CLM Clinical Chemistry

Chapter Four
BASIC CLINICAL CHEMISTRY TESTES
05/30/2024 Clinical laboratory method 1

Learning Objectives
2
At the end of this chapter, the student will be able to:

 List the liver function tests
 List the renal function tests
 Identify method of plasma protein determination
 List test for the diagnosis of Carbohydrate metabolism disorders
 Identify common interferences for tests
Clinical laboratory method 05/30/2024

1. Introduction and sample type for clinical chemistry
3
Clinical chemistry tests include:-

1. Liver function tests
2. Renal function test
3. Carbohydrate metabolic disorder test
4. Lipid profile tests
5. Enzyme tests
6. Electrolyte tests
7. Gastrointestinal and pancreatic function test
8. Tumor marker
9. Hormone analysis
Introduction and sample type for clinical chemistry
4
Defined as an area in laboratory sciences that deals with chemical

(biochemical) analysis of body fluids such as blood, urine, spinal fluid as
well as feces, tissue, calculi, and other material
Samples for clinical chemistry
Serous fluids (Pleural, pericardial & peritoneal)
CSF
Feces
Urine
Whole blood
Other specimens including saliva, sweat, synovial fluid, amniotic fluid etc

sample type…..
5
Majority of clinical chemistry samples are :
 serum or plasma
Blood Specimen can be
-Random specimen
-Fasting specimen
-Timed specimen

sample type….
6
Sample preparation
A. Plasma preparation
 Collect blood in anticoagulated tube (heparin or oxalate)
 Centrifuge the anticoagulated whole blood to separate plasma from cells at

3000rpm for 5-10 minutes
 Take the plasma out using pasture pipette to another tube

sample type…
7
B .Serum preparation
 Collect the blood in plain tube
 Allow specimen to fully clot
 Place a specimen vertically in a test tube rack as this will speed up the clotting
action
 The clotting should be complete after 15 – 30 minutes depending on the type
of tube used & patient condition
 Centrifuge at 3000 RPM 10 – 15 minutes
 Take the serum out using pasture pipette to another tube

sample type…
8
Specimen Collection precautions

 Do not shake the blood but gently mix it with the anticoagulant.
 Use clean dry glass tubes or bottles
 Centrifuging blood samples for a minimum period of time.
 Not store whole blood samples in, or next to, the freezing compartment of a
refrigerator

sample type….
9
Factors regarding the collection of blood specimens that can
affect the correctness of clinical chemistry test results include: –

A. Incorrect venipuncture technique,
B. Hemolysis of red cells or lipemia,
C. Collection into the wrong container,
D. Instability of some chemical substances in whole blood, serum, and plasma

sample type….
10
Instability of some chemical substances

 Diffusion of potassium and some enzymes
 Diffusion of carbon dioxide off the surface of the blood
 Decrease in the concentration of glucose by glycolysis (at a rate of about

10%/hour)
 Reduction or loss in activity of certain enzymes, for example acid phosphatase
 Decomposition of bilirubin in daylight or fluorescent light.
 Decrease in volume by evaporation (if not stoppered)

1.1 Analytical methods
11
For estimation of analytes, these four methods are common

1. End point
It employs measuring of analyte after reaction is completed once
3. Fixed time mode
 The sample and the reagent pipetted together once and absorbance will be
measured at two different time
4. Kinetic Assay
 Here the rate of reaction will be calculated at two different measurement

2. liver function test (LFT)
12
 LFTs are used for the recognition of hepatic dysfunction and
 For this different diagnostic modalities have been devised to:

 Screen for abnormalities in liver function
 Determine the type of injury
 Assessing severity
 Facilitate prognosis & follow-up of patients with hepatic disease
 Identification of pattern disease

liver function test
13
Classified in 4 groups:
1. Testes that can detect hepatocyte injury
 AST, ALT
2. Testes that can detect synthetic function

 Albumin
3. Testes for cholestasis

 ALP, GGT, 5`-nucleotidase
4. Tests that detect metabolic/detoxification activity of liver

