Control materials and performance

We can think of the concept of stability in two aspects:

• Stability guaranteed by the manufacturer — from the manufacturing process, storage and transportation under recommended conditions, to delivery to the laboratory. Developing and delivering a stable product are the manufacturer's commitments and all of its recommendations must be strictly observed.
• Stability guaranteed by the user — in the storage and use of the product, so that it preserves the characteristics it had when produced and thus fulfills its role as a reference for control.

1.1 Freeze-dried Control Reconstitution

Reconstitution is a very critical process. The inaccuracy of the volumetric material used to reconstitute or dilute the controls will be reflected in the result obtained.

The use of solvent/diluent (demineralized water, saline solution, etc.) of compromised quality, applied in reconstitution, can interfere in an unpredictable way with the results of the analyzes to which this material will be subjected.

Control results will vary from vial to vial of the same batch if reconstitution fails in any way. Therefore, this process must be well standardized and carried out by a qualified and qualified person. Class A volumetric pipette, reagent-type deionized water and manufacturer's instructions for the reconstitution process must be used.

Standardization is key. Only with processes defined with a SOP, with a clear description of the use of the volumetric pipette and other processes, can good results be achieved.

The laboratory must create a SOP that establishes the rules for storage, preparation and use of control materials, the criteria for introducing new lots of controls and reagents and that defines control limits.¹

1.2 Fractionation and Storage

Fractionation for storage provides rationalization of use according to laboratory needs, preserving the stability of the entire product, especially for materials stored frozen.

The size of the aliquot in each fractionation must be established by the laboratory itself, according to its usage needs, in a rational use that considers the need for the analyzer and the costs. The aliquots must always be stored tightly sealed, under the conditions defined by the manufacturer, observing the different stability times for the different analytes.

The containers used for storage must be appropriate (cryotubes) in order to prevent concentration of the sample by evaporation of the solvent. Samples kept in containers with poor sealing, in any condition (frozen or not), suffer the effects of concentration due to solvent evaporation. Small aliquots should not be stored in large volume containers.

The identification of each rate must be rigorous. It must contain the control name, lot, level and expiration date after reconstitution. As it is difficult to apply all this information in small containers, the professional may find it useful to make records in a From → To table, which contains an acronym or code related to the data on the side.

The small tube would be labeled 1LabQC and the other 2LabQC, identifying the material for level 1 and level 2, for correct positioning in the analyzer.

1.3 Freezing

More than the physical appearance of hardened material, the freezing of different preparations must be guaranteed according to the temperature recommended by the manufacturer. To achieve the stability(s) recommended by the manufacturer, storage temperatures must be strictly respected.

Apparent freezing often occurs at temperatures higher than those recommended, and there is no guarantee of preservation of the material's characteristics.

1.4 Defrost

The sample thawing process can be accelerated using a cold water bath. It should not be accelerated through heating, as this may accelerate the degradation of a component.

Care must be taken to ensure that the samples, after thawing, are adequately homogenized, avoiding excessive agitation at this time. Do not use vortex. Homogenization is more difficult for small volumes stored in small containers. A tube containing frozen material should not be opened until it has been adequately homogenized.

1.5 Sample conditions in the analyzer cuvette

The homogeneity of the sample must be guaranteed before transferring it to the analyzer. The use of non-homogeneous samples leads to inadequate results, leading to random errors. The presence of air bubbles in the control sample in the cuvette may provide incorrect results, with random errors (violation of 1:3s and R:4s rules).

1.6 Environmental exposure

Samples kept uncovered are subject to the evaporation process, which will increase the concentrations of the analytes. The intensity of evaporation depends on the environmental conditions (low relative humidity) and the container where the sample is contained (opening width of the container). Well-ventilated and/or air-conditioned environments allow sample concentration by evaporation of the solvent. The analytical run must be started as soon as the controls are uncapped and placed in the analyzer cuvettes.

Avoid leaving the vial on the bench after transferring it to the cuvette. If kept open, concentration occurs — deviation towards more, on the graph. If kept closed, degradation occurs — downward deviation.

Control residues after use by the equipment must not be returned to the original vials or even stored for reuse.

When planning for the use of control materials, one should consider purchasing sufficient volume for six months — or more, if possible — of the same batch. This is justified as a cost reduction factor, as it eliminates the need to frequently carry out the preparation phase (the laboratory's own values) in a superposition process before finalizing the batch in use. This strategy may require planning with the supplier, logistics for partial delivery or storage by the laboratory, in addition to adjustments in the payment method.

The mean and standard deviation values vary from batch to batch, even from a given manufacturer, which requires a new preparation phase to determine the laboratory's own values. So the laboratory will have several vials of materials from the same batch and it is understood that these vials will provide material with adequate stability.

