Dayyal Dg.

Dayyal Dg.

Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

Why is this test performed?

This test is evaluated in different conditions and diseases such as nephrotic syndrome, liver diseases, kidney diseases, in a patient suspected of malnutrition, and patients severe burn. See also: Procedures for the collection of blood for hemotological investigations.

Collection of sample

For the estimation of serum albumin, patient's serum is needed. A random blood sample may be used for the test, however, fasting sample is preferred.

About 3 to 5 ml of blood is collected in a plain test tube and blood is allowed to clot to get clear serum. The blood sample is centrifuged for 5 to 10 minutes and the serum is separated for the test. The patient's serum is may be stored at 4° C which may be stable for 72 hours.


  • Fasting blood sample is preferred.
  • Specimen with lipemia or hemolysis should be avoided.


  1. This is the richest protein in the blood.
  2. Plasma proteins are separated into three major groups:
    (a) Albumin
    (b) Globulins
    (c) Fibrinogen
  3. The most common method is electrophoresis. It forms 5 bands named as:
    (a) Albumin
    (b) α1 fraction
    (c) α2 fraction
    (d) β fraction
    (e) γ fraction
  4. Total protein is made up of 40 to 60% of albumin.
    (a) Because of its low molecular size and high concentration in blood, it is found in urine, cerebrospinal fluid (CSF), interstitial fluid, amniotic fluid, and most of the extravascular fluids.
    (b) Due to its negative charge at normal pH, it is highly water soluble.
    (c) The half-life of albumin is about 15 to 19 days.
    (d) It is extremely sensitive to liver damage.
    (e) It helps to maintain the osmotic pressure in the blood vessels, without which the fluid will leak out.
    (f) It synthesized in the liver, that is why it reflects the functions of liver and kidney.
    (g) Albumin binds calcium, bilirubin, free fatty acids, and a number of drugs.
    (h) Low level of albumin causes Edema.
    (i) In acute or chronic liver diseases, malignancy, Amyloidosis, and malnutrition decreased synthesis in the liver is seen.
    (j) The role of albumin in transporting calcium, bilirubin, bile acids, metal ions, and drugs will be the variation in its concentration.
    (k) The presence of albumin in the urine represents kidney diseases.
    (l) Dehydration causes an increase in the level of albumin (Hyperalbuminemia).
    (m) The albumin performs a role of carrier protein for calcium, bilirubin, progesterone, and drugs.
    (n) Albumin provides nutrition to the tissues and binds various molecules like vitamin, hormones, and drugs.



It is defined as a condition in which the level of albumin in blood/serum is lower than the normal values.


It is defined as a condition in which the level of albumin in blood/serum is higher than the normal values.

Normal Values

  • Normal range 3.4 to 5.5 g/dl (35 to 55 g/L)
  • Male 4.2 to 5.5 g /dl
  • Female 3.7 to 5.3 g/dl
  • Cerebrospinal fluids 15 to 45 mg/dl
  • Urine (Spot Urine) 1 to 14 mg/mL
  • 24-hour Urine 15 to 45 mg /24 hours
  • Newborn 2.8 to 4.8 g/dl

Why is this test performed?

This hormone test is evaluated in different conditions, such as Hyperaldosteronism, Cushing's syndrome and Virilizing syndrome.

Lab tests for various adrenal abnormalities

Primary Adrenal Insufficiency (Addison's disease):

This is due to the diseases of the gland.

  • Level of sodium in serum is low.
  • Cortisol level is decreased.
  • Glucose level is decreased.
  • ACTH level is raised.
  • Potassium, calcium, and blood urea level are raised

Adrenal Hyperfunction leads to:

  • Increased level of Aldosterone causes Hyperaldosteronism.
  • Increased level of Cortisol causes Cushing's syndrome.
  • Increased level of Androgens causes Virilizing syndrome.

Primary Hyperadrenalism:

  • Level of Cortisol is raised.
  • ACTH level decreases.

