Sample Collection

In Biochemistry, venepuncture, venopuncture or venipuncture is the process of obtaining intravenous access for
the purpose of intravenous therapy or obtaining a sample of venous blood

The equipment consisting of a plastic hub, a hypodermic needle, and a vacuum tube. Under certain
circumstances, a syringe may be used, often with a butterfly needle, which is a plastic catheter attached to a
short needle. In the developing world, a needle and syringe are still the most common method of drawing blood.

Blood is most commonly obtained from the median cubital vein, which lies within the cubital fossa anterior to
the elbow. This vein lies close to the surface of the skin, and there is not a large nerve supply

The tubes in which blood is transported back to the laboratory contain a variety of additives or none at all. It is
important to know which tube the individual laboratory requires for which test as reagents vary between
laboratories and may be affected by different additives. In general whole blood needs to be mixed with EDTA
which chelates calcium to prevent it clotting, unless the clotting time is the test to be measured in which citrates
are used. The majority of biochemistry tests are performed on serum and so either a plain tube or a clotting
accelerator is used. This clotting accelerator can interfere with some assays and so a plain tube is recommended
in these cases but will obviously delay the result. Some assays may also require whole blood but are interfered
with by EDTA and in this case Lithium Heparin is an alternative.

With the vacuum tube system, the needle pierces the top of the sample tube and will potentially come into
contact with the additives in the tube. As it is a hollow needle some of this can be carried into the next tube and
contaminate it. The most likely additive to cause trouble is EDTA which will affect the coagulation time assays
and by chelating some of the metal ions may interfere with some of the biochemistry results (especially
potassium). Thus EDTA samples should be drawn last in most cases and plain tubes drawn first.

Steps for sample collection

(1) Use surgical gloves for sample collection

(2) Apply tourniquet patient should close hand
(3) Select vein puncture site
(4) Clean site
(5) Inspect needle
(6) Anchor pull skin taut with thumb grasp arm with 4 fingers
(7) Align needle(bevel up) with vein and insert at 15’ angle
(8) Grasp holder tightly with opposite hand anchor fingers against arm
(9) Push tube onto needle fill
(10) Release tourniquet; patient should open hand
(11) Gently remove needle
(12) Apply pressure

Complications

Immediate: Haematoma and sycope

Hematoma can be avoided by applying adequate gentle pressure at site

 Syncope a physician should be brought to take care

Late-local complications: Thrombosis of the vein rarely thrombophlebitis may occur

In Biochemistry, venepuncture, venopuncture or venipuncture is the process of obtaining a sample of venous

blood. Blood is most commonly obtained from the median cubital vein, which lies within the cubital fossa
anterior to the elbow. This vein lies close to the surface of the skin, and there is not a large nerve supply

The equipment consisting of a plastic hub, a hypodermic needle, and a vacuum tube. Under certain
circumstances, a syringe may be used, often with a butterfly needle, which is a plastic catheter attached to a
short needle. In the Pakistan, a needle and syringe are still the most common method of drawing blood.

The tubes in which blood is transported back to the laboratory contain a variety of additives or none at all. It is
important to know which tube the individual laboratory requires for which test as reagents vary between
laboratories and may be affected by different additives. In general whole blood needs to be mixed with EDTA
which chelates calcium to prevent it clotting, unless the clotting time is the test to be measured in which citrates
are used. The majority of biochemistry tests are performed on serum and so either a plain tube or a clotting
accelerator is used. This clotting accelerator can interfere with some assays and so a plain tube is recommended
in these cases but will obviously delay the result. Some assays may also require whole blood but are interfered
with by EDTA and in this case Lithium Heparin is an alternative.

