Basic clinical chemistry tests

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Clinical chemistry tests
 Clinical chemistry tests
 laboratory tests demonstrating the presence of
physiologically significant substances in a
specimen.
 Application ranges from
 simple chemical tests for various components
---To--
 use and measurement of enzyme activities,
electrophoresis, and immunoassay.
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 Clinical chemistry tests
 Clinical chemistry tests performed depend on:
 The health needs of the community
 The training and experience of laboratory staff
 Availability of appropriate and affordable
equipments
 Whether the numbers of tests performed are
sufficient to avoid;
o reagents
o standards
from being wasted due to their expiry
o controls
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Clinical chemistry tests

 Tests
 Whole blood, serum, or plasma tests
o Albumin : to investigate
 disorders of water balance
 liver diseases
 protein energy malnutrition
o Creatinine -to investigate disorders of renal
function 4
 Whole blood, serum…..
o Glucose- to diagnose and monitor diabetes
o Bilirubin- to diagnose and monitor
jaundice
o Alanine aminotransferase -investigate
liver disease.
o Na+ & K+ sodium-investigate & monitor
electrolyte disturbances.
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Urine clinical chemistry tests

o Protein -diagnose and monitor proteinuria

o Glucose- screen and monitor diabetes
o Bilirubin- Dx of hepatocellular and obstructive
jaundice
o Urobilinogen- investigate hemolytic jaundice
o Ketones- detect and monitor ketonuria
o Hemoglobin- investigate intravascular Hemolysis
o Nitrite & leukocyte esterase- diagnosis of UTI

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 Fecal clinical chemistry tests

o Occult blood- to investigate bleeding lesions of GIT

o Lactose- to investigate lactase deficiency
o Examining feces for excess fat to investigate
disorders of fat absorption
 Cerebrospinal fluid (c.s.f.) clinical chemistry tests
 Protein- to investigate meningitis and African
trypanosomiasis
 Glucose- occasionally needed to assist in
diagnosing meningitis
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 Measurement of serum or plasma creatinine

 Analyte: Creatinine is a nitrogenous waste product

formed from the metabolism of creatine in skeletal
muscle.
 it diffuses freely throughout the body water & filtered
from the ECF by the kidney and excreted in the urine.
 Excretion is mainly renal and normally, relatively
constant.
 Increasingly used to investigate HIV associated renal
disease and to monitor nephrotoxic antiretroviral drugs.

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Creatinine….
 Value of test
o Measurement of serum or plasma creatinine is an
important test of kidney function.
o Recommended in preference to the measurement of
serum urea
 better indicator of overall renal function
 Serum creatinine levels are less affected than urea
levels by age, dehydration, and catabolic states
 Creatinine levels are also less influenced by changes
in diet
 Creatinine….

Jaffe-Slot alkaline picrate method

o Principle: Creatinine reacts with picric acid in an alkaline
medium.
 The absorbance of the yellow- red color produced is
measured in a colorimeter at 490nm wavelength.
 other compounds similarly react with picric acid giving
artificially high results for creatinine
 A second reading is made after making the solution acid.
 The color produced by creatinine is quickly destroyed by
acid
 By subtracting the second reading from the first reading, the
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color produced by the true creatinine can be obtained
Jaffe-Slot alkaline picrate method
Two sets of tubes are required Blank Standard Control Test/s
Pipette into each set of tubes as follows: (B) (s) (C) (T)
Creatinine working reagent (fresh)…… 2ml 2ml 2ml 2ml
Distilled water………………… 0.2ml
Standard ,200 µmol/l………………….. 0.2ml
Control serum……………………….. 0.2ml
Patient’s serum/plasma…………………… 0.2ml
Mix well & Leave at room temperature for 90 minutes
READ ABSORBANCES OF FIRST SET OF TUBES, i.e. Reading 1 colorimeter: 490mm
Zero instrument with blank solution in tube B (first set of tubes)
Pipette into second set of tubes: 50µl 50µl 50µl 50µl
Acetic acid, 60% v/v.............

Leave second set of tubes for 6 minutes.

READ ABSORBANCES OF SECOND SET OF TUBES, i.e. Reading 2
Subtract Reading 2 from Reading 1.
Report patients’ results Males (0.7–1.4 mg%) Females: 0.4–1.2 mg%

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 Measurement of serum or plasma bilirubin
 Bilirubin:
 formed from the breakdown of erythrocytes and
other haem containing proteins such as myoglobin
and cytochromes.