Secondary Hypoadrenalism (secondary or tertiary Adrenal insufficiency):

This is due to external factors which lead to under activity of the glands.

  • ACTH level is low.
  • Cortisol level is decreased.

Adult adrenogenital syndrome:

  • There is an elevated level of:
    1. Urine 17-ketosteroids
    2. DHEA
    3. ACTH
  • There is a decreased level of:
    1. Cortisol

Congenital Adrenal Hyperplasia:

  • Decreased level of:
    1. Cortisol
    2. Aldosterone
  • There is an elevated level of:
    1. ACTH
    2. Androgens level

Primary Hyperaldosteronism (Conn's syndrome):

  • There is a decreased level of:
    1. Renin
    2. Potassium
    3. No response to aldosterone suppression test
  • There is an elevated level of:
    1. Serum sodium
    2. Aldosterone urine and blood

Cushing's syndrome:

  • There is an elevated level of Glucose, Sodium, and Cortisol.
  • There is no diurnal variation of ACTH and it is absent.
  • There is a decreased Potassium level.

Collection of sample

For the estimation of Adrenal Gland Hormones, patient's serum is needed. The 24-hour urine sample is also collected along with blood sample.

For the estimation of Cortisol level, patient's serum is required. 24-hour urine sample is also collected in a plastic container containing boric acid. The serum is stable for 2 days if kept at 2° to 8° C.

For catecholamines (Epinephrine and Norepinephrine), plasma in EDTA or heparin is needed. The sample is transported along with an ice-pack, centrifuged immediately at 4° C, and plasma is separated. Plasma is freeze until the test is run. 24-hour urine sample may also be collected in a plastic container containing 6 ml of 20% HCl and refrigerate during collection.

Estrogen can be estimated in serum. The blood sample is collected in a plain test tube, centrifuged immediately, serum is separated and refrigerated until the test is performed. 24-hour urine sample is also collected in a plastic container containing boric acid.

Aldosterone test can be done from the plasma (EDTA, citrate or heparin). Patient's blood serum may also be used for the test. Patient must be upright for 2 hours before the sample is taken. 24-hour urine sample is also collected in a plastic container containing boric acid.

Precautions before performing the Adrenal gland tests

  • Fasting blood sample is required.
  • Avoid herbal medicines and any medication which interfere with the test.
  • Avoid nuclear scan before this test.
  • Avoid exercise or physical activity.
  • Reduce the stress before performing this test.
  • Aldosterone AM sample is higher than PM.
  • Cortisol highest level is 8 AM and >50% less at 8 PM.

Normal Values

  • Plasma Epinephrine <50 pg/mL
  • Norepinephrine 110 to 410 pg/mL
  • Cortisol serum 5 to 20 µg/dL
  • Urine cortisol (free) 20 to 90 µg/ day
  • Aldosterone (recumbent) 50 to 150 ng/L
  • Aldosterone (upright) 150 to 300 ng/L


  • Male 20 to 80 pg/mL
  • Female Luteal phase 160 to 400 pg/mL
  • Follicular phase 60 to 200 pg/mL
  • Postmenopausal <130 pg/mL

Biological Oxygen Demand (BOD)

It is also known as Biochemical Oxygen Demand (BOD). It is defined as the amount of oxygen consumed during the process of degradation and eventual stabilization of unstable organic substances by the biochemical activities of aerobic and other microbes. This degradation of the chemical complex is a desirable process and the final product is called stabilized wastewater. The aerobic bacteria consume oxygen during the process of oxidization of the organic and other oxidizable inorganic substances. The immensity of biochemical degradation depends on the population of bacteria. An actively growing population of bacteria will consume more oxygen to quickly decompose unstable complexes. Biological/Biochemical Oxygen Demand (BOD) is reduced with the decrease in the quantity of these complexes in the wastewater. Therefore, it can be surmised that BOD is directly proportional to the level of degradable chemical complexes; high concentration of chemical substances will result in the high BOD.

