2.2.4. Evaluation

Evaluation is performed from IQC and EQA results. IQC results are obtained from every assay while EQA results are obtained from every cycle of the program. Laboratory performance was also determined by conducting evaluation using the Six Sigma quality metrics which focused on the laboratory achievement as compared to the world-class level of Sigma-6 [10]. Evaluation should be done periodically.

#### 2.2.5. Improvement

Corrective actions are made upon every occurrence of non-conformance. Corrective actions are meant to troubleshoot problems and prevent them from being repeated. Relevant IQC rules [11] are to be obeyed and corrective actions are done accordingly to improve quality of test. Improvement may lead to better laboratory practices and the cycle of the Quality System (Figure 3) continues as it gets better throughout time.

A well-managed laboratory quality system will enable good laboratory practice, assessment of method, instruments and laboratory performance, and will help the interpretation of respondent results by knowing the accuracy of the method used for Urinary Iodine measurement.

In defining the scope of method, all method validation data should be noted [9] including the expected precision and accuracy and method robustness. The type of equipment to be used as listed in the instrument maintenance section should be noted. The method is applicable to all laboratories possessing the three main instruments, i.e. the heating block, microplate reader, single channel and multichannel micropipette. It is also important that the heating blocks are placed in a fume hood during sample digestion at 100C for 1 hour so that any fume accumulated can be channeled out from the laboratory for safety purposes. Another vital issue is the skills and competency of the operators which determine the high precision and accuracy of results especially on the pipetting which ensures excellent replicates.

#### 2.3. Urinary iodine analysis

#### 2.3.1. Urinary iodine micromethod

Urinary iodine is a biochemical indicator in monitoring iodine deficiency disorders (IDD) [12]. Urinary iodine is measured using the urinary iodine micromethod (UIMM) which was modified to improve method used in the urinary iodine laboratories in the country [9]. The modified method offers minimal expenditure for new devices, usage of less hazardous chemicals and lesser amount of chemical waste produced. Through method validation, comparison plot and difference plot [11] had been prepared for UIMM against the urinary iodine measurement method proposed by the World Health Organization (WHO) [12]. From the comparison plot (Figure 4), we are ensured that the UIMM works well and it is comparable to the WHO method with excellent correlation coefficient (r) of 0.9428. From the difference plot (Figure 5), the performance of UIMM is shown with not much difference from the WHO method with

only two out of 50 readings with biases of more than �22 μg/l. Other method validation includes (i) sensitivity: 13.809 μg/l, (ii) intra-assay precision: 5–13%, (iii) inter-assay precision:

Figure 5. Difference plot between the modified method and the WHO method (image reproduced with permission of the

Mainly, there are three main solutions used in the UIMM, namely ammonium persulfate, arsenious acid and ceric ammonium sulfate solutions (Table 1). The former oxidizes the urine samples

The main steps in the UIMM are sample digestion and Sandell-Kolthoff reaction [reaction formulas (1) and (2)]. Urine digestion eliminates the interferences which may cause false positive in the analysis [13]. Arsenite in the presence of iodine reduces yellow-colored ceric ions to colorless cerous ions. Thus, by spectrophotometrical measurement, the absorbance is

Urinary iodine determination incorporation of two steps of action, i.e. urine digestion at high

Successful analytical procedures are supported by good pre-analytical (involves documents, chemicals, consumables and glassware) and post-analytical processes (involves records and

! 2Ce3<sup>þ</sup> <sup>þ</sup> I2 ð Þ colorless

As<sup>3</sup><sup>þ</sup> <sup>þ</sup> I2 ! As<sup>5</sup><sup>þ</sup> <sup>þ</sup> 2I� (1)

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(2)

7–15%, (iv) linearity: correlation coefficient (r) = 0.993, and (v) recovery: 106–114% [9].

and the two latter solutions contribute to the execution of the Sandell-Kolthoff reaction.

inversely correlated with the concentration of urinary iodine.

rights holder, Tropical Biomedicine).

temperature and iodine measurement in Sandell-Kolthoff reaction of:

2Ce4<sup>þ</sup> <sup>þ</sup> 2I� ð Þ yellow

reports). These processes are discussed further in the subsequent subsections.

The procedural steps for UIMM comprised of four steps as depicted in Figure 6 [9].

Figure 4. Comparison plot of the modified method versus WHO method (image reproduced with permission of the rights holder, Tropical Biomedicine).

In defining the scope of method, all method validation data should be noted [9] including the expected precision and accuracy and method robustness. The type of equipment to be used as listed in the instrument maintenance section should be noted. The method is applicable to all laboratories possessing the three main instruments, i.e. the heating block, microplate reader, single channel and multichannel micropipette. It is also important that the heating blocks are placed in a fume hood during sample digestion at 100C for 1 hour so that any fume accumulated can be channeled out from the laboratory for safety purposes. Another vital issue is the skills and competency of the operators which determine the high precision and accuracy of

Urinary iodine is a biochemical indicator in monitoring iodine deficiency disorders (IDD) [12]. Urinary iodine is measured using the urinary iodine micromethod (UIMM) which was modified to improve method used in the urinary iodine laboratories in the country [9]. The modified method offers minimal expenditure for new devices, usage of less hazardous chemicals and lesser amount of chemical waste produced. Through method validation, comparison plot and difference plot [11] had been prepared for UIMM against the urinary iodine measurement method proposed by the World Health Organization (WHO) [12]. From the comparison plot (Figure 4), we are ensured that the UIMM works well and it is comparable to the WHO method with excellent correlation coefficient (r) of 0.9428. From the difference plot (Figure 5), the performance of UIMM is shown with not much difference from the WHO method with

Figure 4. Comparison plot of the modified method versus WHO method (image reproduced with permission of the

results especially on the pipetting which ensures excellent replicates.

