BIOCHEMISTRY OF DISEASES 1

OUTLINE:

1. Blood Sampling Technique Serum And Plasma Separation

2. Estimation Of Total Plasma Protein (BIURET METHOD)

3. Determination Of Serum Albumin

4. Blood Glucose Estimation Method(Fasting & Random)

5. Glycosylated Hemoglobin Hba1c

6. Lactose Tolerance Test

7. Liver Function Test

8. Renal Function Test

9. Determination Of Lipid Profile

10. Serum And Urine Electrolyte

BIOCHEMISTRY OF DISEASES 2

1. BLOOD SAMPLING TECHNIQUE SERUM & PLASMA SEPARATION

Blood sampling is basically crucial step for biological understanding for treatment and
diagnosis identification and prevention of , it includes collection of venous and arterial blood.

MATERIAL:
Syringe, Vacutainer, Vials, Test tubes, Alcohol swab,
Dressing.
TYPES
There are three blood sampling techniques;

1. Arterial Sampling is common method required for analysis of metabolic, respiratory

and acid base disorders.
2. Venipuncture Sampling is collection of blood from spherical vein in upper limb.
3. Fingerstick Sampling is minimally invasive procedure using a lancet to draw a drop or two
of capillary blood from a finger.

PROCEDURE
1. Wash hands and put on apron.
2. Apply tourniquet, palpate the vein.
3. Put on gloves, clean the area with swab in circular motion.
4. Prepare the strings tight the needle, remove needle cover and turn the bevel up.
5. Secure the vein and insert the needle, angle should be 20-40°.
6. Let the entire syringe to get filled.
7. Remove tourniquet and needle carefully.
8. Pour the blood into vacutainer carefully.
BIOCHEMISTRY OF DISEASES 3
9. Discard the needle and apply dressing on patient.

RESULT: Blood sample has been collected

(AFIP LAB MANUAL FOR READING PURPOSE)

1. Make the patient to sit comfortably in the phlebotomy chair. Identify the patient by asking his
particulars and compare them with the request form.
2. Inform the patient about the specimens to be collected. Always ask if he or she has undergone
blood tests previously. In case of any history of abnormal reactions to blood collection, inform
MO I/C lab/Pathologist before phlebotomy and then follow his instructions.
3. Thoroughly check the request form for the number and type of the investigations. Prepare
proper labels and paste them on appropriate containers before obtaining specimens. In case of
any doubt, check the authenticated test list where information regarding type, quantity,
preservative and storage of the specimen is given for various blood tests. If still there is any
doubt, ask the senior colleague/NCO/JCO in-charge or the Pathologist.
4. Select syringe of appropriate size so that the quantity of blood required can be obtained in
single prick. If multiple samples are required, or >15 ml of blood is to be collected use a
butterfly needle or a canula. Select appropriate vein (preferably antecubital) from forearm.
Cleanse the skin over the venepuncture site in a circle approximately 5 cm in diameter with
70% alcohol/spirit swab, scrubbing the area vigorously.
5. If the sample is to be collected for blood culture then skin is to be thoroughly sterilised rather
than simple cleansing.
6. Starting in the centre of a circle apply 2% iodine (or povidone-iodine) in everwidening circles
until the entire chosen area has been saturated with iodine.
7. Allow the iodine to dry on the skin for at least 1 min.
8. Completely remove1 the iodine with 70% alcohol/spirit swab following the pattern of
application.
9. Apply a tourniquet tight enough to obstruct venous flow only and relocate the vein to be
punctured but don’t touch the proposed site of needle entry or the needle itself. Ask the patient
to clench the fist to make the veins prominent. If the vein is not visible, palpate it with fingers.
In case the veins of forearm are not visible/palpable, other sites such as dorsum of the hand
may be selected.
10. Insert the needle into the vein and withdraw blood till the required quantity of blood is
obtained. Do not try to withdraw the piston too forcefully (hard pulling) as it can collapse the
vein and it may cause frothing/ hemolysis of the specimen.
11. Release the tourniquet once the needle has entered the vein.
12. Apply pressure with thumb on antiseptic swab at puncture site for 2-4 min till the blood ooze
stops. Only then patient should 1 This is important to wipe of iodine to prevent iodine
sensitization. 69 be allowed to move away from the specimen collection chair. The antiseptic
swabs should be disposed off in designated baskets.
13. Remove the needle from the syringe.
14. The blood from syringe is distributed to appropriate, labeled containers.
15. Inform the pathologist promptly under the following circumstances:
1. If patient feels unwell after specimen collection, ask him to lie down on couch,
reassure and give him hot drink.
BIOCHEMISTRY OF DISEASES 4
2. Some patients collapse when the skin is punctured or at the sight of blood. In such
cases withdraw the needle immediately and ask the patient to lie down in supine
position. Raise the legs of the patient.
3. If specimen is not drawn in first prick.
4. In case of children below the age of one year.
5. In case of very sick patients/special blood specimen collection.