The BOD is a very useful measure of the efficiency of methods of wastewater treatment. A method in which amount of BOD reduced quickly is considered as most effective and efficient method. Therefore, exactly stabilized effluent, when discharge in the body of water, does not cause reduction of oxygen in the water.

Wastewater Disposal Methods

It is a well-known fact that the wastewater should be treated properly and effectively before its disposal into receiving water bodies. Disposal of wastewater may be accomplished with or without treatment.

Disposal of Wastewater by Treatment Methods

There are different methods available for the removal of microorganisms and stabilized the putrescible organic and inorganic chemicals in the wastewater. These methods are known as wastewater treatment methods. It is a very interesting fact that usually microorganisms are used to reduce the large burden of wastewater, which is organic matter. With few exceptions, wastewater treatment plants are integrated with physical, chemical and microbiological methods to concern with the different problems related to wastewater.

According to distinct types of treatment, they are divided into four types. Each type of treatment process has a special purpose, targeting the removal of all sources of materials and reducing the burden of microorganisms from the wastewater.

Primary Treatment of Wastewater

This process is mainly designed to remove the total solids from the wastewater by sedimentation and render it adequately free from pathogenic bacteria by chlorination. Initially, large objects are removed by bar screens from the wastewater flow. It removes a significant amount of particulate matter. The collected objects are then put into the grinder and released back into the wastewater flow.

The wastewater is then allowed to flow to a series of large primary settling compartments in which most of the organic matters and dense inorganic particles such as grit and sands are removed. Usually, there are two types of settling compartment, (a) grit compartment and (b) sedimentation tank or quiescent settling compartment. In grit compartment, wastewater flows very slow which permits large and heavy particulate matter to settle out. In the next step, the municipal and industrial wastes (particulate organic matters) in wastewater are removed in the sedimentation tank. In sedimentation tank, wastewater is allowed to stay for 1 to 3 hours during which most of the suspended organic matter settles out. The sedimented material is in the form of a semi-solid mass called sludge. The efficiency of sludge formation can be increased by the addition of various chemicals to coagulate the suspended particles which enhanced the sedimentation rate. The sludge is not allowed to remain in the bottom of sedimentation tank for a long period because of anaerobic bacteria produce gases during metabolism that tend to resuspend the settled material and increased the odor. Therefore, the sedimentation tank is equipped with scrapper mechanisms that occasionally removes the bottom sludge to a collection hopper. The underflow sludge becomes a waste product of the process. The remaining liquid portion of the wastewater which leaves the tank is called effluent.


To be continue...

There are certain bacteria which cannot be stained by Gram's method. In 1882, Paul Ehrlich developed a method of staining such type of bacteria. This method was named, and still known as acid-fast staining and the bacteria were named as acid-fast bacteria. In the same year, Ehrlich's method was improved by Zehil and Neelsen. Nowadays, Ziehl-Neelsen method is believed as most important differential staining procedure used for the identification of acid-fast species of Mycobacterium, Actinomyces, and Nocardia. There are many acid-fast bacteria which are pathogenic, such as M. tuberculosis (tuberculosis), A. israelii (actinomycosis), M. leprae (leprosis), and N. asteroides (nocardiosis).

Acid-fast bacteria may be defined as those cells which keep the color of the primary dye (carbol fuchsin) even after the process of decolorization by the acid-alcohol solution. Those bacteria which fail to do so are known as non-acid-fast bacteria.

Acid-fast bacteria are coated with a thick waxy material, mycolic acid, which makes the bacterial cells highly resistant to the penetration of dyes. The penetration of dye is promoted by using heat as mordant. The heat invades the dye through the waxy coat and into the cytoplasm.


  • Differentiation between acid-fast and non-acid-fast bacteria.
  • Diagnosis of pulmonary tuberculosis from sputum smear. See also: Examination of Sputum



Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.


