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Oral Glucose Tolerance Test (OGTT): Oral Glucose tolerance test is used to evaluate the ability to regulate glucose metabolism. It is used to identify patients at high risk of developing type-2 diabetes based on clinical risk factors, with a view to beginning preventive therapy.

Indications:
1. Plasma fasting glucose between 5.6 mmol/l (100 mg/dl) and 6.9 mmol/l (125 mg/dl).
2. To screen for impaired glucose tolerance, which is associated with an increased risk of developing diabetes mellitus
3. Patients with family history of diabetes mellitus.
4. To screen for gestational diabetes mellitus at 24-28 weeks of gestation in all pregnant women not known to have diabetes
5. To screen for diabetes mellitus at 6-12 weeks postpartum in women with a history of gestational diabetes mellitus, using non-pregnant OGTT criteria

Requirements for OGTT:
1. Duration of fast: 10 – 16 h
2. Prior carbohydrate intake: Unrestricted diet containing at least 150 g of carbohydrate per day for three consecutive days prior to test.
3. Medications: Discontinue medications known to affect glucose tolerance like thiazides, oral contraceptives, corticosteroids
4. Intercurrent illness or trauma: The test should not be performed on hospitalized, acutely ill or inactive patients

Administration of Glucose:
Form of glucose: Anhydrous or monohydrate
Quantity of glucose ingested: Recommended load for adults is 75 g and for children 1.75 g/kg up to 75 g maximum
Volume in which administered: 300 ml of water
Rate of ingestion: Ingested over 5 minutes

Precautions during the test:
1. Time of the day; Test should begin between 7 AM and 9 AM
2. Posture; Patient should remain seated during the test
3. Smoking; Smoking is not allowed during the test
4. Caffeine; Patient should not take tea or coffee during the test
5. Anxiety; Patient should be stress free

Sample tube: Sample should be taken in sodium fluoride tube

Procedure:
1. Sample is taken for Fasting Plasma Glucose
2. Second plasma sample should be taken exactly TWO hours after glucose load.

Interpretation: (Based on 2 h Post Glucose Load):
1. Normal: < 7.8 mmol/l (140 mg/dl)
2. Impaired Glucose Tolerance: 7.8 – 11.1 mmol/l (140-200 mg/dl)
3. Diabetes Mellitus: > 11.1 mmol/l (200 mg/dl)

Contraindications:
Plasma Fasting Glucose > 7.0 mmol/L (126 mg/dl)
Bone Marrow Aspiration: Patient’s preparation
i. The test is done on all working hours from 0800 – 1300hrs.
ii. Before bone marrow examination full breakfast meal is recommended for adults.
iii. In children <12 years eating and drinking is prohibited four hours prior to procedure.
iv. After procedure use of fruit juices is recommended.
v. If patient is using any medication (Heparin, warafrin, Aspirin) or having any drug allergy to analgesic drug, he/she should inform the doctor

Brief description of test procedure
i. Consent is taken from the patient before the procedure.
ii. Sterilization of site(PSIS) is done.
iii. Local anesthesia is given before procedure.
iv. Aspiration and Trephine biopsy taken from selected site after 5 min of local anesthesia.
v. After procedure proper dressing is applied to the site.
vi. This procedure requires 10 – 15 min.
vii. In children, this test is performed by minimal general anesthesia.
viii. Pressure dressing should be removed after one day.
ix. For pain simple analgesics (Ponston or Paracetamol) are recommended.
Hyperglycemia in Pregnancy: Diabetes mellitus (DM) has dreadful complications in pregnancy. It can present with more than one ugly faces:

a. Pre-gestational DM: This term is sometimes used for women who are known cases of DM and become pregnant. Diagnosis of these cases is similar to the DM discussed in previous guidelines.

b. Overt DM: Relatively newer class describes women who had pre-existing diabetes but were unaware of it. Full blown DM diagnosed at any stage of pregnancy is called Overt DM. The incidence of this category is increasing with worsening life styles and increased prevalence of type 2 DM in women. WHO has endorsed this category in 2013 but with the term ‘Diabetes is Pregnancy’ (without the word ‘Overt’)

c. Gestational DM (GDM): Women with onset or first recognition of abnormal glucose tolerance during any stage of pregnancy (other than those in overt category).

Identification of high risk patients:
1. Ethnicity (e.g. Sub-continent countries)
2. Strong family history of diabetes
3. Prior history of GDM
4. Previous delivery of a baby > 4.1 kg
5. Manifestations of Insulin Resistance e.g. Metabolic Syndrome or PCOS
6. Other manifestations of glucose intolerance

Recommended screening protocol:
Screening at first ante-natal visit: If TWO or more risk factors are present then screening should be done at first visit (before 24 weeks) by protocols used for non-pregnant adults (see below). If overt DM or GDM is diagnosed at this stage patient should be managed not only for her glycemic control but also for diabetic complications.