With the vacuum tube system, the needle pierces the top of the sample tube and will potentially come into
contact with the additives in the tube. As it is a hollow needle some of this can be carried into the next tube and
contaminate it. The most likely additive to cause trouble is EDTA which will affect the coagulation time assays
and by chelating some of the metal ions may interfere with some of the biochemistry results(especially
potassium). Thus EDTA samples should be drawn last in most cases and plain tubes drawn first.
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 GLUCOSE LEVEL INDICATION

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From 100 to 125 mg/dL (5.6 to 6.9 mmol/L) Hyperglycemia

Border line

126 mg/dL (7.0 mmol/L) and above Diabetes

Fasting Blood Glucose

GLUCOSE LEVEL INDICATION

From 70 to 99 mg/dL (3.9 to 5.5 mmol/L) Normal fasting glucose

From 100 to 125 mg/dL (5.6 to 6.9 mmol/L) Impaired fasting glucose (pre-diabetes)

126 mg/dL (7.0 mmol/L) and above on more than one testing occasion Diabetes

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 Bilirubin

Bilirubin (formerly referred to as hematoidin) is the yellow breakdown product of normal heme catabolism.
Heme is found in hemoglobin, a principal component of red blood cells. Bilirubin is excreted in bile and urine,
and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, urine (via its
reduced breakdown product, urobilin), and the yellow discoloration in jaundice

Chemistry

Bilirubin consists of an open chain of four pyrrole-like rings (tetrapyrrole). In heme, by contrast, these four rings
are connected into a larger ring, called a porphyrin ring.

Bilirubin is very similar to the pigment phycobilin used by certain algae to capture light energy, and to the
pigment phytochrome used by plants to sense light. All of these contain an open chain of four pyrrolic rings.

Like these other pigments, some of the double-bonds in bilirubin isomerize when exposed to light. This is used
in the phototherapy of jaundiced newborns: the E,E-isomer of bilirubin formed upon light exposure is more
soluble than the unilluminated Z,Z-isomer.

Some textbooks and research articles show the incorrect geometric isomer of bilirubin.[2] The naturally
occurring isomer is the Z,Z-isomer.

Function

Bilirubin is created by the activity of biliverdin reductase on biliverdin, a green tetrapyrrolic bile pigment which is
also a product of heme catabolism. Bilirubin, when oxidized, reverts to become biliverdin once again. This
cycle, in addition to the demonstration of the potent antioxidant activity of bilirubin, has led to the hypothesis
that bilirubin's main physiologic role is as a cellular antioxidant

Metabolism

Unconjugated (indirect)

Erythrocytes (red blood cells) generated in the bone marrow are disposed of in the spleen when they get old or
damaged. This releases hemoglobin, which is broken down to heme as the globin parts are turned into amino
acids. The heme is then turned into unconjugated bilirubin in the reticuloendothelial cells of the spleen. This
unconjugated bilirubin is not soluble in water. It is then bound to albumin and sent to the liver.

Conjugated (direct)

In the liver it is conjugated with glucuronic acid by the enzyme glucuronyltransferase, making it soluble in
water. Much of it goes into the bile and thus out into the small intestine. Some of the conjugated bilirubin
remains in the large intestine and is metabolised by colonic bacteria to urobilinogen, which is further
metabolized to stercobilinogen, and finally oxidised to stercobilin. This stercobilin gives feces its brown color.
Some of the urobilinogen is reabsorbed and excreted in the urine along with an oxidized form, urobilin.

Principle

Direct bilirubin is determined by the reaction with drazotied sulphanhlic acid.

Total bilirubin is determined in the presence of caffeine by the reaction with diazotized sulphanilic acid
Clinical Significance

Elevated levels of bilirubin are usually associated with hepcitocellular damage or biliary tract obstructions.

Sample

Serum: Heparinised plasma or EDTA plasma

Serum should be protected from direct light at all times.

