CLINICAL CHEMISTRY LABORATORY ELECTROLYTE METHODOLOGIES OUTLINE • Electrolytes • Cations o Sodium o Potassium o Calcium o Magnesium • Anions o Chloride o Bicarbonate o Phosphate • ELECTROLYTES • Charged ions • Cations - positively charged ions • Anions - negatively charged ions • Electrolytes are ions carrying an electric charge either anion or cations. An important component in numerous process in the human body that affects the metabolic physiology and regulations of each electrolytes particularly distribution of water intracellular or extracellular. It influences the either side of the cell membrane as an osmoregulator. CATIONS SODIUM • Major extracellular cation • Sodium-potassium ATPase pump - regulates sodium levels extracellularly & intracellularly o 2 potassium out = 3 sodium out • Regulation • Pre-analytical considerations o Slight hemolysis does not affect the testing process o Significant hemolysis may dilute the sample causing lowered results o Serum, plasma, urine can be used as samples o Avoid additives on plasma that contains sodium (sodium EDTA, sodium heparin, etc.) • Measurement/Analysis o Reference method: Atomic Absorption Mass Spectrophotometry (AAS) o ISE electrode - most common procedure ▪ Uses membrane component ▪ Membrane: glass membrane electrode ▪ Direct - undiluted sample ▪ Indirect - diluted sample (sample diluent needed) o Chemical method - Albanese-Lein method (cupric sulfate and NaOH) ▪ Uses magnesium uranyl acetate ▪ End product: yellow complex o Flame Emission Photometry: obsolete method ▪ Sodium emits yellow flame o Determination of concentration ▪ Conventional methods instead of kinetic ▪ Cu (mg/dL) = Au/As x Cs • Cs = given METHOD, PRINCIPLE, & MATERIALS • Method Use o Photometric Determination of Serum Sodium Mg-Uranylacetate Method, Colour test. • Principle of the Test o Sodium is precipitated with Mg-uranyl acetate; the uranyl ions remaining in suspension form a yellow-brown complex with thioglycolic acid. The difference between reagent blank ( without precipitation of sodium ) and analysis is proportional to the sodium concentration. • Materials o Reagents, composition or contents ▪ PREC. Precipitating solution • Uranyl acetate - 19mmol/l • Magnesium acetate - 140mmol/l ▪ RGT. Colour reagent • Ammonium thioglycolate - 550mmol/l • Ammonia - 550 mmol/l ▪ STD. Standard • Sodium ( Na+ ) - 150mmo o Specimen: Serum/ Plasma can be used ▪ Use Ammonium heparin, Lithium heparin or Lithium oxalate anticoagulant ▪ Very hemolyzed samples should not be used ▪ Urine : 24 hour urine sample o Tubes, parafilm, centrifuge, micropipettes, pipet tips o Assay : ▪ Wavelength: Hg. 365nm; Hg 405nm; 410nm ▪ Optical path: 1 cm ▪ Temperature : 20-250C ▪ Measurement: Against reagent blank. Only one reagent blank per series is required. PROCEDURE, RESULT, & CALCULATION • Procedure o Pipetting Scheme SEMI-MICRO RB:Reagent Blank; STD: Standard RB [ul] STD [ul] SAMPLE [ul] STD --- 20 --- SERUM --- --- 20 PREC. --- 1000 1000 Close tubes and mix well. Allow to stand for 5 minutes. Shake intensively for at least 30 sec. Allow to stand for 30 min. Centrifuge at high speed for 5-10 minutes.