 Bilirubin
liver function test….
14
Liver chemistry test Clinical implication of abnormality
ALT Hepatocellular damage

AST Hepatocellular damage
Bilirubin Cholestasis, impair conjugation, or biliary obstruction
ALP Cholestasis or biliary obstruction

PT Synthetic function
Albumin Synthetic function
GGT Cholestasis or biliary obstruction
Bile acids Cholestasis or biliary obstruction
5`-nucleotidase Cholestasis or biliary obstruction
15
1. Alanine aminotransferase (ALT)

 Is found principally in the liver with only small amounts being
present in other organs.
 While both ALT and AST are raised with hepatocellular injury,
ALT is more specific for detecting liver cell damage.
 Value of test
 Performed to investigate liver disease
 To monitor patients receiving antiretroviral drugs

16
Alanine aminotransferase (ALT)

 Serum or plasma reference ranges vary with method
Adult male <45 U/L, Adult female <34 U/L

 Interpretation of serum or plasma ALT results
 The most important cause of raised ALT activity hepatocelluar
injury.

17
2. Aspartate aminotransferase (AST)

Large amounts of AST are present in the liver, kidneys, cardiac
muscle, and skeletal muscle
Value of test
 Mainly used to the evaluation of hepatocellular disorders and
skeletal muscle

18
Aspartate aminotransferase (AST)

 Serum or plasma reference ranges vary with method
Adult male <35 U/L, Adult female <31 U/L

 Interpretation of serum or plasma ALT results
 Elevated AST activity is myocardial infarction and others
(disease, muscle disorders)

19
 Cholestasis is a condition where bile cannot flow from the liver to

the duodenum
 Release of ALP into the circulation
 Cholestasis may cause ALP increased 3-10 X the normal levels
 Serum total and direct bilirubin are increased

20
Alkaline phosphatase (ALP)

Enzyme localized to biliary & sinusoidal membranes of hepatocytes
(also found in bone, intestine & placenta).
↑ ALP in : Biliary disease (esp. cholestatic disorders), hepatic

infiltrative diseases, pregnancy, Paget’s, bone tumor

21
Reference range= 53 -128 U/L
Interpretation of result
o 2x or more increases in serum or plasma:

Bone cancer, bone disease (such as Paget’s)
Hepatobiliary disease such as cholestasis

3. Bilirubin
22
Bilirubin is a yellow pigment produced from senescent RBCs.

 The hemoglobin will be liberated from the RBcs

Bilirubin….
23
 Protoporphyrin –biliverdin----- biliverdin reductase-------billirubin
 The bilirubin bound to albumin is transported to liver.
 In liver, it will be converted to billirubin glucoronide
 This conjugate bilirubin is then reduced by the intestinal microflora in to 3
compounds collectively known as urobilinogens.

 In the lower intestinal tract the three urobilinogens (stercoblinogen ,
mesonblinogen and uroblinogens) which are orange brown in color and they are
major pigments of stool

Bilirubin…
24
N.B. Total billirubin = Conjugated +Unconjugated

 Conjugate billirubin = Direct billirubin, water soluble
 Unconjugated bilirubin = indirect bilirubin, not water soluble, not excreted in
urine and bound to albumin for transport.
Value of the test

 To investigate the causes of liver disease, jaundice, and
 To monitor a patient’s progress, e.g. an infant with serious neonatal
jaundice
Bilirubin…
25
Jaundice : It Is an abnormal accumulation of bilirubin in the blood

Causing a yellow pigmentation of the skin, sclera of the eye, nails and
other mucus membranes
Types of jaundice
 Pre – hepatic jaundice
 Hepatic jaundice
 Post- hepatic jaundice

Bilirubin…
26
1. Prehepatic (hemolytic)
 More bilirubin is produced than the liver can metabolize, e.g. in
severe hemolysis
 The excess bilirubin which builds up in the plasma is mostly of the

unconjugated type
 Therefore not found in the urine.