Screening at 24-28 weeks: All women who have not been diagnosed as GDM or overt DM earlier should undergo either
1. One step approach with 75 g Oral Glucose Tolerance Test (OGTT) at 24-28 weeks of gestation.
Or
2. Two step approach

1. One Step Approach:
a. OGTT with 75 g glucose (Plasma glucose is measured fasting and at 1 and 2 h post glucose load)

b. Diagnosis of GDM at any stage of pregnancy is made if any one of the following is present following a 75 g oral glucose load:
Fasting: Between 5.1-6.9 mmol/l (92 -125 mg/dl)
One hour: >10.0 mmol/l (180 mg/dl)
Two hour: Between 8.5 – 11.0 mmol/l (153-199 mg/dl)

2. Two Step Approach:
a. Glucose challenge test with 50 g glucose
• 50 g oral glucose load is given without regard to time of day
• Plasma glucose is measured after 1 hour
• If plasma glucose ≥7.2 mmol/L, 100 g OGTT is performed

b. OGTT with 100 g glucose
• Patient would be in fasting state (not having had anything to eat or drink for the past 8 hours.
• A fasting blood glucose would be done.
• Patient would drink 250 ml of a glucose solution containing 100 grams of glucose.
• Blood glucose level will be tested again one, two and three hours after drinking the solution.
After drinking the glucose solution, patient would likely to remain in the lab. After the glucose tolerance test, patient can return to her usual activities immediately.

Diagnosis is confirmed when two or more Plasma Glucose levels meet or exceed:
• Fasting: 95 mg/dL or 105 mg/dL (5.3/5.8)
• 1 hour: 180 mg/dL or 190 mg/dL (10.0/10.6)
• 2 hour: 155 mg/dL or 165 mg/dL (8.6/9.2)
• 3 hour: 140 mg/dL or 145 mg/dL (7.8/8.0)
hCG Stimulation Test: Indications:
1. Ambiguous genitalia
2. Delayed puberty
3. To determine testicular reserve

Preparation:
No special patient preparation is required

Requirements:
hCG injection- Dose is 100 IU/Kg or 1500 IU/m2 body surface area for infants or 5000 IU for children over 2 years

Test protocol:
Day 0:
1. Patient is required to report at endocrine clinic of AFIP and blood sample for serum testosterone, LH and FSH are taken .
2. Inj 1500 IU or 5000IU hCG IM is given
Day 3:
1. Blood sample for testosterone is taken
2. Samples are analysed for testosterone

Interpretation:
1. A doubling of testosterone concentration over base line with testosterone > 5.2 nmol/l is consistent with normal testicular reserve.
2. 2-9 times response is either normal response or is suggestive of Androgen Insensitivity Syndrome
3. >10 times response is suggestive of 5-alpha reductase deficiency.
Evaluation of Hypercortisolism: Initial diagnostic tests for hypercortisolism should be highly sensitive, even though the diagnosis may be excluded later by more specific tests.

Dynamic Function Tests:
1. Low Dose Dexamethasone Suppression Test (LDDST)
2. High Dose Dexamethasone Suppression Test (HDDST)

1. Low-Dose Dexamethasone Suppression Test
There are two forms of the Low-Dose Dexamethasone Suppression test:
 1.0 mg “overnight” DST
 Two-day 2.0 mg DST

Patient preparation:
There should have been no treatment with glucocorticoid drugs (including topical preparations) for several weeks.
Mineralocorticoids do not interfere with this test. The test may be performed on inpatients or outpatients.

Indication:
To diagnose Cushing’s Syndrome

Procedure:

Protocol for 1.0 mg overnight DST:
• At 2300 h Dexamethasone (1.0 mg) is given orally.
• A venous blood sample is taken at 0800 h on the following day for serum Cortisol.

Protocol for two-day 2.0 mg DST:
• Dexamethasone 0.5 mg is given orally at 6 hours interval for four doses: 0800 h, 1400 h, 2000 h and 0200 h-total of 2.0 mg
• Blood sample is taken at 0800 hours next day for Cortisol

2. High-Dose Dexamethasone Suppression Test
There are two forms of the high-Dose Dexamethasone Suppression Test:
 8.0 mg “overnight” DST
 Two-day 8.0 mg DST

Patient preparation:

Patient can be admitted to hospital. There should be no treatment with Glucocorticoids drugs (including topical preparations) for several weeks; mineralocorticoids do not interfere with this test.