It is estimated that, if there is standardization in the reconstitution process for each vial, the variation in results from vial to vial in the same batch corresponds to a small fraction of the total variation that can occur — which is almost entirely due to measurement imprecision.

This stability is ensured if the reconstitution of each vial follows strict standards, carried out with the utmost care. If there is a failure to reconstitute a vial, the product's characteristic of little variability from vial to vial is lost.

Assayed control material is the most common in Brazil. It is provided with a specification of which analytes for what it was designed to control and with so-called package insert values, for average (target value) and recommended range for variation.

For quantitative, statistical internal control, the range is not suitable, making it necessary to calculate the standard deviation. The standard deviation value is fundamental to statistics and is the basis of all computer programs for IQC.

The manufacturer's values should only be used in the initial stage (preparation phase), until the laboratory's own values are determined, as they were obtained in analytical environment conditions different from those existing in the laboratory.

In a system of good quality and stability, the variability measured by the laboratory is smaller than that suggested by the range offered by the manufacturer, which is usually wide. Performing the IQC with limits within the range provided by the manufacturer (label values) is not considered a good practice, as it is based on limits that are not those of the laboratory and is therefore not very sensitive for detecting errors.

The unassayed material differs in that it does not present the manufacturer's average and range values. It can be used in the same way as assayed control material, by previously determining the laboratory values and then adopting the limits for the Levey-Jennings graph. The use of unassayed material is not yet common in Brazil.

The same method as for the use of unassayed material applies to materials produced with pool of stable serum, which would be an alternative form. It is necessary to guarantee the stability of the material used, which is a critical point of high relevance. It is necessary to highlight for stable serum pool the need to adopt precautions, as we are dealing with potentially infectious material.

We often refer to control levels when talking about Internal Quality Control, and this has special meaning. When planning IQC, the laboratory must consider that it is important to monitor its system so that it is stable in detecting concentrations at levels of medical interest. In clinical chemistry, two levels are most often used, one at the reference range and the other at the pathological level. The CLSI² document suggests a minimum of two levels, in general, but at other times an additional level may be necessary to adequately monitor performance, such as in hematology.

Each level of control is ensured by a material, since we are dealing with analyte concentrations. That's why two materials are needed — one for each level — if we are going to work with two levels. If we take Glucose as an example, we must have a material with a value of around 80 mg/dl (level 1) and another material with a value of around 140 mg/dl (level 2). There would then be two control determinations per day for Glucose, one in the sample of each material.

Developing IQC with at least two levels allows for greater sensitivity of the method for detecting nonconformities, as well as greater effectiveness when identifying the type of error and the root cause.

A control material, depending on the substances included in its composition, can exhibit different results compared to different methodologies, depending on the formulation of the reagents and equipment used in the measurement.

These differences occur due to the effects of the matrix used — material of human origin or other origin —, incorporated additives, freeze-drying or other processes used in its production.

It is considered that ideally the control material has the same matrix as the specimen to be tested. Materials prepared from human sources have been used, but there are cited preferences for using materials of bovine origin, given the potential for infectious risks with the former.³

There are other variables due to the specificities of the methodology that require the use of specific materials for specific techniques. An example is what can happen with CKMB through the immunoinhibition method, given the refinement of the technique. In cases like this, the laboratory must adopt control materials recommended by the reagent manufacturer.

The matrix effect cannot be eliminated, but its impact on the assessment of imprecision tends to disappear when the laboratory determines its own values in a phase before judging the performance of the analytical system — the so-called preparation phase. This reinforces the need for the laboratory to determine its own mean parameters and limits and not use the manufacturer's range as a reference to judge their systems.

Historical context of the revoked standard. According to RDC 302 item 9.2.2 and 9.2.2.1, the text in force at the time allowed the use of commercial control samples, regularized with ANVISA/MS, or alternative forms described in the literature as long as they allowed the assessment of the precision of the analytical system.⁵

The choice of supplier must take into account several factors and must be part of quality planning. The selection of materials and respective suppliers must be based on analyzes involving costs, material stability, ease of use, matrix, the analytes present in the material and the possibility of obtaining the same batch for a long period of time — if possible, for a year.

A good relationship with the supplier and the latter's forecast for its customers' material consumption will help it to have a supply plan, ensuring good service.

Some suppliers offer data analysis services, software for IQC, intra-group comparisons, etc. It is clear that the costs of software and services are added to the costs of purchased materials and these "packages" usually result in some degree of lock-in, making it mandatory to purchase the entire line from the same supplier. The laboratory will always judge whether this business model is appropriate for its IQC, or whether it will be more convenient to avoid lock-in.

Suppliers are constantly evolving in the production and availability of materials that can meet the growing control needs of laboratories. Good interaction with your supplier helps the laboratory manager to make the best choices.