BLOOD SPECIMEN FOR SEROLOGY

Serological tests are required in most of the bacterial, viral and parasitic diseases. A clotted blood
specimen is preferred.
1. A vacuum collection system is both convenient as well as reliable.
2. Paired specimens are to be collected during acute and convalescent phases of illness in certain
viral and other infections to document a diagnostic rise in antibody titre (see VIRAL
SEROLOGY on page 204)
3. Protect blood specimens from extremes of heat and cold during transport.
4. Specimens must be refrigerated. Whole blood is to be stored at 4°C. Serum can be frozen at -
20°C or lower temperature and can be sent frozen to the reference laboratory.
5. Sera for serology cannot be kept below 0°C, instead should be kept at 2-8°C.

2. ESTIMATION OF TOTAL PLASMA PROTEIN (BIURET METHOD)

SIGNIFICANCE:
Proteins are constituent of muscle , enzymes, hormones and several other key functional and
structural entities in the body. They are involved in the maintenance of the normal maintenance of the
normal distribution of water between blood and the tissues. Consisting mainly of albumin and
globulin in the fractions vary independently and widely in diseases. Increased levels are found mainly
in dehydration. Decreased levels are found mainly in malnutrition, impaired synthesis, protein losses
as in hemorrhage or excessive protein catabolism.

PRINCIPLE:
Proteins, in an alkaline medium, bind with the cupric ions present in the biuret reagent to form a blue-
violet colored complex. The intensity of the color formed is directly proportional to the amount of
proteins present in the sample.

REACTION:
Total Protein + Cü Violet Complex

CONTENTS:

• Reagent 1: Biuret Reagent

BIOCHEMISTRY OF DISEASES 5
• Reagent 2: Protein Standard 6g/dl

MATERIALS:

Clean and dry glassware, Micropipettes & tips, Bio-Chemistry Analyzer Samples,
Serum, Heparinized/EDTA Plasma Proteins are reported to be stable in the sample for 6days at 2-8⁰C.

PREPARATION OF REAGENT & STABILITY:

All reagents are stable till the expiry date mentioned on the label at room temperature.
Standard vial once opened should be stored ar 2-8⁰C, it is stable till the expiry date mentioned on the
vial.
All reagents are in ready to use form

GENERAL SYSTEM PARAMETERS:

• Reaction type : End point

• Wave length : 546nm (530_570nm)
• Temperature: Room temperature
• Incubation: 5 minutes
• Reagent Volume : 1.0ml
• Sample Volume : 10Micro litre
• Standard Concentration : 6gm/dl
• Zero setting : Reagent blank
• Light path : 1cm

PROCEDURE:

Pippete into clean dry test tube labeled as Blank (B), Standard (S) and test (T).
BIOCHEMISTRY OF DISEASES 6

Addition Sequence B S T
Biuret Reagent 1ml 1ml 1ml
Standard - 10 -
Sample - - 10

Mix well, Incubate for 5 minutes at room temperature.

Measure the absorbance of the standard Abs. S and sample Abs. T against the reagent blank, within
60 minutes.

CALCULATION:
Total Protein Conc. (gm/dl) = Abs.T ÷ Abs.S ×6
NORMAL VALUE:
Serum 6-8 gm/dl
It is recommended that each laboratory establish its own normal range.

LINEARITY:
This procedure is linear upto 10gm/dl. Samples above this concentration must be dilluted with normal
saline and results should be multiplied with dilluted factor.

LIMITATIONS & PRECAUTION:

• Storage conditions mentioned on the kit must be adhered.

• Do not in any case freeze or expose reagent to high temperature as it may effect the
performance of the kit.
• Before the assay bring all the reagents to room temperature.
• Avoid contamination of the reagents during the assay process.
• Use clean glassware free from dust and debris.

3. DETERMINATION OF SERUM ALBUMIN

PRINCIPLE:
23% of sodium sulphate and ether, Globulin in serum get precipitated with react to 23% of sodium
sulphate and ether.
Rest of globulin ------(reagent)-------blue color compound-------(spectroscopy/530nm)---O.D

METHOD:
This method is based on dye binding method. In this albumin react with bromocresol. Green as a
result green color obtain, which is directly proportion of O.D.
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PREPARATION OF REAGENT:
a. 8.8g of succinic acid, add I 200ml of water.
b. 85mg of bromocresol green (that is already dissolved in 5ml of 0.1ml of NaOH)
c. Dissolved in 800ml of water.
d. Ph: pH adjustment adding NaOH, until it will reach to 4.2 PH.
e. Storing temperature: at 4-8C
f. Using: before use, pre warm

PROCEDURE:
1. Take 2 test tubes, 1 containing sample and other containing blank (blank means sample
comparing)
2. Then add prepared reagent.