2.3. Urinary iodine analysis

72 Quality Control in Laboratory

rights holder, Tropical Biomedicine).

2.3.1. Urinary iodine micromethod

Figure 5. Difference plot between the modified method and the WHO method (image reproduced with permission of the rights holder, Tropical Biomedicine).

only two out of 50 readings with biases of more than �22 μg/l. Other method validation includes (i) sensitivity: 13.809 μg/l, (ii) intra-assay precision: 5–13%, (iii) inter-assay precision: 7–15%, (iv) linearity: correlation coefficient (r) = 0.993, and (v) recovery: 106–114% [9].

Mainly, there are three main solutions used in the UIMM, namely ammonium persulfate, arsenious acid and ceric ammonium sulfate solutions (Table 1). The former oxidizes the urine samples and the two latter solutions contribute to the execution of the Sandell-Kolthoff reaction.

The main steps in the UIMM are sample digestion and Sandell-Kolthoff reaction [reaction formulas (1) and (2)]. Urine digestion eliminates the interferences which may cause false positive in the analysis [13]. Arsenite in the presence of iodine reduces yellow-colored ceric ions to colorless cerous ions. Thus, by spectrophotometrical measurement, the absorbance is inversely correlated with the concentration of urinary iodine.

Urinary iodine determination incorporation of two steps of action, i.e. urine digestion at high temperature and iodine measurement in Sandell-Kolthoff reaction of:

$$\text{As}^{3+} + \text{I}\_2 \rightarrow \text{As}^{5+} + 2\text{I}^- \tag{1}$$

$$\text{2Ce}^{4+} + \text{2I}^{-} \rightarrow \text{2Ce}^{3+} + \text{I}\_{2} \tag{2}$$

The procedural steps for UIMM comprised of four steps as depicted in Figure 6 [9].

Successful analytical procedures are supported by good pre-analytical (involves documents, chemicals, consumables and glassware) and post-analytical processes (involves records and reports). These processes are discussed further in the subsequent subsections.


beakers, glass marbles) is required to avoid carryover of the leftovers of iodine in the glassware into subsequent assay. Current practice of soaking glassware in distilled water overnight for two consecutive days after washing with detergent is adequate to remove iodine residues and

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During the digestion procedure of the urinary iodine assay, it is essential that heat be distributed evenly across the heating block on every test tube. Check for even heat distribution can be achieved by placing twenty test tubes filled with 2 ml oil or sand and inserted with a thermometer each. Heating block should achieve 100�C before placing the test tubes and heated for 20 minutes. Temperature of each thermometer should be recorded and mean � SD is calculated. The temperatures recorded across the digestion unit should fall within the manu-

The uniformity absorbance reading of the microplate reader maintenance checking is done by pipetting 200 μl of 1:1500 green food coloring in water in the first row (Row A) of a 96-well microtiter plate. Mean, SD, 1.5SD and CV of the readings are then determined. A scatter plot should be graphed for the individual readings with horizontal lines for mean and mean � 1.5SD. The number of readings outside mean � 1.5SD is determined and its percentage is calculated. Percentage error should be ≤20%. If it is not achieved, the maintenance check should be repeated and a request for calibration or repair should be lodged if problem persists. Maintenance check

To check the micropipette performances, a maintenance-check-up every three-monthly is performed. Three points of volume should be tested, i.e. within the lowest, middle and highest ranges. For example, if the micropipette volume range is 100–1000 μl, then the pipette should be checked at 100 and next time around at 500 or at 1000 μl; it is up to the operator to decide. Water with the chosen volume should be pipetted into 10–20 clean disposable test tubes (LP3 or LP4 tubes). The weight of the tubes with and without water is recorded accordingly. Other information that should be recorded includes the brand and model of pipette, its code number, date of maintenance check-up and name of the person carrying it out. Mean, standard deviation (SD) and coefficient variation (CV) of the readings are determined. Inaccuracy is also

Mean � 100%

The maintenance check-up is repeated if the CV is >5% and inaccuracy is >10%. If the problem persists, request for instrument check-up and calibration should be lodged for further action.

Set Volume � 100%

facturer's stated temperature distribution range. CV should be �5%.

to ensure cleanliness for usage in the next assay.

2.3.3. Maintenance of equipment

2.3.3.1. Heating block

2.3.3.2. Microplate reader

2.3.3.3. Micropipette

determined as follows:

• % Coefficient of Variation (CV) = SD

• % Inaccuracy = ð Þ calculated mean�set volume

up every three-monthly has to be performed.

Table 1. Purpose of each chemical addition in the urinary iodine micromethod (UIMM).

Figure 6. Diagram showing steps in UIMM assay.

#### 2.3.2. Chemicals, consumables and glassware

Chemicals to be used for urinary iodine measurement should be more than or equivalent to analytical reagent-grade. Consumables to be used are non-sterile while the 96-well microtiter plates can be used either of flat- or round-bottom polystyrene ones. Microtiter plate lids should be covered with aluminum foil to prevent direct light onto reaction mixture. Sandell-Kolthoff reaction is sensitive to heat [14]. Thorough washing of glassware (test tubes, volumetric flasks, beakers, glass marbles) is required to avoid carryover of the leftovers of iodine in the glassware into subsequent assay. Current practice of soaking glassware in distilled water overnight for two consecutive days after washing with detergent is adequate to remove iodine residues and to ensure cleanliness for usage in the next assay.
