Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

General Introduction

     Preparation of low-level QC serum

Start with 500ml of clear serum. Measure potassium level. Calculate the final volume, to which 500 mg of this serum must be diluted to adjust the potassium level to 3.5 mmol/L.

Initial volume 500 ml

Final volume (x) ml

Initial K+ value (e.g) 6.3 mmol/L

Final K+ 3.5 mmol/L

Therefore, 500 x 6.3 = 3.5 x (x)

x= 500 x 6.3/3.5 = 900 ml

15% of 900 ml = 135 ml

Extra volume to be) = 400 ml
added (900-500

Ethanediol = 135 ml

Therefore, distilled water
to be added (400-135) = 265 ml.

In actual practice, although potassium concentration will be diluted to 3.5 mmol/L, the levels of some other constituents will become too low. Therefore the levels of these need to be raised to some extent so that these will be maintained at low level but not too low. Out of the 265 ml distilled water, 25 ml may be used for dissolving desired quantities of such of those constituents that need to be added to the serum to raise their amount to the desired low levels.

     Adjustment of analytes

Glucose, urea, creatinine, sodium (NaCl) and potassium (KCl): -

Dissolve each of these in 5 ml distilled water and add to the main bulk of serum.

Calcium :- Use dried CaCO₃. Dissolve in 5 ml in 1N HCL and add to the serum.

After adding all the analytes mix the contents well and centrifuge the serum once again for 10 minutes at 3500 rpm. Collect the supernatant serum in a sterile one-litre flask. Mix the contents well and dispense the serum into 5 ml sterile penicillin vials, seal the vials and store at –20⁰C. Laboratories can decide on the volume of aliquots depending on their requirements.

     Construction of Levey Jennings Chart

On each day when analyses are performed a fresh sample is thawed, thoroughly mixed and analysed. (Remember: Ethanediol-treated serum may not freeze completely at –20⁰C; however, the constituents are quite stable). The QC serum is analysed for a period of 20 days or so. [Important to note: Analysis should not be carried out by only one person; all staff should participate in this exercise to determine the true unavoidable error in the laboratory]. From these data, mean and SD are calculated. Levey Jennings chart is then constructed with x + 2SD as warning limits and x + 3 SD as control limits.

Calculate the %CV for each analyte to ascertain whether this is within the acceptable limit (Ideal = < 5%. Must be definitely < 8%). If % CV is found to be high, this will indicate that between-day laboratory precision (variation) is high and the data cannot be used to construct a Levey Jennings chart. It is then essential to identify the causes for this, correct these and then repeat the whole exercise and confirm that the %CV is well within the acceptable limit.

Table 1 shows precision data obtained by a WHO trainee from a developing country for routine biochemical analytes through analysis of ethanediol-treated QC serum for 20 days, while undergoing training at the author’s laboratory recently.

Table 1

     Interpretation of QC data

A.      According to WHO⁽⁶⁾an analytical system is ‘out of control’ if one of the four criteria is met. That is:

     a value lies entirely outside the control limits

     seven consecutive values show a rising tendency

     seven consecutive values show a falling tendency

     seven consecutive values lie on the same side of the mean

If one of these situations arises, the patients’ results must be discarded, the cause of the error sought and removed, and then the batch repeated with a QC serum.

B. Use of two different levels of QC simultaneously in every batch of analysis provided valuable information on the type of errors – whether these are random (precision) errors or systematic (accuracy) errors.

Westgard’s rules for interpreting QC data obtained using a single-level QC as well as two different-level QCs are schematically presented in Fig.1.⁽⁷⁾

     Warning Rule

¹2S One observation > x + 2 SD

Rejection Rules (used if warning rule is exceeded; run rejected if any of the following rules are violated) R= Random error; S = Systematic error

R ¹3S One observation > x + 3 SD

S ²2S Two observations > same limit, that is x + 2 SD or x - 2SD (same control- two consecutive runs, or two different controls – same run)

R ^R4S Difference between two observations within run > 4SD (two different controls – one > x + 2SD and the other > x – 2SD)

S ⁴1S Four consecutive observations>same limit, that is x + 1SD or x - 1SD (same control four consecutive runs )

S ¹⁰X Ten consecutive observations on same side of mean (same control, ten
consecutive runs, or two different controls, five consecutive runs)

     Remedial action

A well-run internal QC system makes possible immediate intervention in the release of patients’ results. In the event of a control system alert, it is advisable to proceed through the following steps in that order.