Procedure

Total Bilirubin

(13) Take Two Test tubes mark them sample blank & sample
(14) Put 200 ul sulphanilic Acid in both test tubes
(15) Put 50ul Nitrite in Test tube Sample
(16) Put 1000ul Caffeine in both test tubes
(17) Put 200ul serum in both test tubes

(18) Mix well and allow to stand for 10 minutes at room

(19) Add 1000ul Tartrate in both test tubes
(20) Measure the absorbance of the Sample against the sample blank at 578nm wavelength

Calculation

Total Bilirubin Conc = 10.8 X Absorbance (mg/dl)

185 X Absorbance (umol/l)

Subtract absorbance of sample blank from absorbance of sample for Total Bilirubin

Direct Bilirubin

1. Take Two Test tubes mark them sample blank & sample
2. Put 200 ul sulphanilic Acid in both test tubes
3. Put 50ul Nitrite in Test tube Sample
4. Put 2ml 0.9% NaCl in both test tubes
5. Put 200ul serum in both test tubes
6. Mix well and allow to stand for 5 minutes at room
7. Measure the absorbance of the Sample against the sample blank at 546nm wavelength

Calculation

Direct Bilirubin Conc = 246 X Absorbance (mg/dl)

14.4 X Absorbance (umol/l)

Subtract absorbance of sample blank from absorbance of sample for Direct Bilirubin

Indirect Bilirubin Con

Subtract Direct Bilirubin from Total Bilirubin for Indirect Bilirubin

Increased Concentration

Total Bilirubin:

Total bilirubin is increased mildly in chronic hemolytic disease (below 5mg/dl) moderately to
severely (10 to 30mg/dl) in hepatocellular disease, and markedly in cholestasis(internal or external
obstruction to bile flow,where the concentration could vary from 10 to 60 mg/dl or 170 to 1030 umol/L)

Esterified bilirubin.

This fraction is increased in both hepatocellular disease and cholestasis.

Nonesterified bilirubin. This increased in bilirubin concentration in hemolytic disease,including HDN,is

almost entirely in this fraction. it also accounts for about 30 to 50% of the bilirubin rise in hepatocellular
disease or cholestasis.

Decreased concentration.

Of no clinical significance.

Question

(1) How is bilirubin formed?

(2) What is vandenbergh’s reaction?
(3) What is reflected by hyperbilirubinaemia?
(4) Define jaundice
(5) What is urobilinogen? What is stercobilinogen?
(6) Under What clinical condition, the test is performed?
 Serum Amylase

Amylase is an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins
the chemical process of digestion. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch
into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with
energy. Plants and some bacteria also produce amylase. As diastase, amylase was the first enzyme to be
discovered and isolated (by Anselme Payen in 1833). Specific amylase proteins are designated by different
Greek letters. All amylases are glycoside hydrolases and act on -1,4-glycosidic bonds.

-Amylase

Alternate names: 1,4- -D-glucan glucanohydrolase; glycogenase. The -amylases are calcium metalloenzymes,
completely unable to function in the absence of calcium. In animals, it is a major digestive enzyme and its
optimum pH is 6.7-7.0

Also found in plants (adequately), fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus)

-Amylase

Alternate names: 1,4- -D-glucan maltohydrolase; glycogenase; saccharogen amylase During the ripening of
fruit, -amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit.

-Amylase

Alternative names: Glucan 1,4- -glucosidase; amyloglucosidase; Exo-1,4- -glucosidase; glucoamylase;

lysosomal -glucosidase; 1,4- -D-glucan glucohydrolase, -amylase is most efficient in acidic environments
and has an optimum pH of 3.

This test measures the amount of amylase in the blood or urine or sometimes peritoneal fluid. Amylase is one of
several enzymes produced by the pancreas to help digest carbohydrates. It is secreted through the pancreatic
duct into the duodenum, where it helps break down dietary carbohydrates. Amylase is also produced by other
organs, particularly the salivary glands.
Amylase is usually present in the blood and urine in small quantities. When cells in the pancreas are injured, as
in pancreatitis, or the pancreatic duct is blocked by a gallstone or rarely by a pancreatic tumor, increased
amounts of amylase find their way into the bloodstream, increasing concentrations in the blood and the urine,
which is the excretion path for amylase from the blood.