  1. Carbol fuchsin
  2. Acid-alcohol solution
  3. Methylene blue


  1. Bunsen burner
  2. Wire loop
  3. Glass Slide
  4. Spirit lamp
  5. Microscope


Acid-Fast staining by Ziehl-Neelsen method

  1. Take a clean glass slide and prepare a thin smear from the specimen using sterile technique. The smear should be extremely thin covering a large area of the slide.
  2. Dry the smear is air and then fix the slide by passing the slide through the flame.
  3. Cover the slide with carbol fuchsin. Keep it for 5 minutes over a spirit lamp with constant heating but not boiling. Do not allow the stain to dry over the slide.
  4. When the slide is cooled, wash it with tap water.
  5. Flood the slide with acid-alcohol and leave it for 3 minutes. Wash the slide with tap water and drain.
  6. Counterstain with methylene blue for 2 minutes.
  7. Wash the slide with tap water and keep it for dry.
  8. Observe the slide under oil immersion objective.


Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved bright red rods. Non-acid-fast cells appear blue.

Acid-Fast Staining by Mobin Method

In 1985, a Pakistani microbiologist, Abdul Mobin Khan developed a method for the staining of acid-fast bacteria. In this method, heating of flooded primary dye on smear is not required. However, initial fixing of the smear over the flame is necessary in order to increase the permeability of the cell wall and promote the newly formulated primary dye to penetrate the cell.



Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.


  1. Mobin stain
  2. 1% H2SO4
  3. Crystal violet


  1. Wire loop
  2. Bunsen burner
  3. Glass slides
  4. Microscope


  1. Using sterile technique, prepare a thin smear from the specimen covering a large area of the glass slide.
  2. Dry the smear in the air and then fix it by passing the slide 20 times through the flame.
  3. Place the smear on the staining rack and cover it with Mobin stain. Keep it for 10 minutes.
  4. Pour off the stain and wash the slide with tap water.
  5. Decolorize the smear by 1% H2SO4 solution till it is light pink.
  6. Wash the slide with tap water and keep it for dry.
  7. Observer the slide under oil immersion objective.


Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved red rods while non-acid-fast bacteria as blue.

In 1883 (originally published in 1884), Dr. Hans Christian Gram (1853-1938) developed a technique for the classification of bacteria into two broad groups, Gram-positive and Gram-negative. It is the most important staining technique for the classification and differentiation of bacteria.

The Gram stain consist of four reagents; crystal violet (use as a primary dye), Gram's iodine (use as a mordant), ethyl alcohol (use as a decolorizer), and safranin (use as a counterstain). The Gram-negative, on the other hand, lose the primary dye (crystal violet) when decolorized and, thus, take the color of counterstain (safranin).

The Gram-reaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 11-22% in Gram-negative cell wall and 1-4% in the Gram-positive cell wall. In the Gram-negative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell wall. The dehydrating result of alcohol cannot fill these pores which cause the liberate of primary stain making the cell colorless. Such cells take the color or counterstain (safranin). On the contrary, the amount of lipid is very low in the Gram-positive cell wall and easily dissolved by in ethyl alcohol, causing the formation of very small pores. These pores are further closed by the dehydrating effect of alcohol which does not permit the primary dye (crystal violet) to leave the cell.


Identification, differentiation, and classification of the bacteria.



Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria or fungi grown and isolated in culture.


  1. Crystal violet
  2. Gram iodine
  3. Ethyl alcohol (95%)
  4. Safranin
  5. Ceder wood oil


  1. Bunsen burner
  2. Wire loop
  3. Glass slides
  4. Microscope


  1. Prepare a smear of the specimen, dry in air and then fix it in low flame.
  2. Flood the smear with crystal violet, and keep it for 1 minute. Wash the smear with running tap water.
  3. Pour Gram's iodine on smear and after 1 minute, wash it with tap water.
  4. Pour alcohol on the smear until the purple color no longer comes from the smear.
  5. Pour safranin on the smear and after 45 seconds, wash it with tap water and keep the smear dry in air.
  6. Observe the stained smear under oil immersion lens and note down the arrangement, shape, and Gram-reaction of the cells.