Indication:
To differentiate pituitary-dependent and ectopic causes of Cushing’s Syndrome

Procedure:

Protocol for 8.0 mg overnight DST:
• Morning (0800-0900 hours) basal sample is taken for serum Cortisol.
• At 2300 h Dexamethasone (8.0 mg) is given orally.
• A venous blood sample is taken at 0800 hours on the following day for serum Cortisol.

Protocol for two-day 8.0 mg DST:
• Dexamethasone 2.0 mg is given orally at 6 hours interval for four doses: 0800 h, 1400 h, 2000 h and 0200 h-total of 8.0 mg
• Blood sample is taken at 0800 hours next day for serum Cortisol
GnRH Stimulation Test: Indication:
1. Precocious puberty
2. Delayed puberty

Preparation:
No specific patient preparations

Requirements:
100ug GnRH preparation. Dose for children is 2.5 ug/Kg body weight to a maximum of 100 ug

Procedure:
0 min: Take 3 ml blood for LH and FSH. Followed by inj GnRH i.v. as a bolus
30 min: Take 3 ml blood for LH and FSH
60 min: Take 3 ml blood for LH and FSH

Interpretation:
In Women; Normal response varies in relation to menstrual cycle
In Follicular Phase; LH peak should be increased at least 2-fold over
baseline or a net change of at least 10IU/L and FSH peak should be
at least 1.5fold over baseline or a net change of 2 IU/L.
In Men; Response may be considered normal if the basal values are in the reference range and there is at least a doubling at 30 min for both LH and FSH.
Saline infusion test for Primary Hyperaldosteronism: Indication:
Confirmation of primary hyperaldosteronism.

Preparation:
• Stop spironolactone and eplerenone for 6 weeks before the test
• Stop beta blockers, calcium channel antagonists, ACE inhibitors and AT2 blockers for 2 weeks before the test.
• Can continue to use alpha blockers to manage hypertension e.g. doxazosin
• Ensure plasma K in normal range (ideally >4) prior to performing test

Procedure:
• Patients stay in the recumbent position for at least 1 hour before test begins.
• Cannulate and take blood for plasma aldosterone, plasma renin concentration, Urea and electrolytes.
• Infuse 2 liters of 0.9% saline over 4 hours, starting at 9.00 a.m.
• Blood pressure, oxygen saturation and heart rate are monitored throughout the test.
• After 4 hours (i.e. 13:00), take further blood sample for aldosterone, urea and electrolytes.

Contraindications:
• This test should not be performed in patients with severe uncontrolled hypertension, renal insufficiency, cardiac insufficiency, cardiac arrhythmia, or severe hypokalemia.
Interpretation:

Plasma Aldosterone Concentration will fall below 139 pmol/L in normal subjects, whereas values above 277 pmol/L are consistent with Primary Hyperaldosteronism
Growth Hormone Evaluation: GH stimulation tests include:
1. Exercise stimulation test
2. L-dopa stimulation test
3. Insulin Tolerance test
For establishing GH deficiency as a cause of short stature, at least two provocative tests should be suggestive of GH deficiency.

1. EXERCISE STIMULATION TEST:

Principle:
Strenuous physical exercise causes GH secretion in normal subjects.

Preparation:
Patient should be fasting overnight (no calorie intake for at least 8 hours).
Test should be performed early in the morning (0800 hours)

Indication:
Short stature

Procedure:
1. Basal venous sample is obtained for GH.
2. Patient is subjected to strenuous exercise on treadmill for 20 minutes.
3. Pulse is monitored during the test.
4. A venous blood sample is obtained 10 minutes after termination of the exercise.
5. Both the samples are centrifuged and GH is estimated in serum portion of both samples.

Interpretation:
In children:
GH level >20 mIU/l, adequate response to exercise stimulation test.
GH levels <20 mIU/l, Inadequate response to exercise stimulation test. It raises the need for a second provocative test.
In adults :
Normal response is > 15 mIU/l

2. LEVODOPA STIMULATION TEST:

Principle:
Levodopa stimulates release to GH from anterior pituitary gland, measurement of which serves as an indicator to anterior pituitary function.

Preparation:
Patient should be fasting overnight (no calorie intake for atleast 8 hours)
Test should be performed early in the morning at 0800 hours.

Procedure :
1. Basal venous sample is taken for GH.
2. L – dopa is administered orally according to the following dosage schedule :
Patient > 30 kg : 500 mg
Patient between 15 – 30 kg : 250 mg
Patient < 15 kg : 125 mg
3. Venous blood sample is collected 60 minutes after L – dopa administration.

