CP: Guide to specimen collection 

• Ideally, the patient should be fasting and smoking should be avoided for at least 6 hours before sample collection unless in emergency situation. The patient should be in non-stressed condition, avoid fist clenching or using tourniquet as it may cause spurious increase in ammonia concentration.

• Please call the lab to notify the sending of the sample.

• A free‐flowing venous blood sample should be collected into EDTA specimen tube (preferably pre‐chilled).

• The sample should be transported on ice to the laboratory immediately, centrifuged and analysed within 30 minutes of collection.

• Delay in separation of plasma, haemolysed or clotted samples will cause falsely elevated ammonia levels.

• Plasma stability after separation from cells: 3 weeks at -38°C

Reference:

1. Hawke L. (2012.2). Ammonia (plasma, blood). The Association for Clinical Biochemistry & Laboratory Medicine. “https://www.acb.org.uk › resource › ammonia”.

2. IFU: NH3L Ammonia for Roche/Hitachi cobas c701/702

• Patient should rest for at least 30 minutes before blood taking as lactate level increases rapidly with physical exercise.

• Ideally, venous specimens should be obtained without the use of a tourniquet because venous stasis will increase lactate levels. If a tourniquet must be used, the blood should be drawn immediately after the tourniquet is applied (less than 30 seconds).

• Please call the lab to notify the sending of the sample.

• Collect the sample in Na‑fluoride/K‑oxalate tube (gray-capped tube) and transport to the laboratory immediately. Centrifugation and separation of the plasma from the cells is recommended within 15 minutes of collection to avoid falsely elevated plasma  lactate concentration.

• Plasma stability after separation from cells: 14 days at 2‑8 °C.

A. Indication: Estimating glomeruli filtration rate.

B. Specimen: 24-hour urine sample and plasma/serum creatinine

C.  Procedure:

• On the day of urine collection, discard and do not collect the first voided early morning urine, begin the collection after this void. Collect all subsequent voided urine for the next 24 hours and the final collection will be the first voided early morning urine on the second day. For example, collection start at 6 am on the first day until 6 am the next day.

• After completed the 24-hour urine collection, label the container appropriately and state the collection date and time.

• Send the collected 24-hour urine to laboratory immediately for processing.

• Plasma/serum creatinine must be collected during the sending of the 24-hour urine bottle.

• Tests profile include CSF Protein, Glucose and Chloride. Please specify other biochemistry test in the request form other than the profile tests such as LDH if needed.

• Specimen should be collected in a sterile manner and placed in Bijou bottle.

• Collect a minimum of 1 mL of specimen.

• For CSF glucose, plasma glucose should be collected at the same time. The normal CSF glucose is about 60% compared to plasma level and must always be compared with concurrently measured plasma values for adequate clinical interpretation.

• Transport the specimen to laboratory immediately for analysis as a delay in analysing CSF may cause falsely low glucose value due to glycolysis.

• Results on bloodstained samples will be unreliable.

Reference:

1. Nigrovic LE, Kimia AA, Shah SS, Neuman MI. Relationship between cerebrospinal fluid glucose and serum glucose. N Engl J Med [Internet]. 2012;366(6):576–8. Available from: http://dx.doi.org/10.1056/nejmc1111080

A.    Indication:  screening test for hypercortisolism. 

B.   Patient preparation:

• Avoid or withhold exogenous glucocorticoid use and medications that may induce metabolism of dexamethasone.

• Ensure patients are not on oral oestrogen therapy (stop oral contraceptive pill for at least 4 weeks since oestrogens increase cortisol binding globulin and total cortisol) and advice barrier contraception as required.

C.   Procedure: 

• Dexamethasone 1 mg (2 x 0.5 mg tablets) to be given to patient at 11 pm.

• Collect serum cortisol at 8 am the next day.    

                                           D.  Interpretation:

1. Normal response: 

Morning cortisol level of < 50 nmol/L after 1 mg dexamethasone administered overnight indicates normal suppression of the hypothalamic pituitary adrenal axis.

2. Abnormal response: 

A morning cortisol of ≥ 50 nmol/L after 1 mg dexamethasone administered overnight suggests hypercortisolism.

Reference:

1. Chiang C, Inder W, Grossmann M, Clifton-Bligh R, Coates P, Lim EM, Ward P, Stanford P, Florkowski C, Doery J. Harmonisation of Endocrine Dynamic Testing - Adult (HEDTA). The Endocrine Society of Australia and The Australasian Association of Clinical Biochemists, Australia, 2021

1. Indication:  To diagnose Adrenal insufficiency. 

2. Patient preparation: 

• Withhold any steroid treatment for 24 hours prior to the test (patients treated with dexamethasone require at least 48 hours of steroid withdrawal) if appropriate

3. Procedure: 

• Procedure should be performed between 8 - 9:30am when cortisol is at the peak level.