Bilirubin…
27
2. Hepatic (Hepatocellular)
 More bilirubin is b/c not transported or excreted by the liver cells because they are
damaged, e.g. in viral hepatitis.
 The excess bilirubin is usually of both the unconjugated and conjugated types
 Bilirubin being found in the urine.

Bilirubin…
28
Posthepatic (Obstructive jaundice)

More bilirubin is b/c its flow is obstructed in the small bile
channels or in the main bile duct. E.g gall stones
The excess bilirubin is mostly conjugated types
Bilirubin being found in the urine.

o The term cholestasis is used to describe this condition (a failure of bile
flow).

Bilirubin…
29
Reference Range:
 Total = 0.2 to 1.2 mg/dL

 Direct = 0 to 0.3 mg/dL
Increased levels of Total Bilirubin associated with liver disease and
hemolytic disorders.
Increased levels of conjugated Bilirubin (Direct Bilirubin) associated with
biliary obstruction and hepatocellular disease.

4. Renal Function Tests (RFTs)
30
Tests that detect glomerular and tubular functions

Uses of RFTs
 Identifyrenal dysfunction
 Diagnose renal disease
 Monitor disease progress
 Monitor response to treatment

Renal Function Tests…
31
Non protein nitrogenous substances (NPN) are waste products formed in

the body as a result of metabolism of nucleic acid, amino acid & protein
 These are compounds that contain nitrogen, but are not proteins
 Kidneys act to excrete metabolic waste

 Amino acids
 Ammonia
 Blood urea nitrogen (BUN)
 Creatinine
 Uric acid
32
Commonly used testes for RFT

 Creatinine
 Urea
 Uric acid
N.B electrolyte (Na & K), Albumin can be also used as RFT

RFT(Urea)
33
 Major excretory product of protein metabolism.
 It is synthesized in the liver from ammonia & CO2.
Protein  amino acids  ammonia [LIVER]  urea
 It is transported by plasma to the kidney to be excreted in urine.
 Azothemia is an excess of urea or other nitrogenous compounds in the blood
 Uremia is increase blood level of urea accompanied by renal failure.

RFT(Urea)…
34
 Urea concentration of blood is often expressed in terms of blood urea nitrogen

(BUN).
Test principle
Berthelot method
 Principle of blood urea nitrogen based on the enzyme urease which decomposes
urea to form ammonia and carbon dioxide.
 Ammonia is then converted dicarboxyl endophenol (blue color)
 Sample: serum , plasma

RFT(Urea)…
35
All reference ranges should be established for each country

and region.
 BUN x 2.14 = urea
BUN Reference Range:

-adults (Serum/plasma)……………….. 6-20 mg/dl
-New borne up to one week( Serum/plasma)…3- 25mg/dl
-Adult over 60 (Serum/plasma) ………..8-23mg/dl
• Urine, 12-20 g/24hrs
RFT(Urea)…
36
Clinical Significance of BUN:

1.Serum/plasma urea levels will vary depending upon
A. Diet
B. Liver function
C. Kidney function
D. State of hydration
Increased urea (BUN)
A. Increased protein intake  increased synthesis
B. Decreased kidney function:  decreased excretion
D. Dehydration
37
Decreased urea (BUN)

A. Decreased protein intake  decreased synthesis
B. Decreased liver function (severe)  decreased ammonia to urea

conversion, leading to increased ammonia levels
C. Increased protein synthesis- during late pregnancy
D. Water overload
 Conditions causing elevations of plasma urea are classified according to

cause into three main categories:
 Prerenal, Renal, Postrenal

38
 Renal causes
Pre renal azotemia
(prolonged increase)
 Reduced renal blood flow  Acute and Chronic renal
 Dehydration failure
 Protein metabolism  Glomerular nephritis
 A high-protein diet  Tubular necrosis, and
other intrinsic renal
 Increased protein catabolism
disease

39
Postrenal azotemia
 Obstruction of the urine flow in the UT by renal calculi
 Tumors of the bladder or prostate, or
 Severe infection.
 The major causes of decreased plasma urea
 Decreased protein intake

 Severe liver disease
RFT(Creatinine)
40
Creatinine is a substance derived from creatine & creatine phosphate and

excreted in the urine.
 Creatine is a product of protein metabolism
 The serum creatinine is a better indicator of renal function than either that of
BUN or uric acid, because cretainine

 Is released into the circulation at a relatively constant rate proportional to an
individual's muscle mass.

