Basic Clinical Tests Guide

Basic Clinical Chemistry Tests
Chemical analysis of body fluids

- blood, urine, cerebrospinal fluid, feces, synovial fluid, .
Link chemistry with human physiology
Purpose
To assess our body physiology (liver, renal function)
To diagnose, follow and monitor diseases like Diabetes
Mellitus
Common Testes
LFT
RFT
Blood
glucose
yitayewb2011@gmail.com
LIVER FUNCTION TEST
Liver is the 2nd largest
The heaviest gland
Located just below the diaphragm
Site of intermediary metabolism and

synthesis of many important compounds.
If liver is diseased it loses partially or totally

all these function
LIVER
Physiology of the liver
Metabolism(CHO,
Lipid and proteins)
Detoxification
Excretion
of bilirubin
Synthesis
of bile salt (800-100ml of bile/day)
Storage
phagocytosis
LIVER
Disorders
of the liver
Jaundice
Cirrhosis
Tumor of the liver
Reyes syndrome
Drugs and alcohol related disorders
LIVER
Jaundice
A yelowish discoleration of the skin, nails etc
due to hyperbilirubinemia
Causes:
Prehapatic
Hepatic and
Post hepatic
Tumor of liver
Primary tumor of the liver also known as
hepatocellular carcinoma, hepatocarcinoma or
hepatoma
Occurs as a result of infection (hepatitus virus)
The origin of Secondary tumor is not the liver
LIVER
Reyes syndromes
A
disorder of unknown cause
Hepatic
destruction
Primary
involves children's /some adults/
Occurs
after the recovery from viral infection
Abnormal
liver function tests
Cirrhosis
Irreversible
Form
scaring
nodular structure
LIVER
Common
LFTs includes:
Bilirubin /total and direct/

ALP
AST
ALT
1. bilirubin
Comes from the degradation of Hgb as well as a

degradation of heme containing substances
(myoglobin, cytochrom, catalase )
The human erythrocytes have life span of

about 120 days at the end of which they are
broken down in the retculoendotheial system;
Bone marrow ,spleen & Liver (Kupfer cells ).
Bilirubin
The
hemoglobin
liberated
from
the
erythrocytes is hydrolyzed in the reteculo

endotheial cells to form heme and globin
Heme
dissociate to iron and porphrin

bilirubin
biliverdin
bound with albumin go to the liver

Known as: unconjugated bilirubin
H2O insoluble
indirect bilirubin
Bilirubin
Take
up by hepatocytes(albumin detached from
bilirubin)
Two
non albumin proteins (Y and Z) bind and
transport
the
bilirubin
to
the
ER
where
conjugation takes place

In
the ER of hepatocyte the enzyme Uridine
diphosphate
glucouronate
transferase
transfers glucuronic acid molecules to bilirubin

and
bilirubin
is
converted
to
bilirubin
diglucuronide
Bilirubin
This
is known as
Conjugated
H2O soluble
Direct bilirubin
Secreted in to bile canaliculi
intestine
Bacterial enzyme act on bilirubin

Converts to mesobilirubin
urobilinogen
10
Bilirubin
Bloo
d
2%-5%
reabsorbed
urobilinogen reenters
circulation and
is excreted in
urine.
Urobilinogen
Liver
Most reexcrete
d in bile
Urobilinoge
n
Kidne
y
Portal
Vein
Intestine
s
Bilirubin + bacterial flora +
alk.
pH = -glucoronidase
Urobilinogen
15%
reabsorbed
85
%
Urobili
n,Fece
s
11
Urobilinogen
1.
Conjugated bili.
15% UBG is
(bilirubin gluconoride)
reabsorbed and
enters the high pH of
returns to the liver
small intestine.
2.
4.
5.
All re-excreted into
Bacteria reduce
bile except 2-5% via
biliribin to
kidneys
urobilinogen (UBG)
UBG is colorless
6.
Most (85%) UBG is

oxidized into
urobilin, the brown
12
Jaundice
A
symptom of yellow staining of
connective tissue from excess

bilirubin.
NV:
0.2-1mg/dl
Prominently
- sclera of the eyes
and skin
Icterus
describes the dark
yellow-brown color of serum

with increased bilirubin
Normal serum
Icteric serum
13
Jaundice
Pre hepatic Jaundice
It
is commonly caused by hemolytic of red blood
cell like hemolytic anemia and acute malaria