Test Principle

A buffered starch solution is incubated with serum for 10 min at 37 C in a 2-point assay. The initial starch
concentration and that present at the end of 10 min incubation are measured by the addition of molecular iodine
which forms a deep blue color with linear starch chains the blue color thereassay.Amylase is activated by Cl
which contained in the substrate solution. The enzyme action is stopped by addition of iodine-EDTA solution
because EDTA chelates Ca2+ an ion required for amylase activity.
Procedure

1. Accurately pipet 1.0 ml of starch buffer solution in each of two test tubes. One for specimen and other
for specimen blank.Treat control sera in same manner as specimens. One additional tube is prepared for
reagent blank.
2. Place all test tubes in a 37 C water bath for 5 min to come up to temperature
3. At exactly 15 or 30 second intervals add 25µL of appropriate serum to specimen tubes Incubate for 10
min
4. At the end exactly 10 min of incubation,stop the reaction by adding 15mL iodine EDTA solution to each
tube in the same 15 or 30 sec sequence
5. Add 15 mL of iodine-EDTA sol to each of the specimen blank tubes and the reagent blank
6. After 3min, read and record the absorbance of each tube against water at 600nm

Calculation

Amylase activity in U/mL = AB - AU X 6.2 X 1

AB 10 0.025

AB - AU X 24.8

AB

AB = Absorbance of specimen blank

AU = Absorbance of unknown

6.2 is µmol starch per mL

10 is incubation time in min

0.025 is serum volume in ml

Reference values

The normal concentration of serum amylase is 0.8 to 3 U/mL

Increase activity

Serum amylase activity is raised considerably in

(1) acute pancreatitis,

(2) obstruction of pancreatic ducts, The rise in serum amylase activity after obstruction of the pancreatic
ducts whether by stone, inflammation, or compression of common bile ducts, by a cancer of the head of
the pancreas
(3) Obstruction of the parotid (salivary) gland.,
(4) Serum amylase is rapidly cleared by the kidney, so measurement of urinary amylase is valuable adjunct
to the serum test. Some types of renal damage may be accompanied by mildly elevated level of serum
amylase because of impaired execration.

Decreased Activity A decreased concentration of serum amylase may be found in acute or chronic
hepatocellular damage but this is not sensitive liver function test.
Questions

1. Do elevated amylase levels always mean that I have a pancreatic condition?

No. Amylase levels may also be significantly increased in people with gallbladder attacks. Urine and blood
amylase levels may be moderately elevated with a variety of other conditions, such as ovarian cancer, lung
cancer, tubal pregnancy, acute appendicitis, diabetic ketoacidosis, mumps, intestinal obstruction, or perforated
ulcer, but amylase tests are not generally used to diagnose or monitor these disorders.

2. Can medications affect the amylase level?

Yes. Some drugs that may cause amylase to rise include aspirin, diuretics, oral contraceptives, corticosteroids,
indomethacin, ethyl alcohol, and opiates (such as codeine and morphine).

3. What is the difference between P-amylase and S-amylase?

Amylase is an enzyme that has several different forms called isoenzymes. Different tissues make different
forms. P-amylase refers to the type of amylase made mainly in the pancreas. S-amylase refers to the type of
amylase made mainly by the salivary glands. P-amylase in the blood increases when the pancreas is inflamed or
damaged. S-amylase in the blood increases when the salivary gland is inflamed or damaged. Measuring
pancreatic amylase, or P-amylase, may be useful in determining if an increase in a total amylase level is due to
acute pancreatitis

4. How is it used?
The blood amylase test is ordered, often along with a lipase test, to help diagnose and monitor acute or chronic
pancreatitis and other disorders that may involve the pancreas. A urine amylase test may also be ordered.
Typically, its level will mirror blood amylase concentrations, but both the rise and fall will occur later.
Sometimes a urine creatinine clearance may be ordered along with the urine amylase to help evaluate kidney
function since decreased kidney function can result in a slower rate of amylase clearance. In certain cases, an
amylase test may be performed on peritoneal fluid to help make a diagnosis of pancreatitis.
Amylase tests are sometimes used to monitor treatment of cancers involving the pancreas and after the removal
of gallstones that have caused gallbladder attacks

5. When is it ordered?
A blood amylase test may be ordered when a person has symptoms of a pancreatic disorder, such as:

• severe abdominal or back pain

• fever
• loss of appetite
• nausea
A urine amylase test may be ordered along with or following a blood amylase test. One or both may also be
ordered when a doctor wants to monitor a person to evaluate the effectiveness of treatment and to determine
whether amylase levels are increasing or decreasing over time