PREC 20 --- --- Clear Supernatant --- 20 20 RGT 1000 1000 1000 Mix well. After 5-30 minutes, measure absorbance of RB (∆ARB). the standard (∆ASTD ) and the sample (∆Asample) against distilled water at 360-410nm (Hg 366 or 405 ). • Result/Calculation o C = 150 x (A RB - ∆A Sample ) [mmol/l] (∆ARB - ∆ASTD ) o mval/l = mmol/l • Calculation o C = 0.075 (u) x 150 = 178.57 mmol/L (High) 0.063 (s) • Normal Range : o Serum/ Plasma: 135-155mmol/l o Urine: 40-220mmol/d, varies with diet o Cerebrospinal fluid: 136-150mmol/l • Important Notes : o Use the semi-micro procedure only if a very efficient centrifuge ( 8,000-10,000 RPM) is available. Otherwise use the Macro procedure to obtain reliable results. o PREC. becomes discoloured when exposed to the light. Store protected from light. A slight turbidity does not affect the determination. o Detergents usually contain high sodium concentration. The equipment- test tubes pipettes, stoppers, cuvettes - must therefore be rinsed carefully with distilled water. Avoid contamination by traces of sodium ( sweat ). o Disposable plastic tubes are recommended for the determination. Use parafilm or plastic stoppers to close the tubes. POTASSIUM • Major intracellular cation • Affects the blood pH indirectly o Movement of K, affect hydrogen and other compounds • Skeletal and cardiac muscle contractions (Resting membrance potential ) • Hyperkalemia • Hypokalemia • Maintains H+ and Na+ concentration • Regulation • Pre-analytical considerations o Serum may have higher levels (ref values) than plasma; platelets are also rich in K ▪ Platelets may release K after coagulation o Avoid: hemolysis, prolonged tourniquet application, excessive opening and closing of fists during phlebotomy, post-exercise ▪ Hemolysis will increase levels ▪ Flicking, clenching of fist induce the release of K due to pressure applied ▪ Post-exercise may increase K levels o Blood should be stored at room temp (25C) ▪ When serum is not separated from the RBC ▪ Cold temperature promote release of K from cells • Measurements/Analysis o Reference method - AAS o ISE - valinomycin membrane o Flame Emission Photometry - purple flame o Chemical method - Lockhead and Purcell method (turbidimetric using sodium tetraphenylboron for ppt) ▪ More turbid = higher conc of K ▪ Turbidity increases absorbance METHOD, PRINCIPLE, & MATERIALS • Method Use : Liquirapid Photometric Turbidimetric Test • Principle of the Test : o Potassium ions in a protein free alkaline medium react with sodium tetraphenylboron to produce a finely dispersed turbid suspension of potassium tetraphenylboron. The turbidity produced is proportional to the potassium concentration and read photometrically. • Materials : o Reagent composition /contents ▪ PREC. - Precipitant ( white cap ) • Trichloracetic acid ( TCA ) - 0.3mol/l ▪ TPB - TPB-Na- Reagent (BLACK CAP ) • Sodium tetraphenylboron - 0.2mol/l ▪ NAOH - NaOH Reagent ( red cap ) • Sodium Hydroxide - 2.0mol/l ▪ STD - Standard • Potassium ( K+) - 5.0 mmol/l o Specimen: Serum and Lithium heparin Plasma o Test tubes, deioinized water, parafilm, centrifuge, micropipettes and pipet tips. PROCEDURE, RESULT, & CALCULATION • Procedure : o Reagent Preparation: ▪ Mix the content of bottle TPB with the content of bottle NaOH, for smaller amounts of working reagents mix TPB and NaOH in a ratio of 1:1. Allow to stand for 15-30 minutes, prior to use. o Assay: ▪ Wavelength: 578 nm, Hg 578nm ▪ Optical path: 1 cm ▪ Temperature: 20-25oC ▪ Measurement: Against reagent blank ▪ Only one reagent blank per series is required. o Pipetting Scheme: ▪ Precipitation ▪ Pipette into centrifuge tubes Semi-micro Specimen 50ul PREC 500ul Mix carefully, centrifuge at high speed for 5-10 minutes.
