Proposed Protocol for Investigation of Metabolic Disease in Acutely Ill Neonates and Young Children

Please submit suggestions for improvement to Professor Hjelm: hjelm@cuhk.edu.hk

ISSUED BY: PROFESSOR N M HJELM
COS OF CHEMICAL PATHOLOGY

Last modified: 7 May, 1997 HKT

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CONTENTS

1. Clinical indicators suggesting an underlying metabolic disease

2. Primary investigations

3. Specimen Collection (tests and specimens required)

4. Handling of specimens outside-office hours

5. Metabolic Investigation Kit (tubes required and special request form)

6. Peri-mortem Protocol

7. Post-mortem Protocol

8. Confirmatory Tests

References

Link to OMIN (Online Mendelian Inheritance in Man)

1. Clinical indicators suggesting an underlying metabolic disease

The clinical presentation of IMD during neonatal period is usually of a baby that is getting seriously ill a few days after delivery and after feeding has started. The best chance to establish the biochemical characteristics of metabolic disease is at the time when deterioration occurs. Therefore samples need to be collected before any therapeutic measures have been instigated.

IMD comprised of heterogeneous groups of entities, thus there is no single diagnostic feature, however, the following pointers may be present,

• family history of similar illness in other siblings or cousins,
• family history of unexplained infant death,
• previous episodes of similar illness in the patient,
• previous episodes of unexplained metabolic derangement, e.g. hypoglycaemia, acidosis, etc.,
• history of change of diet or amount of intake (such as fasting) before the attack,
• history of abnormal growth or development.

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2. Primary investigations

Ensure that the patient is well fed with an adequate protein diet 2-3 hours before carrying out these tests. Neonates should be given a milk feed.

The following screening tests should always be carried out before any other investigations are considered. Positive results, singly or in combination, will provide an indication of the presence of any of twenty five of the most common inherited metabolic diseases

Tests for IMD for immediate analysis.

• Plasma glucose
• Plasma ammonium
• Blood pH and gases
• Urine ketones
• Plasma lactate
• Urine for metabolic screening; reducing substances, glucose,

Tests for IMD for analysis as soon as possible

• Plasma amino acids
• Urine for metabolic screening; organic acids

Tests for other causes of illness for immediate analysis

• Liver and Renal function tests including chloride

If these simple investigations are normal in a patient that is well fed the chance of a metabolic disease as the cause of illness is low.

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3.Specimen Collection

(A set of tubes should be available for these investigations, see Metabolic Investigation Kit under 5)

Requests should be accompanied by a completed information sheet for metabolic screening --see sample in appendix 2

Screening Test	Volume, ml	Container
plasma glucose	0.3	yellow cap(fluoride tube)
plasma ammonium**	0.75	orange cap(heparin tube, sent on ice
blood gases	0.3	heparinised capillary
anion gap		calculated based on routine electrolyte & RFT and blood acid/base results. (Chloride ought to be requested)
plasma lactate**	0.3	yellow cap(fluoride tube, sent on ice)
plasma amino acids	2 x 0.75	orange cap (heparin tube)
urine for metabolic screening	spot urine*	pink top sterile container

* 2 ml is optimal but any urine volume can often be used by arrangement with the laboratory.
** Contact the laboratory to arrange for these tests as there are special sample requirements.

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Plasma ammonia (in ice), amino acids and blood lactate must be sent to the laboratory immediately after collection. It is therefore important to contact the laboratory prior to collecting specimens after hours.

Urine specimens can be kept in the ward freezer/refrigerator overnight.

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A kit should be prepared containing most of the specimen containers used for metabolic screening together with an information sheet for metabolic screening. A simple instruction on the specimen requirements should also be provided.

It is advisable to keep a Metabolic Kit on the paediatric wards for after hours use. This kit should be maintained by the Pathology Laboratory.

It is important that a metabolic screening information sheet as well as the laboratory request form is completed by the clinician.

Suggested contents of the Kit

• 3 x 0.75 ml heparin tubes for L/RFT, chloride and ammonia.
• 2 x 0.3 ml fluoride tube for glucose and lactate.
• 2 x 0.75 ml heparin tubes for plasma amino acid pattern.
• 1 x pink top container for urine collection.

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6. Peri-mortem Protocol

In the event that a patient with suspected inherited metabolic disease dies before a specific diagnosis can be made, the following procedure is suggested.

Perimortem collection: Please state the period after death when the specimen was collection.

(a) Blood collection

• 2 x 5 ml in heparin tube
• 5 ml in EDTA tube
• 2 x 1 ml in fluoride tube
• 10 ml in plain tube (for storage)

(b) Guthrie filter paper for collection of capillary blood.

• Spot 1 to 2 drops of blood onto each of the 4 circles on the right side until the circles are filled completely on both sides of the filter paper. Then leave the blood spots to dry in air.

• Blood spots can now be used for the diagnosis of a large number of inherited metabolic disorders using e.g. mass spectrometry and DNA-techniques. Locally assays for galactose 1-phosphate uridyltransferase and carnitine are provided. Other investigations can be carried out in overseas laboratories on blood spots mailed to them.

(c) Urine collection

• as much as possible and put in pink top sterile container.

(d) Tissue Collection

Skin (for Fibroblast culture)

• Collect a few core of skin biopsy into sterile container. It can be kept at 4C refrigerator before being sent for fibroblast culture.

Liver tissue

• Collect multiple (3-4) cores of liver tissue by perimortem liver biopsy (preferred) or at early post-mortem dissection.
• Put on aluminium foil, snap freeze and store at -70 C.

Skeletal Muscle tissue

• Put on aluminium foil, snap freeze and store at -70 C.

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7. Post-mortem Protocol

Bile

• Can be used for post-mortem diagnosis of organic acidaemias.

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8. Confirmatory Tests:

If any of the screening tests are positive, confirmatory tests need to be carried out. Please contact the laboratory.

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References:

Investigation of metabolic disease

General

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Go to appendix 2: Metabolic screening information sheet