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Lot Conversion for CaCl2?

From Cynthia Hollis on 2-2-24: I had someone ask me why I didn’t do a lot conversion for changing lots of CaCl2 that I use in my APTT assays. I was unable to give them a satisfactory answer. Can you explain it for me, please?

From Geo: Hi, Cynthia, thanks for your question. I checked with my colleague Robert Gosselin, who is a validation expert. [See Bob’s ICSH article.] He says he’s never done lot-to-lots for CaCl2 but apparently, ISO requires all buffers and CaCl2 to be evaluated.

From Dave McGlasson, 2-5-24:

George, I have talked to people from Stago, Diapharma, and Werfen and found nothing about having to validate between kits. However, CAP does have something.  So I talked to Dr. John Olson. He says, “This is one of those questions that you don’t want to ask because you might not like the answer you get.” However, he is going to ask the resource committee to get a ruling on that.  Might take a couple of days. I also talked to Dr. Russell Higgins about this. Manufacturers do not mix different lot numbers with kits. I guess if you are using an LDT you would validate the method each time. I would definitely do it if you were making the CaCL2. However, who does that any more? This is one of those questions where you are looking for a problem. We did not address this issue when we wrote H57-A Protocol for the Evaluation, Validation, and Implementation of Coagulometers; Proposed Guideline. Clinical and Laboratory Standards Institute. 2007.

We’d be interested to learn how others do this as part of their lot-to-lot validations. Comments, please.