• Collect baseline serum cortisol.

• Administer IM or IV Synacthen (Tetracosactide) 250 ug.

• Collect serum cortisol at 30 and 60 minutes after injection as picture above👆.

• Send all samples to the lab as soon as possible.

4. Interpretation: 

• Normal response: 

Serum cortisol level ≥ 500 nmol/L at 30 or 60 minutes

• Abnormal response:

Serum cortisol level < 500 nmol/L at 30 or 60 minutes

Reference:

1. Chiang C, Inder W, Grossmann M, Clifton-Bligh R, Coates P, Lim EM, Ward P, Stanford P, Florkowski C, Doery J. Harmonisation of Endocrine Dynamic Testing - Adult (HEDTA). The Endocrine Society of Australia and The Australasian Association of Clinical Biochemists, Australia, 2021

Procedure for Collection of Dried Blood Spot on Whatmann Filter Paper

Filter Paper

Whatmann 903 filter paper is required which has special thickness and is manufactured solely for blood collection. It is commercially available. Please contact the laboratory for further details

Time of collection

1. Timing of collection is critical. It is recommended that the DRIED BLOOD SPOT sample for Newborn screening of IEM be collected from already fed baby at the age between 48 to 72 hours. If a baby is to be discharged before 48 hours of age, blood can still be collected but a repeat sample at >48-72 hours may be required. This allows for early metabolic changes and the commencement of milk and protein feeds. FAOD is best diagnosed if sample is taken within this period.

2. Samples collected too early may give false negative results. Samples collected too late may place children with health problems at risk of irreversible damage.

3. A blood spot sample for High-risk IEM screening in older baby can be taken from heel-prick, finger prick or venipuncture depending on the age of the baby.

Procedure

1. Making Dried blood spots (DBS) from Heel-prick or finger prick.

1.1 For normal newborn baby without any symptoms, it is advisable to collect blood from heel prick.

a) Label each card with baby’s identifications and date of collection and any other pertinent information.

b) Wear gloves and warm the foot with warm cloth/towel

c) Clean the side area (see photo) with either alcohol swab then dries with a clean gauze or cotton wool swab.

d) Puncture heel with sterile lancet (point <2.4 um) on medial or lateral plantar surface.

e) Allow puncture to ooze and wipe away first drop of blood with cotton swab.

f) Gently massage above the puncture to encourage blood flow and drop a big spot of blood onto each of the 3 circles on the filter paper

g) Continue step e and f until all the circles are filled. One full drop of blood equivalent to 50ul is required to fill each circle. Blood must soak through the card to the other side. Do not drop more than one drop of blood on one circle.

h) Completely dry the card at ROOM TEMPERATURE (25°C to 30°C on a clean, flat, non-absorbent surface or a drying rack designed for the purpose, for more than 4 hours or overnight. Minimum time needed for drying is 4 hours.

i) To avoid contamination of the sample, do not touch the blood spot circle with bare hand.

j) Place dried filter paper in an individual envelope or plastic bag for mailing or transport. It is recommended that the filter paper (dried blood spot) is store away from any source of heat, liquid and organic fumes.

2. Making DBS from blood collected from Venipuncture.

a) Label each card with patient reference, sample collection date and any other pertinent information.

b) Collect blood in heparinized tube (green top). If blood is drawn in a syringe, transfer it into a green tube.

c) Set a hand-held pipettor to 50 ul volume and fill the pipette tip with blood. (Note: If the tube has been sitting still for more than 2 minutes, it must be mixed by inverting it up and down few times before pipetting)

d) Point the pipette tip at the center of a circle on the filter paper card. Gradually discharge the blood and let it soak into the paper, filling the circle completely.

e) Complete steps 1.1 (h) to 1.1 (j)

Note1: The filter paper card(s) must be completely dry before dispatch. Protect them from moisture or condensation at all times.

Note 2: Store at 4°C if the card is transported the next day after it is dried. Freeze at -20°C for long-term storage.

Note 3: The DBS must be prepared at least 4 hours ahead of transportation to allow complete drying

Quality of Dried Blood spot

1. Acceptable blood sample cards

• Each blood spot is checked for acceptability. Blood spot should be dry, the pre-printed circles filled and appear as an even dark color on both sides of the card without lighter discoloration.

