metabolic screening tests for inborn errors of metabolism

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RANDOM URINE SAMPLE (50ml for complete & 10ml for MPS) PRESERVATIVE -1ml 6N HCl for 10 ml of urine

Observe for physical appearanceColour Odour Clarity PH Bloody contaminants

Centrifuge at 5000 Rpm for 15 min

Collect the supernatant

Proceed with chemical tests & Chromatography

URINE MICROSCOPYReagents:2% Aqueous Methylene Blue, 1% Toluidine Blue in 1N acetic acid Method: The test should be performed on fresh urine sample.(renal cells will be destroyed otherwise).Centrifuge the urine and decant the supernatant fluid. Add 2 drops of Stain to the sediment. Examine under high power microscopy after 5 to 10 minutes.

Cellular Pathology: With Methylene Blue stain, Metachromatic granules showblue within renal epithelial cells. In older urine the granules are seen extracellularly as the cells will be ruptured. A positive test requires more than 10% cells to be stained and indicates Sulfatide lipodystrophy (Metachromatic Leukodystrophy). A similar test with 1% Toluidine Blue in1N Acetic acid gives positive test for Mucopolysaccharidosis. Granules in this stain purplish and are smaller. Presence of other cells should be noticed (Hereditary nephritis in Alports disease with deafnessIEM of Proline metabolism) Note: Uric acid crystals dissolve in alkaline pH and Phosphates in acidic pH. Uric acid crystals are also seen in normal urine.

1. BENEDICTS TESTPROCEDURE 2ml Benedicts reagent + 0.2ml urine .Place test tubes in boiling water bath for 3 minutes .Take it out and allow to cool to room temp. OBSERVATION Appearance of precipitates of various colours indicates positive test- green, yellow, red, brick red INFERENCE Reducing sugars are present

Reagent preparation : A: 17.3 gm Copper sulphate (CuSO4 5 H2O) in 200ml water B: Dissolve 173gm Sodium Citrate in 500 ml water (heat if necessary).Add 100 gmanhydrous Sodium Carbonate and dissolve with stirring. After cooling mix A & B

Note : White precipitate -Phosphates may be present

2. NINHYDRIN TESTPROCEDURE To 1 ml of urine ,add 10 drops of ninhydrin .Heat to boiling OBSERVATION Appearance of Blue colour INFERENCE Free amino groups are present .

Principle: Ninhydrin reacts with free Alpha amino groups of proteins to give blue to purple coloured complex called Ruhemanns Purple Reagent preparation : 1. 0.2% Ninhydrin Solution PROCEDURE OBSERVATION INFERENCE Excessive Aminoaciduria is present

Add three drops of Appearance of Blue or urine to 1 ml of purple colour reagent in a clean test tube. Mix .Observe the colour after 3 4 minutes

False positive: Concentrated urine, High Ammonia in urine Principle: Ninhydrin reacts with free Alpha amino groups of proteins to give blue to purple coloured complex called Ruhemanns Purple Presence of single amino acid in excess without overall increase in amino acids may not be detected. Reagent preparation : 1. 1gm of Ninhydrin in 500 ml of 95% Ethanol. Reagent should be kept in Dark brown bottle in refrigerator

3.DINITRO PHENYLHYDRAZINE (DNPH) TESTPROCEDURE OBSERVATION INFERENCE Centrifuge the Appearance of turbidity Keto acids are urine(5000rpm /15 or yellow white present min) .Add 0.2ml clear precipitate urine to test tube.0.8 ml of DNPH reagent. Mix .Allow to stand for five minutes. If turbidity is equivocal , add another 0.4 ml of DNPH reagent

Reagent preparation: 200 mg DNPH in 100 ml 2N HCl. Store at ambient temperature stable for 3 months

4.FERRIC CHLORIDE TESTPROCEDURE Take 1ml of urine (T) and 1ml of reference standard (C) in two test tubes respectively. Add 0.2 ml of ferric chloride reagent to both the test tubes and mix. Note the colour change if occurs immediately & after 2-3 minutes OBSERVATION Appearance of Change in colour ( blue, green, greenish grey , purple, deep green INFERENCE Aromatic amino acids present

Reagent preparation: Dissolve 10 gm ferric chloride (anhydrous) in water and make up to 100 ml. Store in dark bottle at 40C.

5.METHYL MALONIC ACID TESTPROCEDURE OBSERVATION Label 2 test tubes as T (test)& C (Control) Appearance of .Add 0.1ml of urine to test tube & 0.1 ml of emerald green colour Methyl malonic acid standard to tube Crespectively. Add 1ml of p- Nitroaniline reagent to each of the tubes and mix .Add 0.25 ml of Sodium nitrite solution(0.5 %) to each of the tubes & mix .Allow tubes to stand at room temperature for a minute.Add 1.5 ml of Sodium acetate buffer,PH 4.3 & mix.Place the tubes in boiling water bath for 3 minutes ,add 0.3 ml of N/8 Sodium Hydroxide & mix INFERENCE Methyl malonic acid present(60 mg/dl)

Reagent preparation :1. MMA reference std(0.025 M): 147.5 mg Methyl malonic acid(MW 118.1) in 50 ml of water and add a 6N HCl to render it acidic. Store in refrigerator at 40C.Stable for six months 2. P-Nitroaniline reagent : Dissolve 100mg p-Nitroaniline in 100 ml of 0.16 N 3. HCl .Store in dark bottle at room temperature. 4. Sodium Nitrite (0.5%) :Dissolve 0.5g of Sodium nitrite in water & male up to 100 ml .Stable for 2 months in refrigerator. 5. Sodium acetate buffer 1 M pH 4.3 :Dissolve 13.6 gm Sodium acetate trihydrate in distilled water &adjust the pH to 4.3+0.02 make up the final volume to 100 ml .Keep it in refrigerator at 40C.Stable for 2-3 months 6. NaOH (8N) : Dissolve 32 gm of NaOH in water, while keeping it cold in a ice bucket, and adjust volume to 100 ml. Store in plastic bottle with screw cap at room temp.