Indication: Short stature

Interpretation:
In children-
GH level >20 mIU/l, Adequate response to test GH level < 20 mIU/l, Inadequate response to test, needs confirmation by second provocative test
Adults :
Normal response is > 15 mIU/l

3. INSULIN TOLERANCE TEST :

Indications:
1. Assessment of GH and cortisol/ACTH reserve
2. Short stature
3. Assessment of hypothalamic pituitary axis

Contraindications:
1. Age > 60 years
2. IHD with abnormal ECG
3. Epilepsy
4. Morbid obesity
5. Severe pan hypopituitarism, hypoadrenalism (9 00 hours cortisol < 100 nmol/L)

Principle:
ACTH and GH are both released as a part of stress mechanism triggered by hypoglycemia. GH hormone is measured directly. Cortisol is measured as an indicator of ACTH response.

Pre-requisites:
1. Detailed history, physical examination and measurement of height and weight (percentiles)
2. Baseline investigations:
Blood CP and ESR
Renal Function Tests
Liver Function Tests
Thyroid Function Tests
3. Morning serum Cortisol levels
4. X-Ray wrist for bone age
5. Exercise or levodopa stimulation test result or serum IGF or IGFBP3 levels.
6. A specialist pediatric endocrine unit is required for this test. Parents should be communicated regarding the procedure and side effects.
Written consent by parents should be taken before the procedure.
7. Physician should be in attendance throughout the test.
8. Medicines required are:
50 ml 50% dextrose solution
Glucagon administration IM in case IV access is lost
Glucose test strips and lancets with glucometer
Indwelling cannula gauge 19 to 22
Sodium Fluoride and plain test tubes
9. Resuscitation equipment

Preparation:
a. Patient should fast overnight (no calorie intake for at least 8 hours) and should be recumbent during the test.
b. Test to be performed around 8 00 am.
c. Weight of the patient.

Procedure :
1. Place an indwelling cannula in the ante cubital fossa with good access for administration of glucose if required.
2. Check fasting plasma glucose by glucometer. Take 3.0 ml blood in plain tubes for GH/ cortisol and 3.0 ml in fluoride tube for glucose levels (for basal levels and at induction).
3. Calculate Inj Insulin as follows and administer:
0.15 U/kg for subjects with normal pituitary function.
0.10 U/kg for subjects with hypopituitarism
0.2 to 0.3 kg for subjects with acromegaly, Diabetes and Cushing's Syndrome.
4. Observe signs of hypoglycemia, like tachycardia, palpitations, sweating, headache, confusion, drowsiness, loss of consciousness, abdominal pain and monitor plasma glucose levels with glucometer.
5. Adequate hypoglycemia is fasting plasma glucose < 2.5 mom/l or 50 % of the basal glucose value which is symptomatic.
6. Take 3.0 ml venous blood in plain tubes for GH / Cortisol and 3.0 ml in fluoride tube for glucose and give sweet drink to child.
7. Repeat sampling at 60, 90, 120, 150 and 180 minutes. Maximum response will be at 2-3 hours.
8. Patient to be given a supervised meal after completion of the test.

Emergency management of hypoglycemia:
Hypoglycemia to be reverted if there are symptoms.
If necessary, IV 50 & dextrose 0.4 ml/kg should be administered
and blood sampling continued as protocol.
Glucagon only to be given if venous access is lost.

Interpretation :
1. An adequate cortisol response is defined as a rise in cortisol of greater than 550 nmol/L or > 200 nmol/L from basal level.
2. GH response : An increase in GH response of > 20 mIU/l indicates adequate pituitary GH reserve.
3. GH response in adults should be > 15 mIU/l.
GH Suppression Test for Acromegaly: Indication:
Used when clinical diagnosis of acromegaly is suspected.

Preparation :
1. Patient should be in fasting state, after at least 10 hours of fasting
2. Test should be performed at 08 00am

Procedure :
1. Take blood sample for GH (yellow top Vacutainer) and glucose at T = 0.
2. Administer 75 grams oral glucose in 300 ml water over about 10 minutes.
3. Take blood for GH and glucose at T = 30, 60, 90 and 120 minutes.

Interpretation:
In normal individuals, GH levels are suppressed following oral glucose, At least one of the samples during the test should have undetectable GH levels (i.e. < 1mcg/l).
Failure of suppression or a paradoxical rise in GH suggests acromegaly
Short Synacthen Test: High Dose Short Synacthen Test:

Indication
Screening test for suspected adrenal insufficiency.

