Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Sodium and Potassium -Flame Photometry

     Introduction

Sodium, the major extracellular cation, plays a role in fluid distribution among body compartments. The ingested sodium is filtered in the renal glomerulus and approximately 70% is reabsorbed in the proximal tubule. Further reabsorption occurs in the loop of Henle and <5% is reabsorbed distally under the influence of aldosterone.

About 65-70% of the total body sodium is in its exchangeable form. The exchangeable sodium is made up of extracellular and intracellular sodium. The intracellular sodium concentration isabout 10 mmol/L and the extracellular, i.e. the plasma sodium concentration, isabout 140 mmol/L. Sodium maintains the osmotic pressure of the extracellular fluid and helps in retaining water in the extracellular compartment. Along with other cations it is also involved in neuromuscular irritability, acid base balance, maintenance of blood viscosity and resting membrane potential.

A high plasma sodium concentration of more than 145 mmol/L is referred to as hypernatremia. This can occur due to simple dehydration, excess sodium intake, steroid therapy as well as in diabetic insipidus. Hyponatremia, with plasma sodium concentration less than 130 mmol/L, can occur due to diuretic medication, kidney disease, excessive sweating, congestive heart failure or gastrointestinal disorder.

Potassium is the major intracellular cation. It is widely distributed in the body in muscle tissue, nerve tissue, blood cells and plasma. It is filtered in the glomerulus, absorbed in the proximal tubule and finally excreted by exchange for sodium in the distal tubule. Potassium influences muscular activity, cardiac function and nerve conduction process.

In hyperkalemia the plasma potassium concentration exceeds 5.5 mmol/L. Acute hyperkalemia is a medical emergency. In hypokalemia the plasma potassium level will be less than 3.5 mmol/L. This can occur due to excessive loss in gastrointestinal secretions and urine, and also in renal tubular acidosis.

     Principle of the method

When a solution of an inorganic salt such as sodium chloride is sprayed into the flame, the elements in the compound are partly converted into the atomic state. Due to the heat energy of the flame a very small proportion of these atoms is excited and the electrons move to a higher energy level. The proportion of the atoms that are excited depends upon the concentration of the particular element and on the temperature of the flame. In the excited state the electrons are unstable and they rapidly revert back to their former lower energy level. As they change from the excited state or higher energy level back to the lower energy level, they emit the light in the form of a fixed wavelength, to produce a spectrum. Under carefully controlled conditions the amount of light emitted is directly proportional to the number of atoms that are excited, which in turn is proportional to the concentration of the substance in the sample.

     Specimen type, collection and storage

Both sodium and potassium are stable in serum for several hours at 25-35° C and for 3 months at -20° C. Anticoagulants containing sodium or potassium salts are not suitable, but lithium heparin may be used as an anticoagulant.

If whole blood is left unseparated for >3hours or refrigerated, potassium will leak out of the red cells giving falsely increased values.

     Reagents

All chemicals must be Analar grade.

Sodium chloride (NaCI) and potassium chloride (KCI) should be dried for 2-3 hours at about 100° C before use. Before weighing, the chemicals must be allowed to cool to room temperature either in a desiccator or in a container with a tight-fitting lid with a small air space.

     Stock Sodium 1000 mmol/L

Weigh out 29.25 g dried NaCl, dissolve in about 400 ml of distilled water taken in a 500 ml volumetric flask and then make up to 500ml with distilled water. Store in a pyrex glass bottle at 25-35° C. Stable for one year.

     Stock Potassium 100 mmol/L

Weigh out 0.746 g dried KCI, dissolve in about 80 ml of distilled water taken in a 100 ml volumetric flask and then make up to 100 ml with distilled water. Store in a pyrex glass bottle at 25-35⁰C. Stable for one year.

     Working Standards

     Low standard for Sodium 100mmol/L: Dilute 10 ml of stock sodium to 100 ml with distilled water. Stable for 6 months at 25-350C.

     Combined standard for sodium and potassium 140Na+/5K+ mmol/L: Dilute 14 ml of stock sodium and 5 ml of stock potassium together to 100 ml with distilled water. Store in a pyrex bottle at 25-350C. Stable for 6 months.

     Aspiration standard for sodium - 1.0 mmol/L: Dilute 1.0 ml of working standard to 100 ml with distilled water. Prepare fresh each time.

     Combined aspiration standard for sodium 1.4 mmol/L and potassium 0.05 mmol/L. Dilute 1.0 ml of working standard (combined standard for Na+/K+140/5mmol/L ) to 100ml with distilled water. Prepare fresh each time.

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP.

     Procedure

The protocol of the procedure is described below.

     Sample dilution

Dilute each serum sample 1:100 with distilled water by mixing 0. 1ml sample with 9.9 ml distilled water.

     Procedure for simultaneous measurement of Na+ & K+ in the flame photometer (digital flame photometer)

     Switch on the flame photometer. Digital display should turn on.

     Turn the set ‘(full scale) F.S. coarse and fine controls’ into maximum clockwise position.

     Select appropriate filter with the help of filter selector wheel (Na+ on the left side and K+ on the right side).

     Switch on the compressor and check the air pressure. Adjust it to read between 0.4 and 0.6 k g/cm2.

     Open the gas cylinder, remove the trapper at the rear of the flame photometer and ignite the flame.