o Determination: ▪ Pipette into cuvettes: STD SAMPLE Working Reagent 1000ul 1000ul STD 100ul ---- Supernatant ---- 100ul To produce a homogenous turbidity, the STD or the clear supernatant have to be added to the center of the surface of the working reagent in the cuvette. Mix each cuvette carefully before proceeding to the next sample. Allow to stand at least 5 min Measure absorbance of the standard ( ASTD) and the sample ( Asample ) against working reagent blank between 5 and 30 minutes. • Result/ Calculation of the Potassium Concentration o C = 5 X Asample [mmol/l] or [meq/l] A STD o Using the above formula, compute the given data to get the potassium level. Interpret your computed value. o Absorbance of Sample : 0.054 o Absorbance of Standard: 0.066 o Note: Samples with higher concentrations have to be diluted 1+1 with physiologic saline and multiply result by 2 • Normal values : o Serum 3.6-5.5mmol/l o Plasma 4.0-4.8mmol/l o Urine (24 hr) 25-125 mmol/d • Calculation o C = 0.054 x 5 = 4.09 mmol/L 0.066 o C = 0.273 x 5 = 15 mmol/L 0.090 ▪ >10 is erroneous; can’t be accurately detect ▪ Dilute & retest ▪ C = 0.137 x 5 = 7.61 x 2 = 15.22 mmol/L 0.090 • Multiply by dilution actor (1:2) • Important notes : o Recommended to use HumaTrol or Serodos for quality control o Do not use hemolyzed sample o Red blood cells contain about 25 times the amount of potassium, they have to be separated from the serum within one hour after blood collection. otherwise, falsely elevated potassium concentrations will be found. o Unseparated samples should NEVER be stored on ice or refrigerate. o Traces of detergents produce turbidity which leads to falsely elevated potassium concentrations, thus have to be avoided. o Contaminated glassware is the greatest source of error. Therefore, should be thoroughly rinsed with deionized water. Disposable plastic ware may contain softeners which react with the reagent and not recommended. CALCIUM • Most abundant cation in the body o Include calcium in bones • Distribution o Bones = 98-99% o Blood (extracellular) = 1% o Cytosol (intracellular) =<1% • Forms (in blood) o Ionized (Ca 2+ ) = 45% ▪ Functional form; usually measured o Protein bound = 40% (bound to albumin) o Complexed = 15% ▪ Bound to lactate, citrate, bicarbonate o Test kits measure ionized calcium or total calcium • Function: o Myocardial contraction o Muscle contraction • Regulation • Methods of Analysis o Every calcium test requires magnesium shelter ▪ Magnesium shelter - prevents Mg interference ▪ 8-hydroxyquinolone/8-hydroxyquinoline - commonly used Mg shelter o Dye binding methods ▪ O-cresolphthalein complexone (570 nm) ▪ Arsenazo III dye (530 nm) ▪ Result: purple complex o AAS - reference method o Chemical method - Clark & Collip method (redox titration) o Sulkowitch (urine calcium) METHOD, PRINCIPLE, & MATERIALS • Method Use : PHOTOMETRIC/COLORIMETRIC TEST FOR CALCIUM • Principle of Test : o Calcium ions react with 0-cresolphthalein-complexone in an alkaline medium to form a purple coloured complex. The absorbance of this complex is proportional to the calcium concentration in the sample. • Materials : o Reagents/Contents ▪ BUF. - Buffer Solution • Lysine buffer ( pH11.1) - 0.2mol/l • Sodium azide - 0.095% ▪ RGT - Colour Reagent • 8-Hydroxyquinoline - 14mmol/l • 0-cresolphthalein - complexone - 0.1mmol/l • Hydrochloric acid - 40mmol/l