 Haem →spleen → biliverdin (biliverdin reductase)

→ bilirubin (unconjugated (indirect) bilirubin)

 Insoluble in water and cannot be excreted in urine

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Bilirubin
 Fate of unconjugated bilirubin-
 It is bound to albumin and transported in the blood to the
liver
bilirubin + glucuronic acid (glucuronosyltransferase)
bilirubin glucuronides =conjugated (direct)
bilirubin(water-soluble) ↓
Bile duct
↓
Intestine
 reduction (bacteria) urobilinogen
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 Bilirubin…
urobilinogen
 most excreted in the faeces as stercobilinogen
 Some of the urobilinogen from the intestine is reabsorbed to liver
where it re-enters the intestine in the bile and is excreted in the
faeces.
 A small amount of reabsorbed urobilinogen is transported to the
kidneys where it is excreted in the urine as urobilin.
 Note: In a healthy individual about 5% of the total plasma
bilirubin is conjugated and 95% unconjugated.
 Urine contains a trace of urobilinogen and no bilirubin 14
 Bilirubin…
 Jendrassik and Grof method
o Principle :
 The azobilirubin produced by the reaction between bilirubin
and the diazonium salt of sulfanilic acid in the presence of
reaction accelerator Caffeine sodium benzoite shows
maximum absorption at 600 nm alkaline tartrate medium.
 The intensity of the color produced is proportional to the
quantity of bilirubin which has reacted.
[sample] = AT x [standard]
AS where: AT= Absorbance of test
AS = Absorbance of standard 15
Summary of Total Bilirubin Method
Pipette into Standard Standard Control Control Tests Test
(S) blank(SB) (C) blank(CB) blanks
tubes as follows
Caffeine-benzoate 1ml 1ml 1ml 1ml 1ml 1ml
reagent .....
Standard serum...... 0.1ml 0.1ml - - - -
Control serum...... - - 0.1ml 0.1ml - -
Patient’s serum - - - - 0.1ml 0.1ml
Diazo reagent (fresh) 0.5ml - 0.5ml - 0.5ml -
Sulphanilic acid, 5 g/l - 0.5ml - 0.5ml - 0.5ml
Mix well and leave at room temperature for 5 minutes
Pipette into tubes 1ml 1ml 1ml 1ml 1ml 1ml
Alkaline tartrate
reagent
READ ABSORBANCES (Blanks first): Colorimeter: 590 nm
Report patients’ results Adults: 0.2–1.3 mg/dl Newborns: 0.5–4.0 mg/dl
 Measurement of serum albumin

o Analyte: 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

 Albumin also has important binding and transport

functions
 Albumin diffuses easily through damaged
membranes
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Albumin…
o Value of test
 Serum albumin is mainly measured to investigate
 liver diseases
 protein energy malnutrition
 disorders of water balance
 nephrotic syndrome
 protein losing gastrointestinal diseases

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Albumin…
 The bromocresol green (BCG) binding method
o Principle:
 Bromocresol green is an indicator which is yellow
between pH 3.5–4.2.
 When it binds to albumin the color of the indicator
changes from yellow to blue green.
 The absorbance of the color produced is measured
in a colorimeter at 590 nm wavelength.

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 Summary of Albumin Method
Pipette into tubes as Blank Standard Control Test
follows:
Bromocresol green 4ml 4ml 4ml 4 ml
(BCG) reagent ……
Distilled water…… 20µl _ _ _
Standard, 30 g/l….. _ 20µl _ _
Control serum……. _ _ 20µl _
Patient’s serum…. _ _ _ 20µl
Mix well…..
Read absorbance within 5 minutes at 590 nm
Report patients’ results Reference range: 30–45 g/l

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Albumin…
 Interpretation
o Increases
 rarely raised (e.g. diarrhea or prolonged vomiting)
o Decreases
 pregnancy
 Pathological causes include:
 protein energy malnutrition
 Liver disease
 nephrotic syndrome
 AIDS
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 Liver enzymatic tests

a) Aspartate aminotransferase (AST) /glutamate

oxaloacetate transaminase (GOT)
b) Alanine aminotransferase (ALT)/glutamate pyruvate
transaminase (GPT)
 Large amounts of AST are present in the liver,
kidneys, cardiac muscle, and skeletal muscle.
 ALT is found principally in the liver with only small
amounts being present in other organs.
→When there is liver cell damage the serum or plasma
levels of both enzymes are raised. 22
Enzymatic tests…
Measurement of serum/plasma ALT activity
o Value of test

 Mainly performed to investigate liver disease

 Increasingly ALT is being measured to monitor

patients receving hepatotoxic antiretroviral drugs.