False positive: Children on Valproic acid treatment for seizures

6.NITROSONAPHTHOL TESTPROCEDURE OBSERVATION To a test tube add 1 ml Appearance of orange nitric acid ,0.1 ml of to deep red colour sodium nitrite solution within five minutes and 0.1 ml of Nitrosonaphthol solution and mix Add 0;.3 ml of urine and mix. Wait for five minutes . INFERENCE Tyrosine and its metabolite present

Reagent preparation:1. Nitric acid(2.6N): Add one volume Conc HNO3 to 5 volumes of water 2. Sodium nitrite solution : Dissolve 2.5 g Sodium nitrite in water & make up to 10 ml .Store at 40C. 3. 2- Nitrosonaphthol reagent: Dissolve 10 mg of Nitrosonaphthol in 100 ml ethanol(95% v/v) .Store at 40C. False positive : Patients with GI disorders due to hydroxyl phenyl acetic acid due to bacterial overgrowth in intestine.

7.SULPHURIC ACID TEST FOR INDOLE DERIVATIVESPROCEDURE Add 0.5ml of reagent to 5 ml of urine. Observe the colour after ten minutes OBSERVATION Appearance of strong red or violet colour indicates positive test INFERENCE Hydrindic acid is present

False positive : Drugs like Phenothiazines Reagent preparation: Conc Sulphuric acid

8.ISATIN TESTPROCEDURE Dip some filter paper (Whatmanns No 3 or Equivalent)into reagent A.Allow it to dry at room temperature.Add one drop of urine and allow it to dry at 100o C for ten minutes.Dip in reagent B and wash it with water. OBSERVATION Appearance of deep blue colour of urine spot indicates a positive reaction INFERENCE Proline is present in excess

Reagent preparation:A: 20 mg Isatin ,96 ml Acetone,4 ml of Glacial acetic acid.The reagent will be stable for three months if kept in refrigerator B:1N HCl

9.ROTHERAS TEST FOR KETONE BODIESPROCEDURE Take 1 cm length solid Ammonium Sulphate in a test tube .Add 5 ml urine .Shake to dissolve and saturate. Some solid should remain at the bottom .Add 1 drop of freshly prepared solution of Nitroprusside solution. Mix well. Add 0.5 -1 ml of liquor Ammonia gently along the sides of the test tube. OBSERVATION Appearance of purple ring indicates positive reaction INFERENCE Ketone bodies are present

Reagent preparation:1. Sodium Nitroprusside solution : Freshly prepared 5% solution 2. Solid Ammonium Sulphate 3. Liquor Ammoni

10. EHRLICHS ALDEHYDE TEST FOR PORPHOBILINOGENPROCEDURE Add 1ml of reagent to 5 ml of fresh urine Examine for colour after ten minutes If positive add 5ml of Chloroform with shaking OBSERVATION Appearance of pink colour indicates positive reaction Pink colour is extracted into chloroform layer INFERENCE Porphobilinogen/Urobilinogen present Urobilinogen present (porphyrins are not extracted into chloroform layer)

False positive: Indoles in urine can give positive test Reagent preparation:1. Ehrlichs Reagent: 2%p-dimethylaminobenzaldehyde in 2N HCl

11.O-TOLUIDINE TEST FOR COPPERPROCEDURE Put 2 drops of urine on thick filter paper(Whatmanns no 3 or equivalent).Allow it to dry and then add one drop of freshly made reagent. OBSERVATION Appearance of blue colour within 30 seconds suggests positive reaction INFERENCE copper is present in excess Wilsons disease to be considered

Reagent preparation: O-Toluidine 0.1 gm, Ammonium Thiocyanate 0.5 gm, Acetone 5 ml. Mix just before use

12.Tests for Mucopolysaccharidosis1. Spot test for MPSPROCEDURE Mark three spots on Whatmann filter paper no 3 with lead pencil .Spot 5l urine on one spot .10l on another and 5lf of reference std on another spot & dry the spots.Stain the spots by placing in Toluidine blue solution for 45 seconds. Rinse the paper with water to remove the excess stain. Destain for 10 min in washing solution OBSERVATION Appearance of Purplish or bluish spot remaining after destaining is positive test Absence of spot or Metachromatic ring is negative Positive with 10l but negative with 5 l is suspicious INFERENCE MPS is present

Reagent preparation:1. Toluidine blue O Stain : 200mg Toluidine Blue O (MW 305.8) in 100 ml of 80% Acetone 2. Washing Solution : Water :Methanol:Glacial acetic acid(200:20:1 v/v) 3. Chondroitin Sulfate ( ref Std) : Dissolve 10mg of ChondroitinSulphate B in 10 ml water , Store in refrigerator.

Tests for Mucopolysaccharidosis4. Cetyl Pyridinium Chloride Citrate Turbidity TestPROCEDURE OBSERVATION INF


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