The
increased destruction of red cell result in large
load of bilirubin- albumin complex to the liver than

the liver can handle and the liver can not conjugate
all bilirubin loaded to it
As a result of this the predominating plasma

bilirubin is the unconjugated bilirubin
Normal
urine
Direct bilirubin
UBG
14
Hepatic Jaundice
It is a type of jaundice associated with liver

disease
The liver has some problem and cant metabolize

bilirubin coming to it
Problems associated with the liver can be:A) Conjugation Failure: - due to the absence of
UDP Glucuronyl transferase enzyme(Crigler-Najjar
syndrome) or inhibition of this enzyme by drug,
poisons.
In this case the predomination bilirubin is ????
15
Hepatic
B) Bilirubin transport problem:- which can be
preconjugation transport failure (as Gilberts
disease) or post conjugation transport
failure(Dubin Johnson disease)
C) Diffuse hepatocelluer damage or necrosis:which can be due to viral hepatitis, toxic
hepatitis (drugs) or cirrhosis
In this case liver is unable to conjugate

bilirubin. So the predominating serum bilirubin
will be the unconjugated bilirubin
16
Post hepatic jaundice
This type of jaundice is mainly due to

obstruction of the common bile duct by stones
Since conjugated bilirubin is normally excreted

as a component of bile, bilirubin accumulates
in circulation, leading to jaundice
normal unconjugated detected in both
serum and urine
In post hepatic jaundice the predominating

serum bilirubin is the conjugated bilirubin
17
Laboratory Diagnosis Of Jaundice

Specimen:
Non-haemolysed serum
Heparinized plasma
Fresh urine
Protect from sunlight
Methods:
1.
Icterous Index method
2. Chemical Methods (for direct & indirect bilirubin)

Jendrassik -Grof method
Malloy and Evelyn method
Van den Bergh method
18
Icterous Index method

The
yelowish color of serum is mainly due to
bilirubin
The
intensity of the serum is directly read on a
spectrophotometer compared with a standard

solution of potassium dichromate, that have a
concentration of 10mg.serum that has the
same color as 10mg% potassium chromate
has been assigned II(ictorus index)
II=At/Ast
NV =4-6 units
19
Chemical Methods
Jendrassik-Grof method
Principle
:Bilirubin present in the sample reacts with diazotized sulfanilic
acid in the presence of caffein benzoit forming a purple colored

azobilirubin.
The
reaction is stopped by addition of acetic acid which destroys the
excess diazoreagent.
The
purple colour azobilirubin converted to a blue color azobilirubin by
the addition of alkaline tartarate solution

The
intensity of the blue color is directly proportional to the amount of
total bilirubin present in the sample .The absorbance read at 620nm
Bil
+DR+cafein benzoate
Bil+DR
Total bilirubin
Direct bilirubin
20
Malloy and Evelyn Method

Bilirubin
react with diazoreagent in the
presence of methanol forming a purple

colour azobilirubin. The color intensity is
directly proportional to the concentration of
total bilirubin. The absorbance is read at
550nm.
Bil+DR+
Bil+DR
methanol
Total bili
direct bilirubin
21
Van den Bergh Method

Similar
to Malloy and Evelyn Method except
methanol is replaced by ethanol

NV=0.2-1mg/dl
(serum)
22
Clinical Correlations
**Urobilinogen = UBG; *Total Bili = (conjugated