▪ STD - Standard • Calcium (ll) - 8mg/dl or 2mmol/l • Sodium azide - 0.095% ▪ Reagent and standard are stable until expiry date when stored at 2-25oC. Avoid contamination when handling. o Specimen: Serum or Heparinized plasma (must be analyzed immediately <30 minutes) ▪ Avoid hemolysis; Never use Citrate, Oxalate or EDTA plasma ▪ Stability in serum: 10 days at 2-25oC o Test tubes, parafilm, cuvettes, centrifuge PROCEDURE, RESULT, & CALCULATION • Procedure : o Reagent preparation: ▪ Add RGT to BUF in equal volume as required. Mix and allow to stand for 30 minutes at room temperature before use. This is stable for 7 days at 2-8oC . o Assay: ▪ Wavelength: 570nm, Hg578nm; 546nm ▪ Optical path: 1 cm ▪ Temperature: 20-25oC ▪ Measurement: Against reagent blank. Only one reagent blank per series is required. o Pipetting scheme Pipette into cuvettes Reagent blank Sample / STD Sample/STD ---- 20ul Working reagent 1000ul 1000ul Mix and measure the absorbance of sample ( Asample ) and standard ( Astd ) against the reagent blank within 5 to 30 minutes. • Result/Calculation of Calcium Concentration o C = 8 x Asample ( mg/dl) Astd o C= 2 x Asample (mmol/l) Astd o Compute the Calcium concentration with the given data. Interpret the value obtained. ▪ Sample absorbance Asample= 0.168 ▪ Standard absorbance Astd = 0.137 • Normal values: o Serum or plasma = 8.1-10.4 mg/dl = 2.02 - 2.60 mmol/l • For Quality control: Use HumaTrol or SERODOS. • Important procedural notes: o Contaminated glasswares are sources of error. Disposable plastic ware is recommended for the test. o The test is not influenced by hemoglobin up to 200 mg/dl and bilirubin up to 20mg/dl. o Lipemic and haemolytic samples require a sample blank. By using the same pipetting scheme mix 20ul of sample with 1000ul of distilled water and measure the absorbance ( Asample blank ) against distilled water. Asample blank has to be subtracted from Asample. o BUF and STD contain sodium azide as preservative. Do not swallow. Avoid contact with skin and mucous membrane. o Measurement at 546 nm results in OD values arount 40% lower than 570/578nm. While interference by hemoglobin is more prominent than at 578nm. Thus hemoglobin content should be assess separately. o A slight brownish sediment may develop as BUF ages but does not influence the functionality of BUF. Do not swirl or mix up the sediment during pipetting. o Anaerobic collection to prevent CO2 loss o Urine samples: Acidify using 6mmol/L HCL per 100 ml urine o Use Magnesium shelter ( 8-hydroxyquinoline) MAGNESIUM • Second major intracellular cation • Distribution o Bones = 53% o Muscle and other organs = 46% o Serum and RBC = 1% • Forms o Ionized (unbound, Mg 2+ ) = 55% o Protein bound = 30% o Complexed = 15% ▪ Bound to phosphate or citrate • Functions: o Enzyme COFACTOR o Neuromuscular transmission o Glycolysis o Carbohydrate, Lipid, Protein, and Nucleic synthesis o Hormone response and release • Laboratory methods o Atomic Absorption Spectrophotometry ▪ Reference method o Dye binding methods ▪ Must prevent interference of calcium ▪ Glycol-EDTA - prevent Ca interference ▪ Titan yellow (570-590nm) ▪ Calgamite (reddish violet complex at 532 nm) ▪ Formazan (blue complex at 660 nm) ▪ Methylthymol blue (blue complex at 600 nm) ▪ O-cresolphthalein complexone (purple at 570 nm) METHOD, PRINCIPLE, & MATERIALS • Method Use : o Photometric Colorimetric Test for Magnesium with Lipid Clearing Factor ( LCF ) • Principle of Test : o Magnesium ions in an alkaline medium form a coloured complex with xylidyl blue. The absorbance increase is proportional to the magnesium concentration. In the sample, Glycoletherdiamine-N, N,N',N'-tetraacetic acid (GEDTA) is used as masking agent for calcium ions. • Materials : o Reagents/contents: ▪ RGT - Colour reagent • CAPS - 50mmol/l • GEDTA - 13mmol/l • Xylidyl blue - 0.09mmol/l • Sodium azide - 0.095% • Activators