The Gram-positive bacteria appear in purple color and Gram-negative bacteria appear in pink color.

Monday, 21 May 2018 11:12


Sputum examination refers to the laboratory examination or test of the material or substance coughed out from the lungs, bronchi, trachea, and larynx. Normally, sputum is mainly composed of mucus and also certain cellular and non-cellular components of host origin. During expectoration, sputum gets contaminated with normal bacterial flora and cells from pharynx and mouth.

Friday, 18 May 2018 12:33


Waste products discharged from the digestive tract are composed of up to 75% water, food which is digested but not absorbed, indigestible residue, undigested food, epithelial cells, bile, bacteria, secretion from the digestive tract and inorganic bacteria. Normally an adult human excretes 100-200 grams of feces in a day.

Examination of stool is very helpful in the diagnosis of disease of the gastrointestinal tract as listed below.

Detection of parasites

Stool examination is performed for the detection and identification of worms (adult worms, larvae, segments of worms, ova) and protozoa (cyst or trophozoites). See also: Microscopic Examination of Feces

Bacteriologic examination

Stool culture is performed for the evaluation of bacterial infection such as Clostridium difficile, Yersinia, Salmonella, Shigella or Vibrio. Bacterial toxins (such as those released by Clostridium difficile or Clostridium botulinum) can also be identified. See also: Microscopic Examination of Feces

Evaluation of chronic diarrhea

Chronic diarrhea defined as a passage of three or more liquid or loose stools in a day lasting for more than four weeks. Acute diarrhea refers to the passing of three or more liquid or loose stools in a day for less than four weeks. In diarrhea, stool examination is very important part of laboratory investigations. Depending on the nature of the investigation, either a random stool sample or 72- sample or 48-hour sample is collected. A random stool sample is used for the tests of occult blood, pH, fat, white blood cells, microscopy, or culture. A 72- or 48-hour sample is collected and examined for the weight, carbohydrate, fat content, osmolality, or chymotrypsin activity. Causes of chronic and acute diarrhea are listed in Table 988.1 and Figure 988.1 respectively.

Table 988.1 Classification and causes of chronic diarrhea
1. Watery diarrhea
  1. Osmotic
    • Carbohydrate malabsorption
    • Osmotic laxatives
  2. Secretory
    • Bacterial toxins
    • Bile acid malabsorption
    • Laxative abuse
    • Hormonal disorders: VIPoma, carcinoid syndrome, gastrinoma, hyperthyroidism
2. Inflammatory diarrhea
  1. Invasive bacterial and parasitic infections
  2. Inflammatory bowel disease
  3. Pseudomembranous colitis
  4. Infectious diseases
  5. Neoplasia
3. Fatty diarrhea
  • Malabsorption syndromes


Figure 988.1 Classification of causes of acute diarrheaFigure 988.1 Classification and causes of acute diarrhea


Evaluation of dysentery

Differentiate between bacillary dysentery and amebic dysentery is done by the identification of the causative organism in the stool. See also: Microscopic Examination of Feces

Identification of Rotavirus

In infants and young children, Rotavirus is the most common cause of diarrhea. Rotavirus can be identified by the electron microscopic examination of stool. Other techniques, such as latex agglutination, immunofluorescence, or enzyme-linked immunosorbent assay (ELISA) are also used for the detection of Rotavirus in stool.