REFERENCE RANGE: 35-45 g/l of serum

4. BLOOD GLUCOSE ESTIMATION METHOD(FASTING & RANDOM)

INTRODUCTION:
Glucose is a monosaccharide. It is central molecule in carbohydrate metabolism. Stored as glycogen
in liver and skeletal muscle.
For glucose estimation from any material, blood is collected in fluoride containing vial.
Fluoride inhibits glycolysis by inhibiting enolase enzyme. In CSF, bacteria & other cells are also
present so analyzed immediately.
For glucose estimation from urine, add 5ml glacial acetic acid as preservative to inhibit bacterial
growth.

PRINCIPLE:
Glucose + H₂O + O₂ GOD Gluconic acid + H₂O₂
4 Amino Phenazone + Phenol + H₂O₂ POD Quinonimine – Pink color compound Intensity is
determined at on 505 nm filter.

PROCEDURE:
Test Stans. Blank1:

1: Glucose reagent (ml) 1.0 1.0 1.0

2: serum(ml) 0.01 ------ -----
3: Glucose standard (ml)
____ 0.01 ____
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Mix & keep it for incubation at 37⁰ for 15 mins or at room temperature for 30 mins. Measure the
Intensity of color at 505 nm filter (Green filter)

MEASUREMENT OF GLUCOSE IN URINE METHOD:

1. QUALITATIVE METHOD:
a. It is determination by Benedict test.
2. QUANTITATIVE METHOD:
b. It Is Determination by Hexokinase & Glucose Dehydrogenase
3. SEMI QUANTITATIVE METHOD:
• It is determination by Glucose Oxidase strip test
• E.g., Urine strip

BENEDICT’S TEST:
This is a very simple and effective method of the amount of glucose in the urine.

PRINCIPLE:
c. Glucose(R-CHO) + 2Cu⁺² +2H ₂ O → Gluconic acid(R -COOH) + COOH) +Cu ₂ O +
4H⁺
PROCEDURE:
d. 5 ml of Benedict’s reagent + 8 to 10 drops of urine Boiling the mixture & cool down
it, observe changes color

RESULT & INTERPRETATION ON BENEDICT TEST

e. Blue – sugar absent.
f. Green – 0.5 gm% sugar = +1
g. Yellow – 1.0 gm% sugar = +2
h. Orange – 1.5 gm% sugar = +3
i. Brick red – 2.0 % or more sugar = +4

NORMAL RANGE BLOOD:

j. Random Blood Sugar: < 140 mg/dl

k. Fasting Blood Sugar: 70 to 110 mg/dl
l. Post Prandial Blood Sugar

5. GLYCOSYLATED HEMOGLOBIN HBA1C

PRINCIPLE:
BIOCHEMISTRY OF DISEASES 9
The HbA1c (Hemoglobin A1c) reagent, when used in conjunction with Beckman Coulter Systems,
HbA1c Calibrators, and SYNCHRON and AU Hemalyzing Reagent, is intended for the quantitative
determination of hemoglobin A1c concentration in human whole blood.
The absolute HbA1c and Total Hemoglobin (THb) values generated as part of the HbA1c assay are
intended for use in the calculation of the HbA1c/Total Hemoglobin ratio and must not be used
individually for diagnostic purposes.

SIGNIFICANCE:

Measurement of hemoglobin A1c is accepted as a method to measure long-term glucose control in

patients with diabetes mellitus (a chronic disorder associated with disturbances in carbohydrate, fat
and protein metabolism and characterized by hyperglycemia).

Determination of HbA1c provides an important tool for monitoring the efficiency of dietary control
and therapy during treatment of diabetes mellitus. Long term treatment of the disease emphasizes
control of blood glucose levels in preventing the acute complications of ketosis and hyperglycemia. In
addition, long term complications such as
Retinopathy, neuropathy and cardiovascular disease can be minimized if blood glucose levels are
effectively controlled.
The process of conversion from hemoglobin A to hemoglobin A1c depends on the blood glucose
concentration. Since the average life of a red blood cell is 120 days, measurement of hemoglobin A1c
can reflect the mean daily blood glucose concentration over the preceding two to three months and
provides a much better indication of glycemic control than blood or urinary glucose determinations.