6. What does the test result mean?

In acute pancreatitis, amylase in the blood often increases to 4 to 6 times higher than the highest reference
value, sometimes called upper limit of normal. The increase occurs within 12 to 72 hours of injury to the
pancreas and generally remains elevated until the cause is successfully treated. Then the amylase values will
return to normal in a few days. In chronic pancreatitis, amylase levels initially will be moderately elevated but
often decrease over time with progressive pancreas damage.
 Aspartate transaminase (AST)

Aspartate transaminase (AST) also called serum glutamic oxaloacetic transaminase (SGOT) is similar to
alanine transaminase (ALT) in that it is another enzyme associated with liver parenchymal cells. The
difference being; ALT is found predominately in the liver, with clinically negligible quantities found in the
kidneys, heart, and skeletal muscle. AST is found in the liver, heart, skeletal muscle, kidneys, brain and
red blood cells. As a result ALT is a more specific indicator of liver inflammation than the AST, %

It facilitates the conversion of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate, and vice-
versa.

Oxaloacetate + glutamate aspartate + -ketoglutarate

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Two isoenzymes are present in humans. They have high similarity.

• GOT1, the cytosolic isoenzyme derives mainly from red blood cells and heart.
• GOT2, the mitochondrial isoenzyme is predominantly present in liver.
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It is raised in acute liver damage. It is also present in red blood cells and cardiac muscle, skeletal muscle,
and kidney and brain tissue, and may be elevated due to damage to those sources as well. as AST may
also be elevated in diseases affecting other organs, such as the heart or muscles in myocardial infarction,
also in acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, musculoskeletal
diseases, and trauma.

AST was defined as a biochemical marker for the diagnosis of acute myocardial infarction in 1954.
However the use of AST for such a diagnosis is now redundant and has been superseded by the cardiac
troponins.

AST (SGOT) is commonly measured clinically as a part of diagnostic liver function tests, to determine liver
health
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Female 6 - 34 IU/L
Male 8 - 40 IU/L
Specimen
Collect serum / plasma ( heparin or EDTA)
Test Principle
2-Oxoglutarate + L-Aspartate ----- Glutamate + Oxaloacetate
Oxaloacetate = NADH + H+ ----- Malate = NAD +
Procedure
Dissolve one volume of R2 with 5 volume of R1
Wait at least 5 min before use
This solution is stable 35days at 2-8 C
(1) Take two test tubes mark them test and blank
(2) Add 1ml of reagent in both test tubes
(3) Add 100µl serum in test tube test
 (4) Mix gently for 1 min
(5) Note the absorbance at 340 nm
Calculation
A/min X 1746

1. What conditions other than liver problems can cause increased AST?
Conditions that affect other organs, such as the heart and skeletal muscle, can cause elevations of AST.
Mild to moderate increases may be seen with vigorous exercise and muscle injury or in conditions such as
acute pancreatitis and heart attacks.
2. What other tests may be used to help determine the cause of liver damage?
After a thorough physical exam and evaluation of a person'
s medical history, there are several other tests
that may be performed as follow up depending on what is suspected to be the cause of liver damage.
Some of these include:

• Tests for hepatitis A, B, and C

• Testing for exposure to drugs and other substances toxic to the liver (Drugs of Abuse Testing and
Emergency of Overdose Drug Testing)
• Ethanol level
• Copper and ceruloplasmin for Wilson disease
• Iron tests and genetic tests for hereditary hemochromatosis
• A liver biopsy
3. When is it ordered?
An AST test is ordered along with several other tests to evaluate a person who has signs and symptoms
of a liver disorder. Some of these symptoms include:

• Weakness, fatigue
• Loss of appetite
• Nausea, vomiting
• Abdominal swelling and/or pain
• Jaundice
• Dark urine, light colored stool
• Itching (pruritus)

AST may also be ordered, either by itself or with other tests, for people who are at an increased risk for
liver disease. Some examples include:

• Persons who might have been exposed to hepatitis viruses

 • Those who are heavy drinkers
• Persons who have a history of liver disease in their family
• Persons taking drugs that can occasionally damage the liver
• Persons who are overweight and/or have diabetes

Persons who have mild symptoms, such as fatigue, may be tested for ALT to make sure they do not have
chronic liver disease.