+ unconjugated)
Jaundice
Type
Clinical
Condition
Serum
Conjugated
Bilirubin
Serum
Total
Bilirubin *
Urine
Bilirubin
Urine
(UBG)**
None
Normal
Normal level
Normal
level
Neg
Normal
<1 mg/dL
Pre-hepatic
Hemolytic
anemia
???
???
???
???
Hepatic
Hepatitis
???
???
???
???
Posthepatic
Obstruct-ion
of bile duct
???
???
???
???
23
Enzyme analysis
Enzymes
that are synthesized within cellular
organelles carry out their functions within cells

and are released into body fluids when those
cells become diseased
Enzyme
activity levels in body fluids can reflect
leakage from cells due to cellular injury, or

changes in enzyme production rate
or
actual enzyme induction due to metabolic or

genetic states or proliferation of neoplasms
24
ALP /Alkaline Phosphatase/

An
enzyme present in a number of tissues of

the body (not specific)
Relatively in high concentration:
Liver
Bone
major
Intestinal epithelium
Kidney
Leukocytes
placenta
AlP used for the diagnosis of liver and bone
disease
25
ALP
Elevated
ALP is found:
Children
Healing of bone fractures
Pregnant women
Liver disease
Bone disease
Renal disease
Decline ALP
dwarf (small bone)
26
Determination of ALP
Several
methods with similar principle but,
different substrate
Standard
methods
Two point procedure (two types)

Paranitrophenyl phosphate in glycine
solution
Paranitrophenyl phosphate in methyl
aminopropranol
Continuous monitoring /kinetics method)
27
PNPP in glycine buffre
Principle: PNPP substrate in glycine buffer is incubated with

the ALP present in serum at 370c for 30 min.
PNPP will be hydrolyzed to para nitrophenyl (PNP) and

inorganic phosphate. PNPP and PNP are colorless.
An addition of NaOH the PNP is converted to a yellowish

color quinoid derivative.The intensity of the color is directly
proportional to ALP activity; the absorbance read at 410nm.
The enzyme is measured in Activity unit.The most accepted

unit is IU
1Unit =that quantity of enzyme that catalyze the reaction

of 1micromole of a substrate per minute
28
continuous kinetics method
The
buffer is the diethanol amine
Principle:ALP
hydrolyzes at PH 9.8 PNPP to PNP
and phosphate ion.At this PH PNP is converted

to the yelowish coloured quinoid derivative.
The rate of production of PNP is directly
proportional to ALP activity.The increase in
absorbance is read at 410nm every minuet.
The average change in absorbance in per
minute is calculated.
29
AST
Also
known as glutamate oxaloacetate transaminase
/GOT or SGOT/
Catalyze the transfer of amino group from the amino acid

aspartate to the ketoacid alpha-ketoglutarate forming oxalo
acetate and glutamate
Elevated AST:
hepatites
Cirrhosis
Liver cancer
Hepatocute necrosis
Normal 12 38U
30
Determination of AST
Two
methods
End point and

Continuous method
1. End Point
Principle: AST catalyzes the transfer of
aminogroup from aspartate to alphaketoglutarate forming oxaloacetate and
glutamate. In alkaline PH the formed
oxaloacetate couples with 2,4 dinitrophenyle
hydrazine forming a goldon brown color
derivative oxaloacetate hydrazone. The
absorbanec is taken at 550nm
31
Determination of AST.
2. Continuous monitoring method
Principle: Apartate + alpha-ketoglutarate
AST
oxaloacetate+ glutamate
In the presence of the enzyme malate dehydrogenase
and a coenzyme NADH, oxaloacetate is reduced to
malate and NADH is oxidized to NAD.
The NADH consumption is directly proportional to the
AST activity. The decrease in absorbance is due to
NADH consumption is measured at 340nm every
minute
32
ALT
Also
known as glutamate pyruvate