Contraindications:
• Pregnancy, history of hypersensitivity to ACTH, Synacthen or Synacthen depot.
• Suspected Cushing’s syndrome

Patient Preparation:
1. Test is performed in morning (0800 hours). Fasting is not required.
2. If possible glucocorticoids medication should be ceased 24 hours prior to test to minimize possible suppression.
3. Patient taking glucocorticoids other than Dexamethasone should be shifted to Dexamethasone after medical consultation because other glucocorticoids interfere with assay.

Dosage for Adults:
250 ug of Synacthen (ACTH) injection I/M or I/V
Dosage for Paediatric Patients:
 For children 1 month to 2 yr use a dose of 125 ug
 For children > 2 yr use a dose of 250 ug

Test Procedure:
• This test should be done under supervision of a doctor.
• History of the patient is taken and necessary physical examination is carried out
• Insert a reliable cannula and, if possible, rest the patient for 30 minutes.
• First take the basal sample. Give the calculated dose and take further samples at 30 and 60 min post injection in plain tube

Interpretation:
1. A normal adrenocortical response is defined as:
A post stimulation Cortisol value exceeding the basal value by more than 200 nmol/l. Or
A post stimulation Cortisol value exceeding 550 nmol/l.
2. A failure to respond is consistent with adrenal failure
3. A sluggish response (60 min values > 30 min) is suggestive of adrenal dysfunction secondary to hypothalamic/pituitary disease or steroid therapy.

Low Dose Short Synacthen Test:
Indication :
Gives more sensitive adrenocortical response

Contraindications:
• Pregnancy, history of hypersensitivity to ACTH, Synacthen or Synacthen depot.
• Suspected Cushing’s syndrome
• Viral infections.
• Heart failure.

Precautions:
Patient should be kept under observation throughout the period of this test.

Patient Preparation:
1. Test is performed in morning (0800 hours). Fasting is not required.
2. If possible glucocorticoids medication should be ceased 24 hours prior to test to minimize possible suppression.
3. Patient taking glucocorticoids other than Dexamethasone should be shifted to Dexamethasone after medical consultation because other glucocorticoids interfere with assay.

Dosage for Adults:
1ug Synacthen IV

Test Procedure:
• This test should be done under supervision of a doctor.
• History of the patient is taken and necessary physical examination is carried out
• Insert a reliable cannula and, if possible, rest the patient for 30 minutes.
• Take the basal cortisol levels, give the calculated dose of synacthen I/V then take blood for cortisol levels at 30 and 60 min.

Interpretation:
1. A normal adrenocortical response is defined as:
A post stimulation Cortisol value exceeding the basal value by more than 200 nmol/l. Or
A post stimulation Cortisol value exceeding 550 nmol/l.
2. A failure to respond is consistent with adrenal failure
3. A sluggish response (60 min values > 30 min) is suggestive of adrenal dysfunction secondary to hypothalamic/pituitary disease or steroid therapy.
Platelet Function: Patient’s preparation
i. The test is done on all working days from 0800 – 1300 hrs.
ii. A clinical history and examination is required.
iii. Test should be performed preferably in fasting.
iv. Last platelet transfusion should be at least 5 days ago.
v. The patient should bring all medical documents.
vi. The patient should not exercise 20 minutes before the test
vii. A number of drugs can affect the results of this test. If patient is using any medication he/she should inform the doctor.
viii. Medication that can interfere with a platelet aggregation test include:
• Non-steroidal anti-inflammatory drugs.
• Aspirin (or medications containing aspirin)
• Antihistamines
• Antibiotics (including penicillin, cephalosporin, nitrofurantoin and similar antibiotics)
• Tricyclic antidepressants
• Anti-platelet drug (including clopidogrel, dipyridamole and ticlopidine)
• Theophylline

Brief description of test procedure
i. After aseptic measures, 10 ml of blood is withdrawn from a peripheral vein.
ii. The sample should reach the main lab within 02hrs where it is processed further.
Thrombophilia Screening: Patient’s preparation
i. The test is performed on all working days from 0800 – 1300hrs.
ii. Before performing test patient is asked about the use of any drug as some drugs (Warfarin, Clexane, DOACS) interfere with the results of test.
iii. After consultation with concerned specialist, medicine is stopped temporarily and test is performed after 4 weeks.
iv. It is better to undergo this test before start of anticoagulant therapy.

Brief description of test procedure
i. After taking complete history, blood sample is taken in tri-sodium citrate.
ii. The sample should reach the lab within 2hrs for processing.

SPECIALIZED TEST