When AST is used to monitor treatment of persons with liver disease, it may be ordered on a regular basis
during the course of treatment to determine whether the therapy is effective.
 Alkaline phosphatase

Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme responsible for removing phosphate groups from
many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the
phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective
in an alkaline environment. It is sometimes used synonymously as basic phosphatase.

Alkaline phosphatase is present in all tissues throughout the entire body, but is particularly
concentrated in liver, bile duct, kidney, bone, and the placenta. Humans and most other mammals
contain the following alkaline phosphatase isozymes:

• ALPI – intestinal
• ALPL – tissue non-specific (liver/bone/kidney)
• ALPP – placental (Regan isozyme)

Test Principle

Alkaline phosphatase reacts with p-nitrophenyl phosphate to form p-nitrophenol, the rate of formation of which
is directly proportional to the levels of alkaline phosphatase

Procedure

1. Take two test tube mark them sample and blank

2. put 1000µl reagent in both test tubes
3. put 20µl serum in test tube sample
4. take the reading after 1 min at 405nm

Calculation

ALP conc = Abs X 2760 u/l

Reference values:

Adults 38 to 126U/L

Increase activity

Serum ALP activity is raised in all bone disorders accompanied by increased ostoblastic activy.this includes
pagment’s disease (osreris deformans) osteoblastic tumors with metastases,hyperparathyroidism when there
is mobilization of Ca,rickets,and osteomalacia .serum ALP activity is increased in liver disease, particularly in
disorder of the hepatic biliary treeand during the third trimester of pregnancy owing to the elaboration of a
placental isoenzyme of ALP that is absorbed into the maternal bloodstream.

Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP can help
in the diagnosis of certain conditions.

Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PM),
and the leukemoid reaction

Decreased activity
 Low levels of ALP are found in a rare congenital defect,hypophosphatasemia,in dwarfs because of depressed
osteoblastic activity,in hypothyroidism,and in prenicious anemia.in the two latter conditions,deficiencies of
thyroid hormone and vitamin B12 respectively for the lowered serum ALP activity

The following conditions or diseases may lead to reduced levels of alkaline phosphatase:

• Hypophosphatasia, an autosomal recessive disease

• Postmenopausal women receiving estrogen therapy because of osteoporosis
• Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism, or severe anemia
• Children with achondroplasia and cretinism
• Children after a severe episode of enteritis
• Aplastic anemia
• Chronic myelogenous leukemia
• Wilson's disease

In addition, the following drugs have been demonstrated to reduce alkaline phosphatase:

• Oral contraceptives

Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP can help
in the diagnosis of certain conditions.

Lower levels are found in chronic myelogenous leukemia (CML) , paroxysmal nocturnal hemoglobinuria
(PNH)and acute myelogenous leukaemia

1. What other tests are used to evaluate liver disorders?

Other commonly used liver tests include more enzymes found in liver cells, such as alanine aminotransferase
(ALT) and aspartate aminotransferase (AST). A test for bilirubin, a substance produced by the breakdown of
red blood cells and removed from the body by the liver, may also be done. Sometimes these tests are
performed together as a liver panel.

2. Who is at risk for liver disease and/or damage?

Some of those who are at risk of liver disease include the following:

• People who have been exposed to hepatitis viruses

• Heavy drinkers

• People who take medication that can be toxic to the liver or who are exposed to other liver toxins

• Those who are obese, have metabolic syndrome or insulin resistance

• People with an inherited disorder affecting the liver such as Wilson disease or hemachromatosis
3. What other laboratory tests may be done if I have a bone disorder?
Depending on the cause, your condition may be diagnosed and/or monitored using other tests such as
calcium, phosphorus, vitamin D, or bone markers - a group of tests used to measure bone formation and bone
resorption.
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 Alanine transaminase
Alanine transaminase or ALT is a transaminase enzyme .It is also called serum glutamic pyruvic transaminase
(SGPT) or alanine aminotransferase (ALAT).