 Removed from the circulation by glomerular filtration and excreted in the urine

RFT(Creatinine)…
41
 Plasma creatinine concentration depends on:
 Relative muscle mass

 Rate of creatine turnover
 Renal function
 Concentration in the blood is reasonably stable and constant

RFT(Creatinine)…
42
 Test principle
 Jaffe reaction
 Creatinine + Alkaline picrate = creatinine picrate (golden brown color)
 The intensity of the golden brown color is directly proportional to the concentration of
creatinine present in the sample and absorbance is read at 520nm
 Normal range
 Adult male:- 0.8 - 1.4 mg/dl, Adult females: 0.6 - 1.1 mg/dl, Children: 0.2 - 1.0 mg/dl
o Slight increases with age because values are proportional to

body mass
RFT(Creatinine)…
43
Decreased creatinine
Increased creatinine
Persons with small size,
Renal failure
Decreased muscle mass, or
Muscle diseases
Inadequate dietary protein.
Congestive heart failure
Muscle atrophy
Shock

Renal Clearance Test
44
Extremely useful effective and sensitive way of measuring the

actual excretory capacity of the kidney
 B/C of the concentration of the substance excreted in the urine is
measured and compared to the concentration of the same substance
in the plasma
Renal clearance expresses volume of blood cleared of a
substance per unit of time

Example: mL of substance per minute

Renal Clearance Test…
45
 Standard clearance formula:

ml plasma cleared = Uc V
Pc
 U = urine substance concentration (mg/dl)
 V = total volume of urine collected
 P = plasma substance concentration (mg/dl)

Renal Clearance Test…
46
 Substance used to monitor Clearance Test (GFR )must meet the following
criteria:
 Filtered exclusively by glomerulus
 Not reabsorbed by kidney tubules
 Not secreted by kidney tubules
 Most often used = creatinine clearance test

BUN/Creatinine Ratio
47

RFT (Uric acid)
48
 is the final breakdown product of purine metabolism
 Transported in the plasma from the liver to the kidney
 Reabsorption of 98-100% occurs in the proximal tubules
Disease correlation
 Gout: increased levels of uric acid in the blood then deposited in the joints or
tissue causing painful swelling.
 Patient with gout are risk of developing renal calculi.
 chronic renal disease will also cause elevated level of uric acid.

RFT (Uric acid) ….
49
Reference Range: Interpretation of test result
Adult male: 3.5-7.2 mg/dl

 Increased uric acid
 Gout
Adult female:2.6-6.0 mg/dl
 Increased breakdown of nucleic
Child: 2.2-5.5 mg/dl
acids
Urine 250-750 mg/24 hr  Renal disease
 Decreased uric acid
 Severe liver disease
 Over treatment with
‘allopurinol’
5. Carbohydrate metabolism disorders
50
Glucose test: The main reason to perform plasma or

serum glucose analysis is to screen and diagnose hyperglycemia, usually
caused by diabetes mellitus. (Glucose metabolism)
- Glucose is commonly determined from plasma and serum and rarely from CSF
and urine
- There are different test for glucose test like, random blood sugar, fasting blood
sugar, 2hr post prandial and glucose tolerance test

Test principle
51
GOD-PAP Method
 β-D-glucose in serum (plasma) is oxidized to gluconic acid by glucose oxidase.
When glucose is oxidized proportionally H2O2 is released.
 In the presence of proxidase enzyme the released H2O2 reacts with forming a rose
colored derivative.
 The intensity of color is directly proportional to the amount of glucose present in
sample. Intensity of color will be read at 515nm (if phenol is the chromogen).