     Decision:Immediate decision whether action is necessary.

     Investigation:Check to locate the error.

     Repair:Action to eliminate the error.

If the decision is taken that the method is out of control, the first action is to withhold patients’ results in that batch. Then the analyst should start by checking for the simplest and most frequent faults, and then continue as necessary in a logical order depending on the method and equipment involved.

It is good practice to start by excluding gross errors such as mix-up of control materials, reagents or pipettes, misuse of measuring instruments [wrong filter or aged lamp in the photometer], or failure to follow instructions for a step in the method.

The results obtained on a control specimen may be in error for several reasons, including deterioration due to age, incorrect storage or contamination, wrong identification and mistake in preparation or constitution.

Further action will depend on whether the alert is due to a change in accuracy or in precision. If accuracy has deteriorated, attention should be focused on the possibility of systematic sources of error such as incorrect reaction temperature, calibration errors and faulty devices. If precision has deteriorated, steps in the analytical procedure should be checked, for instance, deproteinization, composition of reagents and reaction mixtures, measuring systems [photometer, flame photometer, etc].

To easily differentiate between systematic and random errors, laboratories are encouraged to construct Youden charts. These are constructed by having x + 2SD & x + 3SD of one level QC in the x axis and that of another level QC in the y axis. If an analyst analyses both levels of QC and plots the data in the Youden chart, a single plot will be obtained. If this falls within the inner square, it means that the value obtained for both QC are well within the acceptable limit, i.e. x + 2SD. On the other hand, if a plot appears outside this limit, it could mean that the data are outside the acceptable limit and the error could be either systematic or random. Fig.2 shows sample data plotted in Youden chart.

Figure 2.

It can be inferred that :-

1.      Plots E, F, G, K, M, N, O & R are acceptable

2.      Plot A values for both QC are > +2SD
Plot C values for both QC are > -2SD

3.      These data indicate ²2S error, i.e. systematic error

4.      Plot H   Values for QC₁ ( x axis) is > - 2SD
             while value for QC2 (y axis) is > + 2SD
This indicates R_4s error i.e. random error

     Prevention of systematic errors

To prevent or minimize systematic errors, the laboratory should adhere to the following points:

     Use of proper calibration technique. Use of pure chemicals, precision balance, quality distilled water. Proper preservative and storage

     Regular checking of photometric filter, bulb, tubing, etc.

     Use of recommended analytical methods

     Calibration of pipettes at regular intervals

     Instrument calibration – photometric check

     Use of calibrated cuvette

     Regular preventive maintenance of equipment – daily, weekly and monthly.

While it is easy to identify systematic errors, it is quite difficult to pinpoint random errors. In order to minimize this possibility, it is important to educate the staff on various aspects that could lead to random errors.

     External quality assessment

Participation in External Quality Assessment Schemes is important for interlaboratory comparison and the maintenance of long-term accuracy and precision of the analytical systems in the laboratory. This gives the laboratory an opportunity to have an appraisal of the methods employed and switch over to better methods.

      References

1.      Nordtest Technical Report from the technical group for quality assurance – Report #187 (1992).

2.      NABL (National Accreditation Board for Testing and Calibration Laboratories, India) criteria for laboratory accreditation – NABL #101 – 2nd Edition (1994).

3.      NABL Specific guidelines for accreditation of clinical laboratories and checklist for assessors (1998).

4.      Kanagasabapathy AS, Swaminathan S. and Selvakumar R. Quality Control in Clinical Biochemistry Indian Journal of Clinical Biochemistry (1996); 11: 17-25.

5.      Browning DM, Hill PG and Olazabal DAV. Preparation of Stabilized liquid quality control serum to be used in clinical chemistry –WHO document LAB/86.4

6.      Stamm D. Guidelines for a basic programme for internal quality control of quantitative analysis in clinical chemistry – WHO document LAB/81.3.

7.      Westgard, Barry PL and Hunt MR. A multi-rule shewart chart for quality control in clinical chemistry. Clin. Chem. (1981); 27:493-501.