ALT is found in serum and in various bodily tissues, but is most commonly associated with the liver. It
catalyzes the two parts of the alanine cycle
%

It catalyzes the transfer of an amino group from alanine to a-ketoglutarate, the products of this reversible
transamination reaction being pyruvate and glutamate.
Glutamate + pyruvate -ketoglutarate + alanine
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It is commonly measured clinically as a part of a diagnostic liver function test, to determine liver health. When
used in diagnostics, it is almost always measured in international units/liter (U/L). While sources vary on
specific normal range values, most show between 5-60 U/L as being normal.

Significantly elevated levels of ALT often suggest the existence of other medical problems such as viral
hepatitis, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy.
For this reason, ALT is commonly used as a way of screening for liver problems. However, elevated levels of
ALT do not automatically mean that medical problems exist. Fluctuation of ALT levels is normal over the
course of the day, and ALT levels can also increase in response to strenuous physical exercise.

When elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed
down by measuring other enzymes. For example, elevated ALT levels due to liver-cell damage can be
distinguished from biliary duct problems by measuring alkaline phosphatase. Also, myopathy-related ALT
levels can be ruled out by measuring creatine kinase enzymes. Several drugs elevate ALT levels, for example,
Zileuton.
& $
5-60 U/L
Specimen
Collect serum / plasma ( heparin or EDTA)
Test Principle
2-Oxoglutarate + L-Alanine ---ALT-- L-Glutamate + Pyruvate
Pyruvate + NADH + H+ ----- L-lactate + NAD +
Procedure
Dissolve one volume of R2 with 5 volume of R1
Wait at least 5 min before use

This solution is stable 35days at 2-8 C

(6) Take two test tubes mark them test and blank

(7) Add 1ml of reagent in both test tubes

(8) Add 100µl serum in test tube test

(9) Mix gently for 1 min

(10) Note the absorbance at 340 nm

Calculation

A/min X 1746

1. What conditions other than liver problems can cause increased ALT?
ALT is more specific for the liver than AST and so is much less affected by conditions affecting other parts of
the body. Nevertheless, injury to organs other than the liver, such as the heart and skeletal muscle, can cause
slight elevations of ALT. For example, small increases may be seen with acute pancreatitis and heart attacks.

2. What other tests may be performed to help determine the cause of liver damage?

After a thorough physical exam and evaluation of a person'

s medical history, there are several other tests that
may be performed as follow up depending on what is suspected to be the cause of liver damage. Some of
these include:

• Tests for hepatitis A, B, and C

• Testing for exposure to drugs and other substances toxic to the liver (Drugs of Abuse Testing and
Emergency of Overdose Drug Testing)
• Ethanol level
• Copper and ceruloplasmin for Wilson disease
• Iron tests and genetic tests for hereditary hemochromatosis
• A liver biopsy
When is it ordered?

A doctor usually orders an ALT test (and several others) to evaluate a person who has symptoms of a liver
disorder. Some of these symptoms include:

• Weakness, fatigue
• Loss of appetite
• Nausea, vomiting
• Abdominal swelling and/or pain
• Jaundice
• Dark urine, light colored stool
• Itching (pruritus)

ALT may also be ordered, either by itself or with other tests, for people who are at an increased risk for liver
disease. Some examples include:

• Persons who have a history of known or possible exposure to hepatitis viruses

• Those who are heavy drinkers

• Individuals whose families have a history of liver disease

• Persons who take drugs that might occasionally damage the liver
 • Persons who are overweight and/or have diabetes

In persons with mild symptoms, such as fatigue or loss of energy, ALT may be tested to make sure they do not
have chronic liver disease.

When ALT is used to monitor the treatment of persons who have liver disease, it may be ordered on a regular
basis during the course of treatment to determine whether the therapy is effective