MATERIAL:

a. SYNCHRON and AU Systems and Hemolyzing Reagent (for use in sample

preparation)
b. 0.9 % saline solution Two levels of control material
c. 0.1M NaOH
REAGENTS:
d. HbA1c Reagent

REAGENT PREPARATION:

e. Total Hemoglobin R1 and HbA1c R1 and R2 are ready for use, and can be placed
directly on board the analyzer. Bring Hemolyzing Reagent to room temperature prior
to use.

PROCEDURE:

1. The HbA1c assay involves the use of four reagents:

BIOCHEMISTRY OF DISEASES 10
f. Total Hemoglobin R1,
g. HbA1c R1,
h. HbA1c R2,
i. Hemolyzing Reagent.
2. In a pre-treatment step, whole blood is mixed with the Hemolyzing Reagent in a 1:100
dilution and the resultant hemolysate is used. Tetradecyltrimethylammonium bromide (TTAB)
in the hemolyzing reagent eliminates interference from leukocytes.
3. The concentrations of both HbA1c and Total Hemoglobin are determined.
4. The HbA1c/Total Hemoglobin ratio is expressed either as mmol/mol or %HbA1c.
5. Total Hemoglobin Reagent is used to measure total hemoglobin concentration by a
colorimetric method. Cha absorbance is measured at 570/660 nm.
6. HbA1c reagent is used to measure hemoglobin A1c concentration by a turbid metric
immunoinhibition me reaction, hemoglobin A1c antibodies combine with HbA1c from the
sample to form soluble antigen-antibody Polyhaptens from the reagent then bind with the
excess antibodies and the resulting agglutinated complex is measured turbidimetrically.
7. Change in absorbance is measured at 340/700 nm.

SPECIMEN STORAGE AND STABILITY:

Samples (non-pretreated) are stable up to 8 hours when stored at 25°C, 7 days when stored at 2.8°C
and up to 3 months when frozen at -20°C. Whole blood samples are stable for 18 months at -70°C.
Frozen samples should be thawed only once.
Hemolyzed (pre-treated) samples are stable up to 4 hours when stored at 15…25°C, up to 24 hours
when 2…8°C, if stored in a sealed container.
Note: All hemolyzed samples should be mixed thoroughly immediately prior to assay.

SPECIMEN COLLECTION AND PREPARATION:

K-EDTA, K-EDTA. Li-Heparin or Na-Heparin whole blood (freshly drawn blood treated with EDTA
is the preferred specimen). Draw enough blood to yield the necessary tube volume. (It is important to
follow the tube Manufacturer’s recommendations).
It is recommended to aliquot the required volume of hemolyzing Reagent from the primary container,
immediately returning the primary container to recommended storage conditions. Allow the aliquot of
the hemolyzing Reagent to reach room temperature (15…25°C) before use, to allow for reproducible
accurate dispensing. If the aliquot has not been brought to room temperature, it is less efficient and
may require a longer period (e.g., 1min) fully hemolysis the whole blood sample.
Pre-treat samples and controls by dilution of whole blood with Hemolyzing Reagent. 1 part sample or
control with 100 parts Hemolyzing Reagent (for example 10 μL of sample or control plus 1000 μL of
Hemolyzing Reagent Cat. To ensure the highest level of pipetting accuracy when pipetting very low
volumes, always choose the pipette with the lowest nominal volume possible and the smallest tip.
Select a pipette whereby the volume required lies in the center of the pipette capacity range. The
pipette should be calibrated and maintained correctly.
1. Thoroughly mix (on a roller if possible) the whole blood sample before the aliquot is taken for
pre-treatment. Settling of the red cells before the aliquot is taken for pre-treatment may cause
altered total hemoglobin (THb) and glycated Hemoglobin (A1C) results. Avoid the formation
BIOCHEMISTRY OF DISEASES 11
of foam. This could lead to unpredictable sampling or localized dried Blood spots around the
inside of the sample tube.
2. Ensure Pipette is measuring accurately to ensure quantity of blood is dispensed accurately.
Ensure outside of the pipette tip is free of excess blood by careful cleaning with a lint free
wipe taking care not to come in contact with the pipette opening as this will decrease the
amount of whole blood being dispensed.
3. Use pipette mixing (carefully aspirating and dispensing several times) to ensure all the whole
blood is dispensed into the hemolyzing solution. Mix thoroughly (on a roller if possible),
avoid foaming and assay the hemolysate after hemolysis complete (allow at least 1 minute for
Hemolysis). This is indicated by a colour change from red to Brown-green. Incomplete
Hemolysis can manifest as significantly lower levels of Total Hemoglobin.
4. Do not pipette directly from the Hemalyzing Reagent bottle; use a disposable tube. This will
avoid potential contamination.