▪ STD - Standard • Magnesium (II) - 2.5 mg/dl • or 1.03 mmol/l • Sodium Azide - 0.095% ▪ Above reagents are ready to use and stable until expiry date when stored at 2-25oC. o Specimen: Serum, plasma (Do not use edta plasma ) ▪ Stability : 7 days at 2-8oC ▪ NOTE: Lipemic specimens usually generate turbidity of the sample/reagent mixture which leads to false high results. Magnesium liquicolor avoids these false high results through its built-in Lipid Clearing Factor ( LCF ). The LCF clears a turbidity caused by lipemic specimens up to 2000mg/dl Triglycerides. o Assay: ▪ Wavelength: 520nm, Hg 546nm ▪ Optical path: 1 cm ▪ Temperature: 20-25oC ▪ Measurement: against reagent blank. Only 1 reagent blank per series is required. o Other: Test tubes, cuvettes, parafilm, centrifuge, micropipets, pipet tips PROCEDURE, RESULT, & CALCULATION • Procedure o Pipetting scheme Pipette into cuvettes Reagent blank Sample / STD Sample/STD ---- 10ul Distilled water 10ul ---- RGT 1000ul 1000ul Mix, incubate for 10 minutes at 20-25oC,measure the absorbance of the sample and STD against the reagent blank within 60 minutes ( A ). o Result/ Calculation of Magnesium Concentration ▪ C = 2.50 x ASample (mg/dl) ASTD ▪ C = 1.03 x ASample (mmol/l ) ASTD o Compute the concentration of Magnesium with the given data . Interpret your values obtained. ▪ Absorbance of Sample = 0.273 ▪ Absorbance of Standard = 2.045 • Normal values: o Serum, Plasma = 1.9 - 2.5 mg/dl = 0.8-1.0mmol/l • Procedural Notes : o Avoid hemolysis o Never use Citrate, Oxalate or EDTA plasma o Urine samples: Acidify using HCl o Use Calcium Shelter ( Glyco-EDTA) ANIONS CHLORIDE • Major extracellular anion • Function: osmolality, blood volume, electroneutrality o Chloride shift phenomenon o Maintains charges • Function is not well defined o Maintaining Electroneutrality o Blood volume o Contributes to blood osmolality • Regulation o Chloride passively enters during sodium reabsorption • Pre-analytical Consideration o Slight hemolysis does not affect testing process o Significant hemolysis may cause dilutional effect ▪ Slightly decreased levels o Serum, plasma (additive of choice: heparin) or sweat can be used as samples o Bromide, cyanide, and cysteine may affect analysis • Methodologies o ISE: tri-n-octylpropylammonium chloride decanol o Gibson- Cooke/Schale’s & Schale’s : violet complex ▪ Gibson-Cooke - for sweat ▪ Schales and Schales • Hg++ + Cl → HgCl2 • Excess Hg++ + diphenylcarbazone → violet complex • Gibson-Cooke if used in sweat chloride analysis o Amperometric-coulometric titration (Cotlove chloridometer) ▪ Complexed with silver-based reagent for measuring silver chloride ▪ Rate of formation is proportional to concentration o o Schoenfeld and Lewellen ( 480 nm) ▪ Cl- + Hg ( SCN) → HgCl2 + SCN- ▪ SCN- + Fe+++ → Fe (SCN)3 METHOD, PRINCIPLE, & MATERIALS • Method Use : Photometric Colorimetric Test For Chloride Using Tptz Method • Principle of Test : o Chloride ions react with a mercury (ll)-2,4,6-tri-(2-pyridyl)-s-triazine (TPTZ) complex to form mercury(ll)-chloride. The liberated TPTZ reacts with iron(ll) ions yielding a blue colored complex. The resulting absorbance change at 590nm is directly proportional to the amount of chloride ions in the sample. • Materials : o Reagents/ contents ▪ RGT - Colour reagent • 2,4,6,-Tri-(2-pyridyl)-s-triazine (TPTZ) (partially as mercury(ll) complex) - 0.986mmol/l • Iron (ll) sulphate - 0.53 mmol/l ▪ STD - Standard • Chloride - 100mmol/l or 355mg/dl