Chemical examination

Chemical tests can be applied on feces to detect excess fat excretion (malabsorption syndrome), occult blood (in ulcerated lesions of the gastrointestinal tract, especially occult carcinoma of the colon) and presence or absence of urobilinogen (obstructive jaundice). See also: Chemical Examination of Feces

Differentiating infection by invasive bacteria (like Salmonella or Shigella) from that due to toxin-producing bacteria (like Vibrio cholerae or Escherichia coli)

Feces is examined for the presence of white blood cells. Increased numbers of polymorphonuclear neutrophils (identified by methylene blue stain from the presence of granules in their cytoplasm) are seen as shown in Figure 988.2. See also: Causes, symptoms, diagnosis, and treatment of Cholera

Figure 988.2 Preliminary evaluation of acute diarrhea. Examination of feces for white blood cells is helpful in narrowing differential diagnosis in intestinal infections in acute diarrhea
Figure 988.2 Preliminary evaluation of acute diarrhea. Examination of feces for white blood cells is helpful in narrowing differential diagnosis in intestinal infections in acute diarrhea

Leukemia is a malignant clonal hematopoietic stem cell disorders characterized by the rapid increase in the number of blast cells in the bone marrow and rapidly progressive fatal course if untreated. Acute leukemia (AL) are primary disorders of the bone marrow, also known as blood cancer.

Why are these tests performed?

  1. These tests are performed for the diagnosis of bleeding disorders.
  2. APTT is performed to distinguished the functionality of the clotting factors I, II, V, VII, IX, X, XI, and XII.
  3. APTT is used to check the treatment of the patient taking heparin or other medicine for blood thinning.

See: Role of laboratory tests is bleeding disorders

Collection of Sample

Venous blood sample is collected from antecubital fossa in a test tube containing trisodium citrate (3.2%), with the anticoagulant to blood proportion being 1:9. See also: Prothrombin time (PT): Collection of Specimen.


  1. Sample handling is very sensitive, false and raised values are obtained if the ratio of blood and anticoagulant is not correct.
  2. Plasma is stable for one hour if kept at 4º C.
  3. Plasma can be preserved for 28 days if frozen.


Activated Partial Thromboplastin Time (APTT)

Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured.

Partial Thromboplastin Time (PTT)

It is one stage test. It distinguishes the functionality of the clotting factors I, II, V, VIII, X, XI, and XII. Both Activated Partial Thromboplastin Time (APTT) and Partial Thromboplastin Time (PTT) have the same clinical significance but Activated Partial Thromboplastin Time (APTT) is more reliable as compare to Partial Thromboplastin Time (PTT) due to its sensitivity.

Prothrombin Time (PT)

Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways.

International Sensitivity Index (ISI) and International Normalized Ratio (INR)

International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR).

International Normalized Ratio (INR) is calculated by the following formula.

INR = PT of Patient ISI
          PT of Control

Purpose of INR: The INR is calculated to evaluate the following conditions.

  1. Atrial fibrillation
  2. Thrombophilia
  3. Cardiomyopathy
  4. Prosthesis (Replacement of heart valve)
  5. Venous thromboembolism
  6. Antiphospholipid syndrome

Normal Values

Technique of APTT, PTT, and PT is different in different laboratories therefore normal values varies with the lab to lab. A normal control is always run with the patient's sample. In general, normal values are below.

  • APTT: 30-40 seconds
  • PTT: 60-70 seconds
  • PT: 11-16 seconds
  • INR: 1-1.5
Table 882.1 Required value of INR in various diseases
Disease Required INR Value
Deep vein thrombosis prophylaxis 1.5 to 2.0
Deep vein thrombosis 2.0 to 3.0
Atrial fibrillation 2.0 to 3.0
Orthopedic surgery 2.0 to 3.0
Pulmonary embolism 2.5 to 3.5
Prosthetic valve prophylaxis 3.0 to 4.0

Critical Values

  • APTT: > 70 seconds (Usually it is considered as panic value. If APTT is greater then 100 seconds, spontaneous bleeding may occur.)
  • INR: > 5.0 (In Deep Vein Thrombosis (DVT) patient on warfarin treatment, an expected value of INR is between 2.0 to 3.0.)