transaminase
Two methods:
End point
Continuous monitoring
End Point
Principle: the enzyme catalyze the transfer
of amino group from the amino acid
alanine to alpah ketoglutarate forming
pyruvate and the amino acid glutamate.In
alkaline solution pyruvate couples with 2-4
dinitrophenyl hydrazone forming a goldon
brown colored derivatives pyruvate
hydrazone at 550nm.
33
ALT
Continuous kinetics method
Principle:
The formed pyruvate by the enzyme reduce in
to lactate and NADH oxidized in to NAD in the presence

of LDH and a coenzyme NADH.
The
decrease in concentration of NADH is directly
proportional to the activity of ALT (340nm)
34
Renal function tests
Major
Structures of Urinary System
Kidney
Ureters
Urethra
Bladder
35
Physiology of Kidney
Urine
formation /secretion, reabsorption and
Excretion/
Regulation
of electrolyte and fluid balance
Regulation
of acid base balance
Hormonal
Protein
function (erythropoietin, ADH)
conservation
36
Renal pathophysiology
Glomerulonephritis
Nephrotic
syndrom
Tubular disease
UTI
Urinary tract obstruction
Diabetic nephropathy
Renal failure
37
Glomerulonephritis
An
inflammation of the glomeruli /acute or chronic/
Acute glomerulonephritis: associate with recent

infection by group A, B-hemolytic streptococci. The
theory is the circulating immune complex will trigger
a strong inflammatory reaction that leads to direct
damage of the glomeruli
abnormal findings include:
hematuria
Reduced GFR
Anemia
Red cell cast: highly suggestive
Hyline cast
Granular cast
38
Glomerulonephritis
Chronic glomerulonephritis: prolonged
inflammation may leads to scarring which
eventual loss of functioning nephron.
Abnormal findings:
slight hematuria
Increase creatinine and urea
Nephrotic syndrom
An abnormal increased permeability of

gromerular basement membrane
39
Nephrotic syndrom
Characterized
by:
massive protienuria
Hypoalbuminemia
Lipiduria
Hypogamaglobulimia
WBC cast
40
UTI
Bacteria
can infect:
Kidney/pyelonephritis/
Bladder/cystitis/
Other
Characterized by:
bacteriuria
Pyuria/WBCs in urine
Hematuria
WBC cast
41
50%
type I diabetic patients can develop deterioration
of renal function after15-20years
Renal calculi
Kidney
stones formed by crystallized substances such
as calcium oxalate, calcium phosphate, uric acid etc
Renal function tests

Renal Clearance test
Determination of Total non protein nitrogenous/NPN/
substances
Determination of urea
Determination of uric acid
42
Renal Clearance test

Used
for the detection of a much earlier stage
of kidney disease
More
sensitive and clinically more useful than
other RFTs
Clearance
=the volume of plasma from which
a measured amount of a substance can be

completely eliminated in to urine per a given
time
43
Renal Clearance test.

Depends
on:
The concentration of the substance in

plasma
Excretion rate of the substance
Renal blood flow
Size of kidney
Ml of plasma cleared/min=UcXVUX1.73
PCXA
44

It
includes:
Inuline clearance
urea clearance
Creatinine clearance
1. INULIN
An exogenous plant polysaccharide
Completely filtered by the glomeruli
Neither reabsorbed nor secreted by the renal

tubules
The most accurate of all clearance tests

Intravenous administration
45

Reference Range
Adult male:
Adult female:
97-137 ml/min
88-128 ml/min
46
Urea clearance test

Endogenous
Freely
Not
metabolic product
filtered by the glomeruli
commonly performed
Creatinine clearance Test

Endogeneous
Synthesized
at a constant rate for a given
individual
Freely
Not
filtered by the glomeruli
reabsorbed, but slightly secreted by the
tubules
47
Non-Protein Nitrogen Compounds

Nitrogen
containing compounds excluding
proteins
Include >15 compounds
Amino acids
Ammonia
Protein
Blood urea
nitrogen
(BUN)
amino
acids
ammonia
urea
Creatinine
Muscle breakdown
product
Uric
acidacid
Nucleic
catabolism
48
Non-Protein Nitrogen Compounds