 While both ALT and AST are raised with hepatocellular

injury, ALT is more specific for detecting liver cell
damage.
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 ALT activity
 Reitman-Frankel ALT method
o Principle:
 ALT is incubated at 37 °C for exactly 30 minutes in a pH 7.4
buffered substrate containing alanine and α-ketoglutarate.
 ALT catalyzes the transfer of the amino group from alanine to α-
ketoglutarate, forming pyruvate and glutamate.

The pyruvate reacts with 2,4-dinitrophenylhydrazine (DNPH) to
form 2,4-dinitrophenylhydrazone which in an alkaline medium
gives a red-brown color.
 The absorbance of the color produced is measured in24 a
colorimeter at 520 nm wavelength.
ALT activity
 pyruvate + 2,4-dinitrophenylhydrazine (DNPH)→2,4-
dinitrophenylhydrazone( red-brown color)
 The absorbance of the color is measured in a
colorimeter AT 520 nm wavelength.
 Reference range
 5–35 IU/L
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ALT activity
 Interpretation
 >> ALT activity- hepatocelluar injury
 Viral hepatitis-both enzymes are usually raised
 Cirrhosis-ALT levels fall below AST levels
 Both ALT and AST are raised in hepatitis caused by
hepatotoxic antiretroviral drugs.
 Obstructive liver disease- small or moderate ALT and
AST rises

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
Measurement of blood or plasma
Glucose provides the energy for life processes.
glucose

 It is the main end product of carbohydrate digestion.

 Glycogenesis:

 Glycogenolysis:

 Gluconeogenesis:
 an increase in the breakdown of fats to provide energy,
results in an increase in the production of ketones. 27
Blood glucose
 Insulin regulates
 the amount of blood glucose
 the rate at which glucose is taken up by the tissues
 conversion of glucose to glycogen
 Value of test
 Plasma or blood glucose is measured mainly in the
diagnosis and management of diabetes mellitus.

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Glucose….

 Good control of blood glucose levels in diabetic

patients helps to prevent or delay the development of
complications such as;
 Blindness
 kidney failure
 Coronary thrombosis
 Bacterial and fungal infections 29
Blood glucose…
 Diabetes mellitus
 Metabolic defect
 An absolute or relative deficiency of insulin
action/secretion
 Rise in blood glucose
 Depression of glucose metabolism in the tissues
 Increased glycogenolysis
 Stimulation of gluconeogenesis
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Diabetes mellitus…

 Symptoms
 “three polys’’(dypsia,uria,phagia)
 Pruritus
 unexplained loss of weight
 Different forms (WHO clinical classification)

a. Type 1 diabetes/insulin-dependent diabetes (IDDM):

 Insulin treatment is required to sustain life
 absolute insulin deficiency due to the immune
destruction of pancreatic beta-cells
 commonest form among children and young
adults/Europe 31
 Diabetes mellitus…
b.Type 2 diabetes /non-insulin dependent diabetes
(NIDDM):
 some secretion of insulin but a decrease in insulin
action (insulin resistance)
 overnutrition with increased intake of saturated
fats, obesity
 decreased intake of dietary fibre
 physical inactivity
 ageing
 high in developing countries 32
Diabetes mellitus…
c. Malnutrition-related diabetes mellitus
 related to malnutrition during natal and early childhood
 related to pancreatic damage
 cyanide toxicities

d. Gestational diabetes mellitus (GDM)

 women without previously diagnosed diabetes exhibit
high blood glucose levels during pregnancy (3 rd
trimester).
 no secretion of enough insulin
 affects 3-10% of pregnancies
 are at increased risk of developing type 2 diabetes
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mellitus
Blood Glucose Meters

o A wide range of portable electronic blood glucose

meters is available.
o Most are designed for home use by diabetic patients
 Advantages
 small and battery-operated
 require only a small volume of blood
 easy to operate
 provide blood glucose values within 30 seconds
 Capillary blood, EDTA or heparinised venous blood can
be used. 34
Selecting a blood glucose meter