2. Unsuitable DBS will be rejected such as follows:

1. Lack of blood coverage (Quantity insufficient for testing – Blood spot’s diameter is too small)

2. Layering of blood

3. Incomplete blood saturation

4. Separation of red blood cells and serum

5. Blood spot contaminated with fungus

6. Blood spot is diluted/contaminated with water

7. Indication:  To diagnose Adrenal insufficiency. 

8. Patient preparation: 

• Withhold any steroid treatment for 24 hours prior to the test (patients treated with dexamethasone require at least 48 hours of steroid withdrawal) if appropriate

9. Procedure: 

• Procedure should be performed between 8 - 9:30am when cortisol is at the peak level.

• Collect baseline serum cortisol.

• Administer IM or IV Synacthen (Tetracosactide) 250 ug.

• Collect serum cortisol at 30 and 60 minutes after injection as picture above👆.

• Send all samples to the lab as soon as possible.

10. Interpretation: 

• Normal response: 

Serum cortisol level ≥ 500 nmol/L at 30 or 60 minutes

• Abnormal response:

Serum cortisol level < 500 nmol/L at 30 or 60 minutes

Reference:

1. Chiang C, Inder W, Grossmann M, Clifton-Bligh R, Coates P, Lim EM, Ward P, Stanford P, Florkowski C, Doery J. Harmonisation of Endocrine Dynamic Testing - Adult (HEDTA). The Endocrine Society of Australia and The Australasian Association of Clinical Biochemists, Australia, 2021

1. Indication: To diagnose diabetes mellitus or gestational diabetes mellitus.

2. Patient preparation:

• Normal carbohydrate diet for three days before testing.

• Start fasting at 10pm the night prior to testing (plain water is allowed).

• Patient must rest throughout the test.

• Only sips of plain water allowed, smoking and eating are not allowed during the test.

3. Procedure: 

(a)  8.00 am: 

• Collect fasting blood sample in Na‑fluoride/K‑oxalate tube and label the tube with “FBS” and patient’s details.

• Patient to drink 75g anhydrous glucose dissolve in 300 ml water (Paediatric patient: 1.75g anhydrous glucose per kilogram weight; 75g maximum).

• The glucose drink should be finished within 5 mintues.

(b) 10.00 am: 

• Collect another blood sample two hours after patient finishes the glucose drink (2 Hour Post-Prandial) in Na‑fluoride/K‑oxalate tube.

•  Label the tube with “2HPP” and patient’s details. 

4.   Interpretation: Please refer the diagram above 👆

Reference:

1. Clinical Practice Guidelines, Management Of Type 2 Diabetes Mellitus (6th Edition), December 2020 .MOH/P/PAK/447.20(GU)-e.

2. Clinical Practice Guidelines, Management Of Diabetes In Pregnancy, 2017 MOH/P/PAK/353.17(GU).

A. Indications

1. For ADULT patients to fulfill at least 2 out of the criteria stated below:

• Age of onset of hypertension less than 40 years old

• Require more than 3 anti hypertensives (resistant hypertensive)

• Exhibit paroxysms of headache, sweating and tachycardia

• Hypertensives with paroxysmal postural hypotension

2. Adrenal mass/incidentaloma work-up

3. Multiple Endocrine Neoplasia (MEN) 2 work-up

4. Suspected phaeochromocytoma

B. General requirements:

1. Request for 24 hour urinary metanephrines test must be reviewed and endorsed by specialist (preferably endocrinologist)

2. For hospitals with chemical pathologist, endorsed requests should go through chemical pathologist before sending to referral lab.

3. PER-PAT 301 form: Should clearly stated detail of patient, test indication/clinical history, detail of requesting specialist/endocrinologist (name, signature & stamp, contact number).

C. Patients preparation

• The urinary level on Biogenic Amines can be influenced by medication and certain foods.

  • • Medications: If possible should be stop medication with active ingredient L-DOPA, tricyclic antidepressant, anti-psychotics and adrenergic receptor blockers at least 1 week prior to testing.

    • Foods: Recommended to avoid all kind of spicy food and caffeinated drink for at least one week prior to testing.

D. Collection instruction

1. Discard the voided urine and note the start time and date

2. Collect all urine voided for 24 hours duration and mixed well every time added the urine into the container

3. Do not void urine directly into the 24 hours container. Please use other clean uine container for urine collection to avoid spillage or acid injury.