Principle:
sample
the protein present in the clinical

is
precipitated
out
by
using
trichloroacetic acid. The NPN present in the

filtrate
will
be
converted
to
NH3+
by
reacting with a hot concentrated H2SO4. The

forming NH3+ will react with nesselers
reagent /KI,HgI/ producing a yellow to orange
brown color product. The color intensity is
directly proportion to the NPN concentration.
The absorbance read at 470nm
49
Determination of Creatinine
Creatine
(liver, pancreas, kidney from arg, gly, met)

muscle, brain
creatine phosphate (alternative energy store)

Creatinine
kidney
Formation
and Excretion
Spontaneously derived from creatine in muscle
High energy ATP storage and use in muscle
Produced at a constant rate day to day
Excreted into urine through glomerular filtration;
not significantly reabsorbed
measure ability of glomerulus to filter
chemicals
50
Creatinine.
Jaffe reaction method

Creatinine
present in the sample react with
an alkaline picrate form a golden brown

complex of creatinine picrate. Intensity of
the color is directly proporational to cratinine
concentration
creatinine: severe renal disease and

extensive muscle distraction
NV:0.5-1.5mg/dl
51
Urea
May
be expressed as blood urea
nitrogen/BUN/
BUN:
is the nitrogen content of urea
Urea=BUNx2.14
Methods
of determination
Bertholet method
Diacetyle monoxin
Nesselerization
UV method
52
Bertholet method
Principle:
Urea present in the sample is
hydrolyzed to ammonium carbonate by the

enzyme urease. By making the mixture
alkaline ammonium carbonate converted to
amonia, carbon dioxide and water. The
formed amonia react with phenol in the
presence of sodium hypochloride forming a
blue color indophenol. The absorbance read
at 560nm.
53
Diacetyle monoxin
In
the presence of strong acid and oxidising
agent urea directly condenses with diacetyl

to form a yellow color diazine derivative. The
absorbance read at 520nm
54
Nesselerization
Principle:
Urea
urease
NH4+Co3=
The forming NH4+ react with nesselrs reagent

forming a yellow color. The absorbance read
at 470nm.
55
UV enzymitic
Urea
The
urease
formed
NH4+
NH4+Co3=
react
with
alpha
ketoglutarate in the presence of the co-enzyme

NADH+
H+ by catalytic activity of glutamate
dehydrogenase. The decrease in NADH+ H+ is

directly proportional to the urea concentration.
The decrease in absorbance is measured at
340nmfor 5 consecutive minutes; The average
change in absorbance per minute is calculated
56
Clinical Significance
Reference Range:
Serum/plasma...
Plasma
6-20 mg/dl
levels are dependent upon
Diet
Liver function
Kidney function
State of hydration
57
Increased urea (BUN):
Azotemia or uremia
Increased protein intake (leads to increased
urea formation)
Decreased kidney function (decreased
excretion into urine results in elevated
plasma levels)
Dehydration (lack of body water results in
increased levels)
58
Decreased urea (BUN):
Decreased protein intake (leads to
decreased urea formation)
Decreased liver function (decreased
conversion of ammonia to urea)
59
uric acid
Formation
and excretion
End product of purine metabolism; Adenine

and Guanosine (by liver)
Purines are precursors to nucleic acids
Readily filtered by glomerulus, but then is
handled by tubules
60
Uric acid
Methods
of determination:
Folin Denis
Uricase peroxidase
FolinDenis Method
Principle:
The color is directly proportional of uric acid

concentration
61
Uricase peroxidase method

Uric
acid + H2O
O2
Allantoin + H2O2
The
formed H2O2 react with O2 acceptor like4-
aminoantipyrin and dichlorophenol in the

presnce of peroxidase forming a rose colored
quinone derivative
measured
at 530nm
NV:3-7mg/dl
62
Increased uric acid hyperuricemia
Gout: gouty arthritis