Reasons for the high results

  1. Disseminated intravascular coagulopathy (DIC )
  2. Factor XII deficiency
  3. Cirrhosis
  4. Hemophilia A and B
  5. Von Willebrand’s disease
  6. Hypofibrinogenemia
  7. Vitamin K deficiency
  8. Malabsorption
  9. Leukemia
  10. Fibrin breakdown products
  11. All congenital deficiencies of Intrinsic system coagulation factors
  12. Drugs

The significance of APTT, PTT, PT, and INR test for the layman

  • Patients, taking medication for blood thinning or on heparin treatment, are advised for these laboratory investigations.
Saturday, 28 April 2018 14:06

Adrenocorticotropic Hormone (ACTH)

Why is this test performed?

  1. This hormone test is evaluated in different conditions, such as Adrenal insufficiency, in Acromegaly and Cushing Syndrome.
  2. In Addison's disease, the level of Adrenocorticotropic hormone (ACTH) is noted more than 1000 pg/ml.
  3. In Adrenal carcinoma, Adenoma, and Adrenocortical insufficiency, the level of Adrenocorticotropic hormone (ACTH) decreases.

Collection of Sample

For the estimation of Adrenocorticotropic hormone (ACTH), patient’s plasma is needed. Blood is collected in a chilled plastic test tube containing EDTA or heparin and blood is placed in cold ice-water.

The sample is centrifuged at 4º C, plasma is separated and stored at -20º C immediately within 15 minutes of the blood collection.

See: Uses of anticoagulants for hematological investigations

Note: For the diagnosis of Cushing Syndrome, the blood sample is collected in between 6 PM to 11 PM.

See: Procedures for the collection of blood for hematological investigations


  1. Collect the blood sample in a chilled plastic test tube containing EDTA or heparin.
  2. Avoid high carbohydrates diet, take the low-carb diet.
  3. Avoid physical activity for 12 hours before the collection of the blood sample.
  4. Stop medication such as corticosteroids, 48 hours before the collection of blood sample.
  5. An anxious collection of the blood sample may increase the level of Adrenocorticotropic hormone (ACTH).

Normal Values

  • 6 to 8 AM: < 80 pg/ml or < 18 pmol/L (SI units)
    6 to 11 MP: < 50 pg/ml or < 11 pmol/L (SI units)
    or less than 120 pg/ml
  • According to another references:
    8 AM:  < 120 pg/mL
    4 to 8 PM: < 85 pg/mL
    Cord blood: 50 to 570 pg/mL
    Newborn: 10 to 185 pg/mL
Table 881.1 ACTH and Cortisol values in various conditions and  diseases.
Disease ACTH Value Cortisol Value
Cushing syndrome Increased/low Increased
Adrenal cancer Low Raised
Adrenal adenoma Low Raised
Ectopic ACTH (Lung cancer) Raised Raised
ACTH- producing Pituitary tumor Raised Raised
Adrenal gland failure ( Infarction, Haemorrhage) Raised Low
Hypopituitarism Low Low
Congenital adrenal hyperplasia Raised Low
Addison's disease

Reasons for the increased level of ACTH

  1. Cushing syndrome
  2. Addison's disease
  3. Stress
  4. Ectopic ACTH syndrome

Reasons for the decreased level of ACTH

  1. Secondary adrenal insufficiency
  2. Exogenous steroid administration
  3. Hypopituitarism
  4. Adrenal adenoma or carcinoma

The significance of Adrenocorticotropic hormone (ACTH) test for the layman

  1. This test is advised in abnormal metabolism of lipids.
  2. This test is advised to the patients of Diabetes Mellitus (DM).
  3. This test is performed for the diagnosis of Cushing syndrome.
  4. This test is advised if there are truncal obesity and thin extremity.
truncal obesity
Figure 881.1 Example of Truncal Obesity
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