4. Keep the closed urine container in cool and dry place.

5. Add the last voided urine into container and note the finishing time and date

E. Rejection criterias

1. Total 24 hours urine volume of less than 1 litre in ADULT (18 year old and above)

2. Leaking specimen

3. Request not approved/endorsed by chemical pathologist /specialist /endocrinologist

Reference: 

Protocol for 24 hour urine metanephrine test provided by Chemical Pathology Unit, Department of Pathology, Hospital Kuala Lumpur

Sample collection must follow the guidelines as in the ‘Pekeliling Ketua Pengarah Kesihatan Bil 1/2021: Garis Panduan bagi Pengesanan Penyalahgunaan Dadah Dalam Air Kencing Versi 2.0’ as below:

1. Sample collection must be supervised by authorised personnel. Laboratory personnel shall not be involved in the sample collection. Collection site must have suitable toilet facilities and are free from soap, cleaning agent or water supply (toilet water is coloured when this is not possible).

2. Personnel at the collection site shall be responsible for collecting, labelling, packaging and transporting of specimen and shall ensure that the collection and storage procedures have proper documentation and security measures in place.

3. The urine volume should be at least 30 mL collected in urine container.

4. The collecting personnel shall ensure there is no attempt to falsify or adulterate the specimen.

5. The urine bottle must be securely capped and sealed with sealing wax.

6.  Bottle must be labeled in front of the donor (patient/suspect) with the following information:

• Donor’s full name

• Full identity card number

• Date and time of specimen collection

• Signature of Donor

• Signature of Supervising Officer

7.  Proper Chain of Custody shall be maintained from urine collection to specimen transportion to the laboratory.

8. Please use ‘Borang Permintaan Untuk Pengesanan Dadah Dalam Air Kencing UPD1’ (Pindaan 2020).

A. Indications

Screening for primary hyperaldosteronism in hypertensive patients with spontaneous or diuretic-induced hyperkalemia.

Suggested candidates for screening:

1.     Patients with hypertension and hypokalemia

2.     Patients with resistant hypertension

3.     Young hypertensive (age <40 years)

4.     Patient with adrenal incidentaloma

B. Patient preparation

1. Attempt to correct hypokalemia

• Blood should be collected slowly with syringe and needle (preferably not vacutainer to minimize risk of spuriously elevated potassium)

• Avoid fist clenching – wait at least 5 second after tourniquet release to insert needle

• Separate plasma from cells as soon as possible or within 2 hours of collection

• Avoid hypoalemia as it suppresses aldosterone secretion. Give potassium replacement (Slow K tabs) sufficient to raise plasma potassium to >4.0 mmol/L

2.   Subject should be normally hydrated and has adequate intake of sodium

3.   Drugs to avoid: Spironolactone (stopped for 6 weeks), amiloride, triamterene, Potassium-sparing diuretics and product derived from licorice root.

4.   If ARR testing is not diagnostic after withdrawing above agents and hypertension can be controlled with non-interfering medications, test again 2 weeks after withdrawing other medications: ACE-I, ARB, βBlockers, methyldopa, clonidine, oral contraceptives (do not withdraw oral contraceptives unless confident of alternative-effect contraception)

5.  Drugs that do not interfere with renin-aldosterone axis include: Prazosin, verapamil, hydralazine and terazosine.

C. Collection instruction

1.     Collect midmorning after patient has been sitting, standing, walking for at least 2 hours, and seated for 5-15 minutes.

2.     Collect blood carefully to avoid stasis and hemolysis during collection.

3.     Collect samples into 2 tubes of EDTA (Please use different tubes for Renin and aldosterone. Minimum volume: 3ml per sample).

4.     Maintain sample at room temperature (not in ice) during transportation to laboratory for centrifugation.

5.     Fill up only single PER-PAT 301 form for Aldosterone Renin Ratio test request.

MANDATORY to include in the request form: Relevant clinical history, drug history, latest potassium (K+) level, patient posture (supine/upright) upon blood withdrawal.

·     Test should be requested by Specialist / Endocrine Specialist only.

·     Sample collection:

• Sampling time: Should be taken between 8am to 10 am

• Supine sample: Sample taken in early morning before subject arises (If feasible)

• Upright sample: Subject should be upright for ≥ 2 hours prior sampling.

D. Interpretation

• High aldosterone and suppressed plasma renin indicate primary hyperaldosteronism

• Some patients with renal disease may give similar results

Reference: 

Protocol for requesting and collecting samples for plasma renin and plasma aldosterone provided by Department of Pathology, Hospital Putrajaya