TESTING PROCEDURE(S):
Total Hemoglobin and HbA1c tests must be performed on each pre-treated sample and control. Refer
to the appropriate Beckman Coulter AU analyzer User Guide/Instructions For Use (IFU) and the
accompanying AU Instrument Setting Sheets for analyser-specific assay instructions.

REPORTING RESULTS

REFERENCE INTERVALS:

Adults:
– 6.0% (NGSP)
20-42 mmol/mol (IFCC units)
Reference Intervals shown above were taken from the literature. Expected values may vary with age,
sex, sample type, diet and geographical location. Results should always be assessed in conjunction
with the patient’s medical history, clinical examinations and other findings. Data contained within
this section is representative of performance on Beckman Coulter systems. Data obtained in your
laboratory may differ from these values.

DYNAMIC RANGE / ANALYTICAL MEASURING RANGE

TOTAL HEMOGLOBIN

The analytical range for Total Hemoglobin is 3.7-13.0 mmol/L (6-21 g/dL). When the result for Total
Hemoglobin is outside the analytical range an “F” or “G” flag is generated and the calculated %
HbA1c should not be reported. Settling of the red cells before the aliquot is taken for pre-treatment
may cause an elevated Total Hemoglobin result. Samples exceeding the upper limit of Total
Hemoglobin may be mixed well and the analyses repeated on a freshly hemolyzed sample.

HbA1c
BIOCHEMISTRY OF DISEASES 12

The analytical range of this assay extends from 0.19 mmol/L (0.3 g/dL) to the concentration of
Calibrator 6. If the HbA1c concentration is outside the analytical range an “F” or “G” flag is
generated and the calculated % HbA1c should not be reported.

Samples exceeding the upper limit of the analytical range for HbA1c should not be diluted, but
instead should be reported as “% HbA1c 15% or HbA1c 140 mmol/mol”.

% HbA1c
The reportable range for the calculated HbA1c is 20-140 mmol/mol HbA1c (IFCC) and

4-15% HbA1c (NGSP), based on a typical Calibrator 6 value of 1.36 mmol/L (2.19g/dL), at a total
hemoglobin of 9.6 mmol/L (15.5 g/dL).

fggNote: a calculated result may be outside the reportable range based on either the Total
Hemoglobin or the HbA1c result being outside of their respective analytical ranges.

6. LACTOSE TOLERANCE TEST

RATIONALE:

Intolerance to milk and other dairy products is caused by a deficiency in the enzyme lactase, which
catalyzes the hydrolysis of lactose to glucose and galactose. By measuring glucose levels after a dose
of lactose, lactase activity can be evaluated. In the lactose tolerance test, a 50 gram dose of lactose is
taken orally by the patient, and blood levels are collected for up to 2 hours afterward to monitor the
physiologic response.

PATIENT PREPARATION:

The patient should have a glucose tolerance test prior to the lactose tolerance test to provide a
comparison. The patient should be fasting overnight, refrain from caffeine and nicotine after
midnight, and the test should be performed in the morning of the following day.

MATERIALS:

50 grams lactose dissolved in water (obtain from pharmacy) Grey-top (potassium oxalate/sodium
fluoride) blood collection tubes.
BIOCHEMISTRY OF DISEASES 13

PROCEDURE:

1. During the test, the patient should refrain from caffeine, nicotine and physical activity.
2. From the pharmacy, obtain 50 g of lactose dissolved in water. For infants and children, a 15 g
dose of lactose in 250 ml water is sufficient.
3. Collect a fasting glucose from the patient in a grey-top tube, gently invert the tube to mix the
blood with the anticoagulant and send the tube to the laboratory promptly.
4. Administer the lactose to the patient.
5. Monitor the patient for abdominal cramping, bloating, gas, and diarrhea.
6. Collect blood in a grey-top blood collection tube at 30, 60, 90, and 120 minutes post dose.
Send tubes to the laboratory promptly.

REFERENCE INTERVAL:

a. Normal: peak glucose rise >20 mg/dL above the baseline level
b. Lactose intolerant: peak glucose rise <20 mg/dL above the baseline level, with
symptoms

7. LIVER FUNCTION TEST

PRINCIPLE:

Liver function tests, also known as liver chemistries, help determine the health of your liver by
measuring the levels of proteins, liver enzymes, and bilirubin in your blood. They can also monitor
the progression or treatment of an existing disease.