Increased breakdown of nucleic acids:
chemotherapy for proliferative disease such
as leukemia, polycythemia
Renal disease
Decreased uric acid (hypouricemia)
Severe liver disease
Over treatment with allopurinol a drug
interfer uricacid meta.
63
Blood glucose determination

Regulation of blood glucose level
Factors
that add glucose to the blood
Intestinal absorption
Glucogenolysis
Glyconeogenesis
Factors that removes glucose from blood

Glycolysis
64
Hormonal regulation of
glucose
Regulated
by:
Insulin
Glucagon
Cortisol
Adrenalin
65
Insulin
A
peptide hormone
Secreted
by B-cells of the pancreas
Responsible
the entrance of glucose to the cells,
except?????
Insulin
production increased after CHO ingestion
Production
and release will be terminated/decrease
when the extra cellular fluid glucose is starts to

decrease
Concentration
is similar to C-peptide if there is no
exogenous insulin medication

Other
hormones include Glucagon, Adrenalin,
cortisol???
66
Disorders of glucose metabolism

May
results:
hyperglycemia
Hypoglycemia
May be due to:

Inability of the B islet cell of the pancreases to
produce sufficient insulin
Reduced number of insulin receptors
Intestinal mlabsorption of glucose
Inability of the liver to accumulate and break
down glycogen
Increase
hormones
amounts
of
insulin
antagonizing
67
Diabetes mellitus
A
group of hyperglycemic disorders
characterized by:
Absolute lack of insulin
A deficiency of insulin or
Insulin insensitivity by cells
Type I DM
An absolute lack of insulin
Juvenile onset DM /early onset/
Insulin dependant DM (IDDM)
Due to B cell destruction

68
Type II DM
Characterized
by a deficiency of insulin or
insulin insensitivity by tissues

The
most common
Adult
Non
onset DM
insulin Dependent DM (NIDDM)
Obesity
is common
69
Gestational DM
Occur
in a women during pregnancy
Related
Risk
to NIDDM
factors:
Age
Family history
Obesity
70
Blood glucose measurement

Samples
Whole blood( 65-95)

Serum(70-110mg/dl)
Plasma(70-110mg/dl)
CSF(40-70mg/dl)
Other body fluids
Normal values may vary from lab to lab
It
can be measured randomly, fasting 2-hr
postprandial blood sugar

71
Random blood sugar (RBS)

Measurement
of blood glucose that is taken
regardless of meal
Conc:70-150mg/dl
Has
little diagnostic value
Fasting blood sugar (FBS)

Measuring
of blood glucose after an over
night fast (10-14hr)

Conce:70-110mg/dl
More
important for the diagnosis of DM

72
Methods of glucose measurement

include:
Alkaline ferric cyanide method

Copper reduction method
Glucose Oxidize Methods
Hexokinase method and
O-Toluidine method
73
Hexokinase method
Principle:
in the presence of ATP, the enzyme
hexokinase phosphorelate glucose forming glucose-6phosphate and ADP. In the presence of hydrogen
acceptor NADP, the enzyme glucose 6 phosphate
dehydroginase
(G6PD)
oxidizes
G6P
forming
phosphogluconate. NADP is reduced to NADPH and the

amount of NADPH forming is directly proportional to the
concentration of glucose present in the sample. The
increase in absorbance is read at 340nm
The absorbance is read every minute for about 5 min

and the average change in absorbance is will be
calculated
74
Glucose tolerance test

The
ability of an organism to respond in maintain
blood
sugar
homeostasis
when
glucose
is
absorbed from intestine in a high quantity or

given IV is termed as the glucose tolerance of the
individual
In
mild diabetes mellitus blood glucose level can
be with in normal ranges.

Therefore
in any suspected cases of diabetes a
glucose tolerance test is recommended.