Comments (3)
Screening Assays
Mar 7, 2024 9:46am

Thanks to Denise Driscoll, Sarah Lieske, Stacy Meyer, and Lyn Wielgos, College of American Pathologists [CAP] for these comments:
Lieske: CaCl2 is used in the PTT test and it starts the clotting process. For the Siemens analyzer, the CaCl2 is a liquid reagent and most laboratories sequester a lot for six months or a year so they will be on the same lot for a while before switching to a new lot. The HEM requirement says that QC must be performed every eight hours and when there is a change in reagents. At the bottom of the note says every eight hours and with new lots/shipments. The onboard stability of the PTT reagents is usually 1–2 days so it is unlikely that the reagents are being changed out frequently. The reagents are generally changed out on a schedule and would require QC before they begin patient testing for the next 8-hour period. The reagents may also be changed out to troubleshoot when QC is out of control.
Quoting HEM.37300: Coagulation Quality Control
Phase II
The laboratory performs controls using two different levels of control material every eight hours of patient testing and each time there is a change in reagents, or more frequently if specified in manufacturers’ instructions, laboratory procedure, or the CAP Checklist. This includes photo-optical, electromechanical, and manual methods.
For manual methods [ie, tilt tube method], controls must be performed by each individual who performs the tilt tube test in the same eight-hour period.
If an internal quality control process [eg, electronic/procedural/built-in] is used instead of an external control material to meet daily quality control requirements, the laboratory must have an individualized quality control plan [IQCP] ) approved by the laboratory director defining the control process, including the frequency and use of external and internal controls. At a minimum, external control materials must be analyzed with new lots and shipments of reagents or more frequently if indicated in the manufacturers’ instructions. Please refer to the IQCP section of the All Common Checklist for the eligibility of tests for IQCP and requirements for implementation and ongoing monitoring of an IQCP.
Evidence of Compliance:
Records of QC results including external and internal control processes AND manufacturer product insert or manual.
1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Medicare, Medicaid and CLIA programs; CLIA fee collection; correction and final rule. Fed Register. 2003(Jan 24):5232 [42CFR493.1269(b)].
2) Steindel SJ, Tetrault G. Quality control practices for calcium, cholesterol, digoxin, and hemoglobin. A College of American Pathologists Q-Probes study in 505 hospital laboratories. Arch Pathol Lab Med 1998;122:401-408
3) Voss EM, et al. Determining acceptability of blood glucose meters. Statistical methods of determining error. Lab Med. 1996;27:601-606
4) Clinical and Laboratory Standards Institute (CLSI). Statistical Quality Control for Quantitative Measurement Procedures: Principles and Definitions. 4th ed. CLSI guideline C24. Clinical and Laboratory Standards Institute, Wayne, PA, 2016.
5) Ye JJ, et al. Performance evaluation and planning for patient/client-based quality control procedures. Am J Clin Pathol.2000;113:240-248
6) LaBeau KM, et al. Quality control of test systems waived by the clinical laboratory improvement amendments of 1988. Perceptions and practices. Arch Pathol Lab Med. 2000;124:1122-1127
7) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24): [42CFR493.1269(b) & 42CFR.493.1269(c)(2)]
8) Department of Health and Human Services, Centers for Medicare and Medicaid Services. S & C: 16-20-CLIA: Policy Clarification on Acceptable Control Materials Used when Quality Control (QC) is Performed in Laboratories. April 8, 2016.
Meyer: Is the CACL2 being used on a Coag instrument? We used to load it on for PTT testing. We did not do anything other than load in the instrument. I can’t remember if we had to run QC with changes of CACL2 or not.
Wielgos: COM.30450 includes content in the note about how the requirements apply to different types of reagents. Typically saline and reagent water are inert ingredients. Buffers could potentially be part of a chemical reaction. If it was used for cleaning, that would be different. CaCL2 sounds like it would be used to produce a chemical reaction. I welcome comments from others if I didn’t quite hit the mark.
COM.30450 New Reagent Lot and Shipment Confirmation of Acceptability – Nonwaived Tests
Phase II
New reagent lots and shipments are checked against previous reagent lots or with suitable reference material before or concurrently with being placed in service. This requirement applies to reagents that provide a chemical or biological reaction to detect and/or measure a target analyte and would not apply to inert ingredients (eg, reagent water, saline) or materials used for specimen preparation. The purpose of this check is to confirm that the use of new analytic reagent lots and shipments (including different shipments of the same lot) do not affect patient results. Matrix interferences between different lots of reagents may impact the calibration status of instruments and the consistency of patient results. Improper storage conditions during the shipping of reagents may have a negative impact on their ability to perform or exhibit the same levels of reactivity as intended.
The minimum extent of the reagent check is described below; however, the check must be at least as extensive as described in the manufacturer’s instructions. The laboratory may determine the number of specimens tested.
Qualitative: For qualitative nonwaived tests, minimum cross-checking includes retesting at least one positive and negative specimen with known reactivity against the new reagent lot. Utilization of a weakly positive specimen is recommended for confirmation of acceptability.
Examples of suitable reference materials for qualitative tests include:
Positive and negative patient specimens tested on a previous lot;
Previously tested proficiency testing materials;
External QC materials tested on the previous lot (eg, antigen testing kit controls, immunohematology antisera and reagent red cells)
Control strains of organisms or previously identified organisms for microbiology reagents used to detect or evaluate cultured microorganisms;
If none of the above options is available, control material provided by the assay manufacturer with the new test kit.
There are more specific requirements in other checklists for some types of qualitative testing, such as:
Flow cytometry antibodies and reagents (FLO.23325)
Microbiology media and stains, disks/strips, antimicrobials, and reagents (eg, MIC.11038, MIC.21540, MIC.21560, MIC.21624, MIC.21626, MIC.21910, MIC.65320)
Immunohistochemistry antibody and detection system reagents (ANP.22760, CYP.04380, BAP.05363).
Quantitative: For quantitative nonwaived tests, patient specimens are preferred for comparing a new lot against the previous lot, when possible. Manufactured materials, such as proficiency testing (PT) or QC materials may be affected by matrix interference between different reagent lots, even if results show no change following a reagent lot change. The use of patient specimens confirms the absence of matrix interference. The following materials may be used:
Patient specimens tested on a previous lot
Reference materials or QC products provided by the method manufacturer with method specific and reagent lot specific target values
Proficiency testing materials with peer group established means
QC materials with peer group established means based on interlaboratory comparison that is method specific and includes data from at least 10 laboratories
Third-party general purpose reference materials if commutable with patient specimens for the method (per package insert or method manufacturer)
QC material in use with the current reagent lot to check a new shipment of the same reagent lot (There should be no change in potential matrix interactions with use of the same lot number of reagent and QC material).
For hematology analyzers, reservoirs containing testing reagents and cleaning/decontaminating solutions must be checked according to manufacturer’s instructions.
Evidence of Compliance:
Records for the introduction of new lots and shipments, including lot number(s) tested and comparison of results to the acceptability criteria
1) Clinical and Laboratory Standards Institute (CLSI). Evaluation of Matrix Effects. 4th ed. CLSI document EP14. Clinical and Laboratory Standards Institute, Wayne, PA; 2022.
2) Clinical and Laboratory Standards Institute. Verification of Comparability of Patient Results within One Healthcare System: Approved Guideline (Interim Revision). CLSI document EP31-A-IR. Clinical and Laboratory Standards Institute, Wayne, PA; 2012.
3) Miller WG, Myers GL, Rej R. Why commutability matters. Clin Chem. 2006;52:553-554
4) Clinical and Laboratory Standards Institute (CLSI). User Evaluation of Acceptability of a Reagent Lot Change. 2nd ed. CLSI guideline EP26. Clinical and Laboratory Standards Institute. Wayne, PA; 2022.

Robyn Coleman
Mar 4, 2024 9:34pm

In practical terms for CaCl2 I would agree that changes in QC should be sufficient to detect any system errors that a different lot of CaCl2 may cause. Lot numbers should be logged.

Feb 8, 2024 4:59pm

I’m with John Olson on this; don’t ask and plead ignorance. If your CaCl2 forms part of your APTT kit, then it would be included in the APTT lot evaluation. If not, the easiest way to validate the CaCl2 is to run APTT assays with the current vs new CaCl2 lots and show ‘equivalence’ by linear regression/Bland Altman plots. I suspect 40-60 samples over a wide range of values would suffice; alternatively, if you could show no changes to the control values (both normal & pathological) across a change of CaCl2, that may also suffice We used to manufacture our own CaCl2 and then it was compulsory to evaluate lot changes. With modern APTT reagents, the CaCl2 may be part of the kit, and thus can be included with the APTT reagent evaluation.

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