Depending on the test, either higher- or lower-than-typical levels of these enzymes or proteins can
indicate a problem with your liver.

RECOMMENDED:
BIOCHEMISTRY OF DISEASES 14

A liver function test is often recommended in the following situations:

To check for damage from liver infections, such as hepatitis B and hepatitis C, especially if it’s
suspected you were exposed to a virus that causes hepatitis
To monitor the side effects of certain medications because some medications are known to affect the
liver, including:
c. NSAIDs Trusted Source
d. Statins
e. Antibiotics
f. Antiseizure medications
g. Tuberculosis drugs

TYPES OF LIVER TEST:

Common liver function tests include:

ALANINE TRANSAMINASE (ALT) TEST:

Alanine transaminase (ALT) is used by your body to metabolize protein. If the liver is damaged or
not functioning properly, ALT can be released into the blood. This causes ALT levels to increase. A
higher result than what’s typical on this test can be a sign of liver damage.
It’s estimated that about 10 percent of people in the United States have elevated ALT levels.

ASPARTATE AMINOTRANSFERASE (AST) TEST:

Aspartate aminotransferase (AST) is an enzyme found in several parts of your body, including your:
h. Heart
i. Brain
j. Pancreas
k. Liver
l. Muscles

When the liver is damaged, AST can be released into the bloodstream. A high result on an AST test
might indicate a problem with the liver or muscles.
Since AST levels aren’t as specific of a marker for liver damage as ALT, it’s usually measured
together with ALT to check for liver problems. For example, a high AST:ALT ratio may indicate
alcoholic liver disease Trusted Source.

ALKALINE PHOSPHATASE (ALP) TEST:

Alkaline phosphatase (ALP) is an enzyme found in your bones, bile ducts, and liver. An ALP test is
typically ordered in combination with several other tests. An ALP test can be used to evaluate the bile
BIOCHEMISTRY OF DISEASES 15
duct system of the liver.

ALBUMIN TEST:

Albumin is the main protein made by your liver. It performs many important bodily functions.
For example, albumin nourishes your tissues and transports hormones, vitamins, and other substances
throughout your body. An albumin test measures how well your liver is making this protein.

BILIRUBIN TEST:

Bilirubin is a waste product from the breakdown of red blood cells. It’s ordinarily processed by the
liver. It passes through the liver before being excreted through your stool.
A damaged liver can’t properly process bilirubin. This leads to an atypically high level of bilirubin in
the blood. Certain inherited diseases can raise bilirubin levels, even when liver function works as
expected.

PROCEDURE:

Patients may have blood drawn in a hospital or at a specialized testing facility. To administer the test:
m. The healthcare technician will clean the skin before the test to decrease the likelihood
that any microorganisms on your skin will cause an infection.
n. They’ll likely wrap an elastic strap on patient arm. This will help his veins become
more visible. They’ll then use a needle to draw samples of blood from his arm.
o. After the draw, the technician will place some gauze and a bandage over the puncture
site. Blood sample will be sent to a laboratory for testing.

RESULTS:

The result is withdrawn according to each type of liver function test, this may include.
LIVER TEST INDICATION
ALT TESTS A higher result than typical on
this test can be a sign of liver
damage.
Very high levels over 1,000
units per liter (U/L) are most
often caused by viral hepatitis,
ischemic hepatitis, or injury
from drugs or other chemicals
TYPICAL AND ATYPICAL
RANGES
An ALT above 25 international
units per liter (IU/L) in females
and 33 IU/L in males typically
requires further testing and
evaluation.

AST TEST A high result on an AST test The typical range for AST is
might indicate a problem with usually up to 36 U/L in adults
BIOCHEMISTRY OF DISEASES
your liver or muscles.
Elevated AST without elevated
ALT may indicate heart or
muscle disease. If ALT,
bilirubin, and ALP are also
elevated, it may Indicate liver
damage.
ALP TEST High levels of ALP may
indicate liver inflammation,
blockage of the bile ducts, or
bone disease
16
and may be higher in infants
and young children.
Children and adolescents may
have elevated levels of ALP
Trusted Source because their
bones are growing. Pregnancy
can also raise ALP levels. The
typical range for ALP in adults
Is usually 20–140 IU/L trusted
Source.

ALBUMIN TEST A low result on this test can
indicate that your liver isn’t
functioning properly. This
occurs in diseases such as
Cirrhosis, malnutrition, and
cancer.
The typical range for albumin is
35–50 grams per liter (g/L).
However, low albumin can also
be a result of poor nutrition,
kidney disease, infection and
inflammation.