Two
types: OGTT and IVGTT

75
Patient Preparation
For 3 days prior to the test, patient should be on an

unrestricted diet containing at least 150 grams of
carbohydrate per day.
The patient should remain physically active in the days

prior to the test.
For 3 days prior to the test, you should discontinue

medications known to affect glucose utilization, such as
salicylates, diuretics, , propranalol, birth control pills
Patient should fast (nothing to eat or drink, with the

exception of water) for 8-12 hours prior to the test.
Do not exercise for 8-12 hours prior

to the test
76
The Oral Glucose Tolerance Test
The oral glucose tolerance test (OGTT) measures the bodys

ability to metabolize glucose, or clear it out of the
bloodstream.
The test can be used to diagnose diabetes, gestational

diabetes or prediabetes
The OGTT it is better able to diagnose high blood glucose

after a glucose challenge than the fasting blood glucose test
A doctor may recommend it if he or she suspects diabetes in

cases where a patients fasting blood glucose level is normal
However, the test is more time-consuming and complicated

than the fasting blood glucose test
77
How Is the Test Conducted
After 8-12hr over night fast blood and urine glucose

is determined
If the glucose concentration normal, a patient must

quickly drink a sugary (glucose-rich) beverage.
Typically, the drink contains 75 grams with 300 ml

of H2O of carbohydrates, although other amounts
are possible
Blood will be drawn at various intervals to measure

glucose levels, usually one hour and two hours after
the beverage is consumed
78
What Does the Test Indicate?
The
test reveals how quickly glucose is metabolized from
the bloodstream for use by cells as an energy source.

After
fasting, the normal blood glucose rate is 60 to 100
mg/dL .
For
75 grams of glucose, normal blood glucose values are:
1 hour: less than 200 mg/dL

2 hours: less than 140 mg/dL. Between 140-200 mg/dL
indicates impaired glucose tolerance (prediabetes).
If test results are in this range, a patient is at an increased

risk for developing diabetes. Greater than 200 mg/dL
indicates diabetes
79
Pregnancy and the Oral Glucose Tolerance Test
Pregnancy
This
affects a womans ability to metabolize blood sugar
test is common during the 24th to the 28th week of pregnancy
Typically,
Normal
Values
the dose of glucose that is given is 50 or 100 grams
values for pregnancy are described below.
above this range indicate gestational diabetes: For the 50-gram
oral glucose tolerance test that is used to screen for gestational

diabetes:
For
the 100-gram oral glucose tolerance test:

Fasting: less than 95 mg/dL
2 hours: less than 155 mg/dL
3 hours: less than 140 mg/dL
80
IVGTT
Glucose
This
administer intravenously
method eliminates the variable factors
involved in the rate of intestinal absorption in

the different individuals
0.5 gram glucose is given per kg body
weight by infusing 20gm% W/V glucose
solution and the infusion will be completed
with in half an hours
81
IVGTT
In
normal individuals the blood glucose
concentration is <250mg/dl at the end of

infusion and falls below this level with in
2hour
In
case of latent and sever diabetes mellitus
glucose concentration pattern will be as oral

GTT
82
Serological Tests
Serological
tests,
unlike
culture
and
microscopic
investigations, provide only indirect evidence of infection by
detecting bacterial antigens, or antibodies produced in

response to them, in clinical material
After
an
initial
infection
with
pathogenic
microorganisms, most patients will produce both IgM

and IgG antibodies.
Serum
collected during the acute phase of the infection,
when the disease is first suspected, is called the acute

serum; serum drawn during convalescence, which is
usually 2 weeks later, is called convalescent serum.
83
Serological Tests.
The
presence of IgG antibodies in a single
serum sample may indicate past exposure to

the agent and therefore cannot be used to
diagnose a current infection
An
antigen may also stimulate production of
antibodies that cross-react with other

antigens
84
Serological tests for syphilis

Syphilis?????
The
serological tests for the diagnosis of syphilis
include non-treponemal and treponemal tests
1. non-treponemal
The
antigens used in these tests are prepared from
non-treponemal antigens, such as cardiolipinlecithin