Interpretation of Results
52
Reference
Fasting blood glucose
 Serum---------------------------70-110mg/dl
 whole blood------------------- 65 -95 mg/dl
 CSF----------------------------- 40-70 mg/dl
2-hour postprandial: less than 140 mg/dl
Random: less than 126 mg/dl

Interpretation of Results….
53
Criteria for diagnosis of DM are met when any of the following results have
been repeated at least two different days

 FBS is 126 mg/dl or higher
 2hr oral glucose tolerance test result is 200 mg/dl or higher
 a random blood glucose test is 200 mg/dl or higher
 Symptoms of diabetes are present

Interpretation of Results….
54
If the patient’s FBS is between 110-126 mg/dl considered to have prediabetes
(impaired fasting glucose )
FBS level below 40 mg/dl in women or below 50 mg/dl in men
 Hypoglycemia :
 Insulinomia
 Tumor

What affects the test
55
Consuming food or drink less than 8 hours before testing will affect the
FBS test result
Medication that can raise or reduce blood glucose level
Illness or emotional stress, smoking
Inappropriate collection, storage, and processing

Glucose tolerance test
56
 It measures the body's ability to metabolize glucose,
 Oral GTT can be used to diagnose prediabetes and diabetes.
 It is most commonly done to check for diabetes that occurs with pregnancy (
gestational diabetes).
 Glucose test can be done after 1 or 2 hour

6. plasma protein
57
 In plasma more than 300 different proteins can be identified
 Most plasma proteins such as albumin,  ,  globulins, and plasma coagulation factors are
synthesized in the liver
 Except immunoglobulin which is produced by plasma cells
Function of plasma proteins
-Transportation (proteins, enzymes, hormones, drugs, vitamins, metals coagulation

factor).
- Maintenance of colloidal osmotic pressure

- Maintenance of acid-base balance
plasma protein….
58
 The total serum protein in healthy
 Ambulatory adult is 6.3 – 8.3 g/dl and

 Adult at rest is 6 – 7.9 g/dL.
 The two general causes that change the plasma protein concentration are changes in the volume of
water.
A. Decrease in the volume of plasma water is reflected as relative hyper-proteinemia

concentration
 The individual’s plasma proteins are increased.
 This is due to dehydration (inadequate water intake, excessive water loss in severe vomiting, diarrhea).

plasma protein….
59
B. Increased plasma water volume is reflected as relative hypo-

proteinemia concentration
 Individual plasma proteins are decreased.
 This is due to severe nutritional deficiency, malabsorption, and sever liver
disease.

plasma protein….
60
Most of the causes of low total protein levels (below 60 g/l) are due to
reduced albumin concentrations or more rarely to severe immunoglobulin

deficiency.
An increase in total protein concentration can occur (greater than 80 g/l)
when there is prolonged venous stasis during venipuncture, or when a

person is dehydrated.

Determination of plasma proteins
61
Plasma protein should be studied
 First by determining total protein contents and

 Then its composition
 Methods:
1. Quantitative measurement of total protein

2. Electrophoresis
3. Specific quantitative assays of proteins
4. Detection and identification of abnormal proteins

Determination of plasma proteins….
62
There are four methods of determination of total protein

1. kjel - Dahl’s method
2. Biuret’s method
3. Dye binding method
4. Electrophoresis
5. Refractometry
1. kjel - Dahl’s method

determination of Nitrogen content of protein

Determination of plasma proteins….
63
2. Refractometry:
 It is quick and reasonably accurate to measure the serum protein.
 It measures the refractive index of solute in serum.
3. Dye binding method

 Dyes bind to protonated amino groups of acid residues in the polypeptide chain
and the absorbance will be measured
4. Electrophoresis
 Refers to the migration of charged solutes or particles (protein)on cellulose acetate
or agarose gel.