BILIRUBIN TEST A high result on the bilirubin The typical range for total
test may indicate that the liver bilirubin is usually 0.1–1.2
isn’t functioning properly . milligrams per deciliter
elevated Bilirubin levels with
elevated ALT or AST may
suggest cirrhosis or hepatitis.

8. RENAL FUNCTION TEST

SIGNIFICANCE

Renal function tests (RFT) are a group of tests that may be performed together to evaluate kidney
(renal) function. The tests measure levels of various substances, including several minerals,
electrolytes, proteins, and glucose (sugar), in the blood to determine the current health of the kidneys.

INDICATIONS
BIOCHEMISTRY OF DISEASES 17

Indications for the assessment of renal function are varied and range from acute emergency to chronic
settings.
1. Primarily, renal function tests are performed to identify the renal disease to determine
appropriate patient management and prevent further deterioration of renal function.
2. Further indications in patients in whom the renal disease has been identified are to stage level
or type of renal disease and to monitor the progression of renal disease to ensure that optimal
management occurs and to monitor response to interventions.

PROCEDURE:

Assessments for renal functions:

There are several clinical laboratory tests that are useful in investigating and evaluating kidney
function.

GLOMERULAR FILTRATION RATE

The best overall indicator of the glomerular function is the glomerular filtration rate (GFR). GFR is
the rate in milliliters per minute at which substances in plasma are filtered through the glomerulus, in
other words, the clearance of a substance from the blood. The normal GFR for an adult male is 90 to
120 mL per minute. The characteristics of an ideal marker of GFR are as follows:
a. It should appear endogenously in the plasma at a constant rate
b. It should be freely filtered at the glomerulus
c. It can be neither reabsorbed nor secreted by the renal tubules.
d. It should not undergo extrarenal elimination.

ALBUMINURIA OR PROTEINURIA:

Albuminuria refers to the abnormal presence of albumin in the urine. Albuminuria is used as a marker
for the detection of incipient nephropathy in diabetics. It is an independent marker for the
cardiovascular disease since it connotes increased endothelial permeability, and it is also a marker for
chronic renal impairment. Urine albumin may be measured in 24-hour urine collections or early
morning/random specimens as an albumin/creatinine ratio. The presence of albuminuria on two
occasions with the exclusion of a urinary tract infection indicates glomerular dysfunction. The
presence of albuminuria for three or more months is indicative of chronic kidney disease. Frank
proteinuria is defined as greater than 300 mg per day of protein. Normal urine protein is up to 150 mg
per day (30% albumin; 30% globulins; 40% Tamm Horsfall protein). Increased amounts of protein in
the urine may be due to:

e. Glomerular proteinuria: Caused by defects in perm selectivity of the glomerular

filtration barrier to plasma proteins (for example, glomerulonephritis or nephrotic
syndrome)
f. Tubular proteinuria: Caused by incomplete tubular reabsorption of proteins (for
example, interstitial nephritis)
g. Overflow proteinuria: Caused by increased plasma concentration of proteins (for
BIOCHEMISTRY OF DISEASES 18
example, multiple myeloma-Bence Jones protein, myoglobinuria
h. Urinary tract inflammation or tumor

Urine protein may be measured using either a 24-hour urine collection or random urine protein:
creatinine ratio (early morning sample is preferred since it is a near representative of the 24-hour
sample).

Normal And Critical Findings:

The normal GFR for an adult male is 90 to 120 ml per minute. A GFR of less than 15 ml per minute
is end-stage renal failure requiring renal replacement therapy, e.g., dialysis. The presence of a normal
GFR does not exclude the presence of renal disease, which may be evidenced by the presence of
albuminuria/proteinuria or imaging.

9. DETERMINATION OF LIPID PROFILE

SIGNIFICANCE:

A complete cholesterol test — also called a lipid panel or lipid profile — is a blood test that can
measure the amount of cholesterol and triglycerides in your blood.

PRINCIPLE:

A lipid panel is a common blood test that healthcare providers use to monitor and screen for your risk
of cardiovascular disease. The panel includes three measurements of your cholesterol levels and a
measurement of your triglycerides.

TESTS:

A typical lipid profile includes the following tests:

a. High density lipoprotein cholesterol (HDL-C) – “good cholesterol” A lipid profile test
or lipid panel is performed to measure:
b. Total cholesterol level.
c. HDL cholesterol (good cholesterol)
d. LDL cholesterol (bad cholesterol)
e. Triglyceride levels.