 Considerations
 Local availability
 affordable price
 Sensitivity of meters to high temperatures
 Ease of accessing the batteries needed to operate
the meter

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 Measuring plasma or blood glucose
a. Fasting Blood sugar
 measures blood glucose levels after the pt has
gone without food for at least 8 hours.
 Normal value:
 70-110 mg/dL

Accu-Chek Advantage meter

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 Colorimetric glucose method

The glucose-oxidase enzymatic method

 is recommended because it is specific for glucose.
 protein precipitation stage is included because this removes
substances such as urate
 Principle: Glucose oxidase catalyzes the oxidation of glucose to
give hydrogen peroxide (H2O2) and gluconic acid.
 In the presence of the enzyme peroxidase (POD), the
hydrogen peroxide is broken down and the oxygen released
reacts with 4-aminoantipyrine and phenol to give a pink
color.
 The absorbance of the color produced is measured in a
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colorimeter at 520 nm
 Oral glucose tolerance test (GTT)
 Measures the ability of the body to tolerate standard
dose of glucose.
 the degree of tolerance to the glucose is mainly
dependent on the rate of glucose absorption and
on the insulin response.
 as the glucose is absorbed, the level of glucose in
the blood rises and the normal response is for
insulin to be released from the pancreas to lower
the glucose level.
 tolerance is reduced when insulin is insufficient or
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absent.
Oral glucose tolerance test…
 Preparation of the patient
 Before the test the patient should be on a diet
containing not less than 150 g of carbohydrate per
day for at least 3 days.
 If the GTT is performed following starvation or a
diet low in carbohydrate, glucose tolerance will be
reduced which will make the results of the test
difficult to interpret.

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Preparation of the patient…..
The patient should be instructed not to eat, drink
(except plain water), for 10–16 hours before the test.
Excessive exercise, excitement, or fear may reduce GT.
75 g of glucose/250–300 ml water
drink in 5 to 15 minutes
Diabetes Diagnosis
Stage Test
Fasting Plasma Oral Glucose Tolerance
Glucose [FPG] test
Diabetes >126mg/dl >200 mg/dl
Pre-diabetes >100 & < > 140 and <200 mg/dl
126mg/dl
Normal < 100mg/dl <140 mg/dl
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Cerebrospinal Fluid Analysis

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Cerebrospinal fluid analysis

 assist in the diagnosis of meningitis & CNS disorders

 Includes
 Measurement of total protein

 Measurement of glucose (rare)

 Pathogens and cells

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CSF….
 Characteristics
 Appearance: clear, colorless
 Total protein: 15 - 45 mg/100 ml
 Gamma globulin: 3 - 12% of the total protein
 Glucose: 50 - 80 mg/100 mL (>2/3 of blood sugar
level)
 CSF cell count: 0 - 5 white blood cells (all
mononuclear)
 no red blood cells
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CSF….
Laboratory investigations
a. Microscopic examination
 Normal CSF is free of cells
 If cells are found
 Inflammatory diseases
 Hemorrhage
 Neoplasms
 Trauma
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Microscopic examination
 Clinical interpretation
 >500 WBCs-purulent infection
 300-500 WBC (↑lymphocytes)-
 viral meningitis
 Syphilis of the CNS
 TB
 WBC count > 40% monocytes -subarachnoid
hemorrhoid
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Microscopic examination

 Increased neutrophils
 Bacterial Meningitis
 Cerebral abscess
 CNS hemorrhage
 CNS infarction

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CSF…
 Biochemical analysis:
o Elevated protein
 Increased permeability of the BBB
 Decreased resorption
 Mechanical obstruction of CSF flow
 Meningitis
 Endocrine/Metabolic disorders
o Decreased protein
 recent LP
 chronic CSF leakage
 acute water intoxication 47
CSF…
 Glucose
 Hypoglycorrhacia-
 impaired transport to CSF
 Active metabolism of glucose by cells/micro organisms
 Elevated glucose
 DM

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CSF…
 Bacteriological tests
 Gram stain
 AFB stain
 Culture
 Indian ink
 serological tests
 VDRL
 Latex cryptococcal Ag test 49