     Adjust the gas regulator to get a maximum height non-luminous blue flame with 10 distinct cones (5 on each side of the burner head).

     Feed distilled water to the atomizer and wait for at least 30 seconds.

     Adjust the 'Set Ref Coarse' and Fine controls' to zero digital readout for K+ only.

     Aspirate 1.0 mmol/L Na+ solution. Wait at least 30 seconds and then adjust the Set Ref Coarse and Fine controls' to a digital read out of I 00 for Na+ only.

     Aspirate the combined standard solution (1.4/0.05, Na+/K+) and wait at least for 30 seconds. Adjust 'F.S control' on Na+ side for readout 140 and that on K+ side for a digital readout of 50.

     Repeat steps 9 and 10 once again. The flame photometer now stands calibrated.

     Now feed diluted test sample / QC to the atomizer for at least 30 seconds before recording the readings for Na+ and K+.

     Calculation

After aspirating the standard solution, the digital reading for Na⁺ is adjusted to 140 and that of K⁺ to 50. This is done in order to represent Na⁺ and K⁺ values in undiluted serum. Since the test sample/QC is diluted initially 1: 100 and then aspirated, the initial standard values for Na⁺ & K⁺ (1.4 & 0.05 mmol/L) must be multiplied by 100 to represent 140 mmol/L Na⁺ and 5 mmol/L K⁺. In the case of K⁺, in order to improve the sensitivity of the assay the digital reading for the standard is further multiplied by 10 to show a reading of 50.

In essence, the test sample/QC digital readings are compared with the standard readings for Na⁺ and K⁺. The digital reading appearing for Na' of the test sample/QC is read as mmol/L value straightaway. On the other hand, the test sample /QC K⁺ value represents 1/10^th of the digital reading.

For example, digital reading for "140 Na⁺ = 140 mmol/L Na⁺; digital reading for 45 K⁺ = 4.5 mmol/L K⁺

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients' results).

Since Na⁺, K⁺ are very commonly analysed parameters in a laboratory, it isrecommended that internal QC (normal QC pool) be included with every batch of samples analysed in a day, irrespective of the number of samples in a batch. Further, even when a single sample is analysed as an "emergency" sample at any time of the day or night, it is essential to include an internal QC. From the QC results obtained for the day, mean, standard deviation and % CV can be calculated to ensure that within-day precision is well within the acceptable limit, i.e. 4%.

The mean value of internal QC for the day can be pooled with the preceding 10 or 20 mean values obtained in the previous days and between-day precision can be calculated and expressed as % CV. Ensure that this is well within the acceptable limit, i.e, 8%.

At least once a week analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

However, care should be taken when operating the flame photometer as the technician will be using liquefied petroleum gas. Leakage of either air or gas during operation will cause explosion. Apply soap solution at the connecting point to check such leakage.

     Hazardous materials

The reagents used are made up of only sodium chloride and potassium chloride. Therefore no precautionary measures are required.

     Reference range and clinical interpretation

The reference ranges by this method are:

     Serum sodium 130 - 145 mmol/L

     Serum potassium 3.5 - 5.0 mmol/L

Elevated levels of serum sodium occur in conditions such as severe dehydration, hyperadrenalism and brain injury.

Low serum sodium values are noticed in metabolic acidosis, salt- losing nephritis, Addison's disease, etc. Increased serum potassium level is observed inanoxia, metabolic renal tubular acidosis and shock or circulatory failure.

Low serum potassium values are observed due to low intake of dietary potassium over a period of time or increased loss through kidney, vomiting or diarrhoea. Increased secretion of adrenal steroids or some diuretics may also promote the loss of potassium.

     Limitations

Avoid using haemolysed serum. This will cause elevated K⁺ level.

Reliability of the results depends on the proper maintenance of the flame photometer, salient features of which are listed below.

     Requirements

     Non-luminous blue flame

     Supply of dry air at a controlled pressure, viz. 10- 15 Kg /cm2

     Regular availability of liquid petroleum gas

     Maintenance

     Disconnect power and gas supply before proceeding to do maintenance.

     Turn the control in both Na+/K+ display fully anti-clockwise.

     Disconnect the drain outlet and the gas and air inlets.

     Remove the top panel and disconnect the photocell.

     Remove the side panel and take out the atomizer.

     Disconnect the air line at the pressure gauge.

     Disconnect the air and gas inlets to the mixing chamber.

     Remove the burner head and remove mixing chamber

     Wash the above well with tap water and distilled water.

     Clean the atomizer with a thin wire and adjust its spray by passing compressed air. This can be done by screwing / unscrewing the two knurled nuts in the atomizer.

     Remove the air tube and flush out any water remaining in the tube due to the cooling of compressed air.

     Do not use oxyacetylene or highly explosive mixture as fuel.

     After cleaning, refix everything carefully.

      Cleaning of various units in a flame photometer

     Atomizer and capillary tube: Flushing with copious amount of distilled water is adequate. If blockage occurs, remove the atomizer from its seating and flush with dry air or clean it using a thin wire. If cleaning of atomizer is done with a wire before and after using it, blockage will rarely occur. If all the above fails, a new atomizer is to be fixed.

     Mixing chamber: Flushing with distilled water is adequate. Do not use detergent or soap solution because it will remain inside if washing is not done properly out and will give erratic reading due to the presence of Na+ / K+ in the soap solution.

     Fault diagnosis