PROCEDURE:

Following steps are done during determination of a lipid profile.

f. A blood sample is taken with a needle inserted into a vein in patient’s arm.
g. Before his blood is drawn, an elastic band is tied around his upper arm to increase
blood in the veins, and the puncture location is wiped clean with an antiseptic
BIOCHEMISTRY OF DISEASES 19
h. A needle blood draw may cause a temporary sting.
i.
Normal Cholesterol Level

The level of cholesterol in our blood is normally measured in milligrams per deciliter (mg/dL). Lipid
profile normal range is-
j. Total cholesterol normal range- lower than 200mg/dL
k. LDL cholesterol range- between 70 to 130mg/dL
l. HDL cholesterol range- between 40 to 60mg/dL

m. Triglycerides- between 10 to 150 mg/dL These are lipid profile normal values.

RESULTS:

If the lipid profile test results or cholesterol test results are outside the normal range of cholesterol,
then one has more risk of heart diseases, strokes and other conditions. In case of abnormal cholesterol
levels, patients need to take other tests such as thyroid test or blood glucose test. A thyroid test is
done to check whether he has an underactive thyroid. A blood glucose test is done to check for
diabetes.

10. SERUM AND URINE ELECTROLYTE

Serum electrolytes: Sodium measurements are used in the diagnosis and treatment of aldosteronism
diabetes insipidus adrenal hypertension, Addison’s disease, dehydration, inappropriate antidiuretic
hormone secretion, or other diseases involving electrolyte imbalance. Potassium measurements are
used to monitor electrolyte balance in the diagnosis and treatment of disease conditions characterized
by low or high blood potassium levels. Chloride measurements are used in the diagnosis and
treatment of electrolyte and metabolic disorders such as cystic fibrosis and diabetic acidosis.
The urine electrolytes sodium, potassium and chloride are principally used as nutritional indicators in
healthy persons. They are infrequently measured in clinical settings, and when they are it is usually in
Intensive/Critical care units. The more important use of urinary electrolyte data is for public health
studies. Urine sodium data is an important biomarker of dietary sodium intake.

PREPARATION OF QUALITY CONTROL MATERIALS

1. SERUM QC POOLS

For serum Na+ , K+ and Cl- , quality control material can be purchased from Roche Diagnostics in
two levels (Precinorm U Plus and Precipath U Plus). The controls are lyophilized and require
reconstitution. Carefully open one bottle 1, avoiding the loss of lyophilizate and pipette in exactly 3.0
mL of diluent (bottle 2). Carefully close the bottle and dissolve the contents completely by occasional
gentle swirling within 30 minutes. Avoid the formation of foam. Stability of Na+ , K+ and Cl- in the
reconstituted controls is 5 days at 2-8°C or 4 weeks at -15 to -25°C when frozen once. Unopened
controls are stable at 2-8°C until the expiration date.
BIOCHEMISTRY OF DISEASES 20

2. URINE QC POOLS

For urine Na+ , K+ and Cl- , commercially prepared quality control material is purchased from
CLINIQA in two levels (LIQUID QCTM 1 and LIQUID QCTM 2). The controls are liquid and ready
for use. Stability of the constituents at 2-8°C is 36 months or until the expiration date, whichever
comes first.

INSTRUMENTATION

1. MODULAR ANALYTICS E170® system (Roche Diagnostics, Indianapolis, IN)

2. Daigger Vortex Genie 2 (VWR, Suwanee, GA)
3. Eppendorf micropipet and tips (Brinkmann Instruments Co., Westbury, NY)

OTHER MATERIALS

The following materials are available from the manufacturer (Roche Diagnostics):
a. Sample trays
b. Sample cups (standard and micro)
c. Sodium cartridge
d. Potassium cartridge
e. Chloride cartridge
f. Reference cartridge E.
PRINCIPLE:

Ion selective electrode (ISE) is an electrochemical sensor that works based on the principle of a
galvanic cell. It converts the activity or concentration of a specific ion present in a solution into
electrical potential.

PROCEDURE:

1. Allow patient samples, calibrators and QC to reach ambient temperature.

2. Ensure that the amount of reagents is adequate for the amount of samples to be run.
3. Make sure the analyzer and the tests required are not masked.
4. Check to see if calibration is required for the tests to be run.
5. If running the same tests on all samples, go to the “Start” global button and set the “default
profile”.
6. Be sure to clear all previously programmed samples from the Data Review screen after
backing up the data.
7. Perform the required maintenance on the ISE Module.

RESULT INTERPRETATION:
BIOCHEMISTRY OF DISEASES 21

If manual dilution is necessary, dilute the specimen with deionized water and re-assay. Multiply the
result obtained by the appropriate dilution factor.
Critical Call Results (“Panic Values”) Serum critical limits are as follows:
• Sodium 158 mmol/L
• Potassium 6.2 mmol/L
• Chloride 126 mmol/L