Albumin
64
Albumin is produced entirely in the liver and constitutes about 60% of
total serum protein

It is important
 In regulating the flow of water between the plasma and tissue fluid
 Binding and
 Transport functions

Albumin
65
Value of test
 Serum albumin is mainly measured to investigate
 liver diseases,
 protein malnutrition,
 disorders of water balance,
 Nephrotic syndrome, and
 protein losing gastrointestinal diseases.
Albumin
66
 Decreases (Hypoalbuminaemia) occur whenever there is

increased plasma volume (e.g. in pregnancy).
 Pathological causes include: –
 Low protein intake as in protein energy malnutrition.
 Malabsorption
 Chronic liver disease

 Oedema
Interpretation of Albumin
67
Albumin = 3.5 – 5.1 g/dl

Hyperalbuminemia ; is presence of increased concentration of
albumin in serum of a given patient.
 Acute dehydration
 Increased production of albumin has no clinical significance ?
Hypoalbuminemia ; is the presence of decreased concentration of
albumin in serum of a given patient.
 Decreased synthesis
 Increased excretion/digestion

68
Thank you for your attention!!!!!

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Chapter Four

BASIC CLINICAL CHEMISTRY TESTES

05/30/2024 Clinical laboratory method 1

At the end of this chapter, the student will be able to:

Clinical laboratory method 05/30/2024

Clinical chemistry tests include:-

Defined as an area in laboratory sciences that deals with chemical

Clinical laboratory method 05/30/2024

Majority of clinical chemistry samples are :

Clinical laboratory method 05/30/2024

 Centrifuge the anticoagulated whole blood to separate plasma from cells at

Clinical laboratory method 05/30/2024

 Allow specimen to fully clot

 Take the serum out using pasture pipette to another tube

Clinical laboratory method 05/30/2024

Specimen Collection precautions

 Use clean dry glass tubes or bottles

 Centrifuging blood samples for a minimum period of time.

Clinical laboratory method 05/30/2024

Factors regarding the collection of blood specimens that can

affect the correctness of clinical chemistry test results include: –

Clinical laboratory method 05/30/2024

Instability of some chemical substances

 Diffusion of carbon dioxide off the surface of the blood

 Decrease in the concentration of glucose by glycolysis (at a rate of about

 Reduction or loss in activity of certain enzymes, for example acid phosphatase

 Decomposition of bilirubin in daylight or fluorescent light.

 Decrease in volume by evaporation (if not stoppered)

Clinical laboratory method 05/30/2024

For estimation of analytes, these four methods are common

Clinical laboratory method 05/30/2024

 LFTs are used for the recognition of hepatic dysfunction and

 For this different diagnostic modalities have been devised to:

Clinical laboratory method 05/30/2024

2. Testes that can detect synthetic function

3. Testes for cholestasis

4. Tests that detect metabolic/detoxification activity of liver

ALT Hepatocellular damage

ALP Cholestasis or biliary obstruction

1. Alanine aminotransferase (ALT)

Clinical laboratory method 05/30/2024

Alanine aminotransferase (ALT)

Adult male <45 U/L, Adult female <34 U/L

Clinical laboratory method 05/30/2024

2. Aspartate aminotransferase (AST)

Clinical laboratory method 05/30/2024

Aspartate aminotransferase (AST)

Adult male <35 U/L, Adult female <31 U/L

Clinical laboratory method 05/30/2024

 Cholestasis is a condition where bile cannot flow from the liver to

Clinical laboratory method 05/30/2024

Alkaline phosphatase (ALP)

↑ ALP in : Biliary disease (esp. cholestatic disorders), hepatic

Clinical laboratory method 05/30/2024

Reference range= 53 -128 U/L

o 2x or more increases in serum or plasma:

Clinical laboratory method 05/30/2024

Bilirubin is a yellow pigment produced from senescent RBCs.

Clinical laboratory method 05/30/2024

 Protoporphyrin –biliverdin----- biliverdin reductase-------billirubin

 The bilirubin bound to albumin is transported to liver.

 In liver, it will be converted to billirubin glucoronide

 This conjugate bilirubin is then reduced by the intestinal microflora in to 3