▪ The above reagents are ready to use and stable up to the stated expiry date provided stored at 2-25oC and not contaminated. o Specimen: Serum, CSF, urine ▪ Stability in serum 2-25C for 7 days ▪ Stability in urine at 2-8C for 7 days o Specimen considerations: ▪ Blood should be immediately processed ( centrifuged within 1 hr. ), otherwise an electrolyte exchange between serum and erythrocytes may lead to a false low chloride determination. ▪ Precipitates in urine may be redissolved by warming up. o Assay: ▪ Wavelength: 590nm ( 560-600nm ), Hg 578nm ▪ Optical path: 1 cm ▪ Temperature: 20-37oC ▪ Measurement: Against reagent blank. Only one blank per series is required. o Others: Centrifuge, test tubes, micropipettes. pipet tips, parafilm cuvettes. PROCEDURE, RESULT, & CALCULATION • Procedure : o For semi-micro method: Predilute STD and the sample 1+50 ml with distilled water ( ex. 20ul STD/Sample +1000ul of distilled water ). o Pipetting scheme: o Use the prediluted STD/Sample for the following pipetting scheme: Pipette into cuvette SEMI -MICRO STD SAMPLE STD 20ul ---- SAMPLE ---- 20ul RGT 1000ul 1000ul Mix, incubate for 5 minutes in the dark and measure the absorbance of sample (Asample )and STD ( ASTD) within 60 minutes against the reagent blank. Do not expose to light. • Result/Calculation : o C = 100 x Asample [mmol/l] ASTD o C = 355 x Asample [mg/dl] ASTD o The result may be converted according to the following equations: o c[mmol/l] = c[mval/l] o c[mmol/l]x3.55 = c[mg/dl] o c[mg/dl] x 0.282 = c[mmol/l] o 24H urine: c[mmol/l] x urine volume [l] / 24 [h] = c[mmol/24H] ▪ c[mmol/24H] x0.0355 = c[g/24H] o Note: Samples with higher chloride concentration must be diluted further 1:1 with distilled water.. Repeat assay and multiply result by 2. • Normal values: o Serum: 95-108mmol/l = 335-383 mg/dl o CSF: 119-130 mmol/l = 442-460 mg/l o Urine: 110-225 mmol/l = 3.9-8 g/24H • Procedural notes : o The test is a very sensitive to chloride detection. Therefore usage of contaminated glassware and skin contact can cause elevated chloride results. Use of disposable plastic ware and gloves is highly recommended. o Hemoglobin, bilirubin, lipids, and ascorbic acid in physiological concentrations do not interfere with the test. o RGT is sensitive to light, especially UV light RGT should be kept light-protected. o Bromide, Cyanide and Cysteine interferes with chemical analysis BICARBONATE • Second most abundant anion in the ECF • Bicarbonate and chloride have inverse relationship o The two share Na in the ECF o Increased bicarbonate, chloride is lowered o Decreased bicarbonate, chloride is increased • Comprises 90% of total blood CO 2 o 10% - dissolved CO2 • Regulation • Pre-analytical considerations o Serum or lithium heparinized plasma is used as specimen o Anaerobic method (closed system) o Tightly capped and analyzed immediately after centrifugation ▪ 6 mmol/L/hr reduced after opening sample • Methods o Enzymatic ▪ Bicarbonate + phosphoenolpyruvate (PEP) → Oxaloacetate + Phosphoric acid (PEP carboxylase) ▪ Oxaloacetate + NADH → Malate + NAD (MD) o Ion Specific/selective electrode (ISE) or Severinghaus electrode PHOSPHATE • Capable of transcellulat shifts • Utilized in biochemical reactions (parts or enzyme activator) • Organic (~12 mg/dL) vs Inorganic (~3-4 mg/dL) • Distribution o 80% - Bone o 20% - Soft tissues o <1% - Blood • Biochemical reactions o DNA and RNA formation ▪ Crucial component o Enzyme activators o Energy (ATP) o Oxygen and CO 2 transport - dictates affinity of RBCs to oxgen and CO 2 • Pre-analytical considerations o Avoid hemolysis - RBCs release phosphate o Never use citrate, oxalate, or EDTA plasma o Circadian rhythm (low in evening, high/peak in late morning); usual reference range is based on morning
• Methods o Ammonium phosphomolybdate (340 nm) o Fiske and Subbarow (600 nm) o Reactions ▪ Phosphorus + Ammonium molybdate → Ammonium phosphomolybdate (purple) ▪ Ammonium phosphomolybdate + aminonaphtholsulfonic acid → Molybdenum blue (reduced ammonium phosphomolybdate) METHOD, PRINCIPLE, & MATERIALS • Method : Photometric uv test/ colorimetric ( fiske subarrow) for the determination of phosphorous ( inorganic phosphate). • Principle of Test o Phosphate reacts with molybdate in strong acidic medium to form a complex. The absorbance of this complex is the near UV is directly proportional to the phosphate concentration. • Reaction Principle: o 7H 3 PO 4 + 12(Mo 7 O 24 ) + + 51H + → 7[P(Mo 12 O 40 )] 3- + 36H 2 O • Materials o Reagents/Contents ▪ RGT. - Reagent • Ammoniumheptamolybdate - 0.3mmol/l • Sulphuric Acid (pH<1.0) - 160mmol/l • Detergent - 1% • Activators and stabilisers ▪ STD. - Standard • Phosphorous - 10mg/dl or 3.2mmol/l ▪ Note: This reagents are stable until the expiry date if stored at 2-25oC. Avoid contamination. o Specimen: Serum ▪ Stability in serum: 7 days at 4oC ▪ Plasma must not be used. ▪ Anticoagulants may cause false low results. o Test tubes, cuvettes, parafilm, micropipettes. pipet tips. o Assay: ▪ Wavelength: 340nm, Hg 334 cm ▪ Optical path: 1 cm ▪ Temperature: 20-25oC ▪ Measurement: against reagent blank, one reagent blank per series is required. PROCEDURE, RESULT, & CALCULATION • Procedure o Pipetting scheme Pipette into cuvettes Reagent blank Sample or STD Sample or STD ---- 10ul RGT 1000ul 1000ul Mix, incubate at least 1 minute at room temperature. Measure the absorbance of the sample and the STD against the reagent blank within 60 minutes (A). • Note: For quality control use HumaTrol or SERODOS. • Result/Calculation of Phosphorous Concentration o C = 10x Asample (mg/dl ) ASTD o C = 3.2 x A sample (mmol/l) ASTD o Using the following Readings compute the Phosphorous concentration and interpret your answer. ▪ Absorbance of Sample = 0.542 ▪ Absorbance of Standard = 0.320 • Normal Value : Inorganic phosphorous o Adults: 2.5-5.0 mg/dl = 0.81-1.62 mmol/l o Children: 4.0-7.0 mg/dl = 1.30-2.26mmol/l • Procedure Notes : o Icteric and slightly lipemic samples require a sample blank. By using the same pipeting scheme, mix 10ul sample with 1000 ul distilled water and measure the absorbance against distilled water. The absorbance ASample blank has to be subtracted from Asample. o Strong lipemic and hemolytic sera should not be used. o Contaminated glassware is the greatest source of error. Disposable plastic ware is recommended for the test. o RGT contains sulphuric acid. If skin or mucous membranes come into contact with the reagent, wash thoroughly with water and consult a doctor.