application of

sample to slide

• 3 days (72 hours) between application to slide and

staining

• 7 days (168 hours) between staining and examination

Example Required QC Samples

Ongoing Precision and Recovery (OPR)

• Must be tested at a frequency of 1 per every 20

samples tested or portions thereafter if more than 20

samples are analyzed in a week.

• Must be spiked with ~100-500 oocysts/cysts (o/c)

each week that samples are tested.

Example (Con’t)

• More than 50% of the o/c must appear

undamaged and morphologically intact

• Must meet %R for C in the range of 33-100% and 22-

100% for G.

• OPR is to be processed through the entire

procedure.

Method Blank • To be processed after the OPR.

• Must be analyzed each week (7 days or 168 hours,

time period begins with the processing the OPR).

• Must be free of o/c and any potentially interfering

organism or material.

• Every field sample must be associated with an

acceptable OPR and MB.

Matrix Spike (MS) Sample Analysis

• Must be performed for one of every twenty field

samples tested from the same individual sources

analyzed.

• Volume of original sample to sample volume used

for the MS must be within 10%.

• Must be associated with an acceptable OPR and

Method Blank (MB) analysis.

• Recovery must meet 32-100% for C and 8-100% for

G.

Sample # MS %Recovery C/G

S. Vol.

MS. Vol.

MS-1 10/12 10.0 12.0

1 N/A N/A

2 N/A N/A

3 N/A N/A

4 N/A N/A

5 N/A N/A

6 N/A N/A

7 N/A N/A

8 N/A N/A

9 N/A N/A

10 N/A N/A

11 N/A N/A

12 N/A N/A

13 N/A N/A

14 N/A N/A

15 N/A N/A

16 N/A N/A

17 N/A N/A

18 N/A N/A

19 N/A N/A

20 N/A N/A

21 N/A N/A

22 N/A N/A

23 N/A N/A

24 N/A N/A

25 N/A N/A

MS-2 11/15 10.0 11.5

What problems do we see with this MS data? *MS samples are not being performed at the correct frequency. *The acceptance criteria is outside range. *The sample volume to MS volume is not within 10%.

Reagents • 1.0 NaOH and 0.1N HCl solutions must be purchased with

a lot specific certificate of analysis retained, and the lot number in use noted in the raw data records (tracking of the origin of use). Normality cannot be adjusted.

• Working elution buffer solutions must be used within one week of preparation.

• Reagents cannot be frozen including bead suspensions. Light sensitive materials need to be stored in the dark between 1 to 10 degrees C.

• Working DAPI solution is to be made daily with 50µl of 2mg/ml into 50ml of phosphate buffer (PBS) solution.

• PBS (pH 7.4 defined in 1623.1, no final pH in 1623) is to be made with saline.

Reagents

• Diluted and unused working reagents must be discarded after 48 hours or following manufacturer’s instructions. Working elution buffer can be stored for one week.

• Several manufacturer’s have been approved for use, any alternative would require an acceptable IDC prior to use with regulatory samples.

• The same elution buffer batch must be used for all samples associated in the sample set. Prepare enough working elution solution to process all samples including the original OPR and MB that was run for the sample set and any additional OPR samples that would be required for >20 samples in the same day of analysis.

• Reagent water must not contain Cd, Cr, Cu, Pb, Ni or Zn in concentrations of >0.05mg/L to be acceptable for use.

Name Lot or Batch ID

Final pH Prep Tech. Exp. Date Storage Prep. Date.

Working DAPI

Batch 1-10-13 NA DW 1-15-13 Freezer 1-10-13

PBS Batch 1-10-13 7.6 DW 1-10-14 4 C 1-10-13

1.0N NaOH Batch 1-10-13 NA DW 5-10-13 Room temp 1-10-13

0.1N HCl Batch 1-10-13 NA DW 5-10-13 Room temp 1-10-13

Working DAPI

Batch 2-3-13 NA DW 2-8-13 On bench in the light

2-2-13

PBS 7.7 DW 4 C 3-10-13

Working DAPI

Batch 1 NA DW 3-15-14 freezer 3-10-13

Easy Stain Batch 4 NA DW 3-15-13 100 µl of 2 mg/ml stock in 50ml PBS-freezer

3-10-13

Elution Buffer Batch 1 NA DW 4-6-13 4 C 3-29-13

Bead Suspensions

Lot number in receipt file

NA Freezer

What is wrong with this Reagent Prep information?

Reagents are inappropriately stored in the freezer. Storage is beyond one week . The pH for the PBS is >7.4 (1623.1) and information is missing for the 3/10/13 batch The lab is preparing the 1.0N NaOH and 0.1N HCl solutions. The working DAPI solution is not made daily and is being stored in the light.

Date Single Limit

Cadmium Copper Lead Nickel Zinc

Jan. 2005 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.02mg/L 0.01 mg/L 0.01 mg/L

Jan. 2006 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.03 mg/L 0.01 mg/L 0.01 mg/L

Jan. 2007 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.03 mg/L 0.01 mg/L 0.01 mg/L

Jan. 2008 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.02mg/L 0.01 mg/L 0.01 mg/L

Jan. 2009 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.02mg/L 0.01 mg/L 0.01 mg/L

Jan. 2010 < 0.05 mg/L 0.005 mg/L 0.001 mg/L 0.02mg/L 0.01 mg/L 0.01 mg/L

Jan. 2011 < 0.05 mg/L 0.005 mg/L 0.07 mg/L 0.04 mg/L 0.01 mg/L 0.01 mg/L

Jan. 2012 < 0.05 mg/L 0.005 mg/L 0.09 mg/L 0.05 mg/L 0.01 mg/L 0.05 mg/L

Jan. 2013 < 0.05 mg/L 0.005 mg/L 0.10 mg/L 0.06 mg/L 0.05 mg/L 0.10 mg/L

What is wrong with this reagent water testing information?

Results for some are <0.05mg/ L and collectively are >0.10 mg/L. The lab is not testing the water for chromium as required.

General Record Keeping • The FITC, DAPI and DIC characterizations and size

measurements must be recorded for all positive results for field samples tested.

• Report zero organisms if no oocysts or cysts are observed.

• Final results are to be reported as oocycts or cysts per liter and are to be adjusted to reflect the number resulting form the analysis of a 10 or 50L sample (or other approved volume).

• Processed packed pellet volume cannot exceed 0.5ml. If total is >0.5 ml then separate 0.5 ml sub-samples must be tested.

• Record the number of subsamples on the bench sheet.

Magnification Required Procedural Step: Minimum Mag. Preferred or Required Mag. • FITC and counting 200X 200X • DAPI 400X 1000X (Note in 15.2 of 1623/1623.1) • DIC 1000X 1000X

• Measurement 1000X 1000X

Typical Size and Shape for Test Organisms

Cryptosporidium 4 to 6 µm round to oval Giardia 8 to 18 µm long by 5 to 15 µm wide, oval to round *Measurements to be recorded to the nearest 0.5 µm.