They
detect the antibody-like substance reagin, which
is present in the sera of many patients with syphilis

Reagin
May
antibody reacts with cardiolipin, a lipid complex
occasionally be detected in the sera of patients
with other acute and chronic diseases

85
non-treponemal
These
tests
are
practical,
inexpensive
although they are not absolutely specific

This
test is superior to the treponemal tests
as a follow-up investigation after treatment

The
non-treponemal tests include the VDRL
and the RPR tests
86
Venereal Disease Research Laboratory

(VDRL)
uses
cardiolipinlecithin-coated cholesterol
particles
Inactivated
serum or cerebrospinal fluid and
VDRL antigen emulsion are mixed using a

rotating machine for a prescribed period of
time
The
VDRL particles will flocculate if reagin is
present in the serum or cerebrospinal fluid

87
VDRL
The
VDRL test is strongly reactive for early
syphilis infection
After
effective
treatment
the
titre
falls
gradually and usually becomes non-reactive

within 12 years
In
the late phase of the disease the serum
may remain reactive at a low titre for many

years, even after effective treatment
88
VDRL
Reactivity may cease spontaneously in about 2030%

of untreated patients during the latent phase of the
disease, and even more often during the late phase
False-positive results may be observed because of

the similarity of the VDRL antigen with normal host
tissue
Prolonged false-positive reactions usually have high

titres that are caused by autoantibodies (rheumatoid
factors)
in
patients
with
lepromatous
leprosy,
tuberculosis
89
VDRL
Any test sample giving a reactive or weakly reactive

result in the absence of clinical evidence of syphilis
should therefore be subjected to a treponemal test
such as FTA-Abs
Materials and reagents provided in the VDRL
test kit
Buffered saline solution
Control sera set (non-reactive, weakly reactive
and reactive)
VDRL antigen
The test can be either qualitative or semi
quantitative
90
VDRL
VDRL qualitative test
1. Using a 1.0 ml pipette, mix the inactivated
serum several times then add 0.05 ml to the
first well of the VDRL glass plate
2. Spread the serum with a circular motion of
the pipette tip so that it covers the entire
inner surface of the paraffin or ceramic well.
91
VDRL
3. Take a syringe with an 18-gauge needle
and, holding it vertically, carefully add 1
drop of antigen to the serum
4. Place the plates on the mechanical rotator
under a humidity cover and rotate for 4
minutes. If a mechanical rotator is not
available rotate with a steady circular motion
for 4 minutes
92
VDRL
5. Examine the plate immediately after rotation
using a microscope with a 10X ocular and a
10X objective
6. Read the reactions as follows:
Medium and large clumps---- R Reactive
Small clumps ------------------W Weakly
reactive
No clumping or very slight roughness--N
Non-reactive
93
VDRL
VDRL semi-quantitative test
1. Prepare a two-fold serial dilution of inactivated
serum in 0.85% saline (1 : 2, 1 : 4, 1 : 8, 1 : 16,
1 : 32)
2. Test each serum dilution using the qualitative
test procedure
3. Report the results in terms of the highest
serum dilution that produces a reactive.
94
VDRL
4.
If reactive results are obtained up to
dilution 1 : 32, prepare further twofold serial

dilutions in 0.85% saline (1 : 64, 1 :128 and
1 : 256) and retest using the qualitative test
procedure
95
RPR test
The
antigen suspension in the RPR test contains
charcoal particles to allow for macroscopically

visible flocculation
The
main differences between the RPR test and
the VDRL test are that it uses a stabilized antigen,

cards instead of plates, and serum as well as
plasma, and the serum does not need to be
heated
In
the RPR test the antigen is ready for immediate
use
96
RPR.
The
RPR test is slightly more sensitive than
the VDRL test and it is easier and quicker to

perform
False-positive
reactions occur slightly more
often with the RPR test than with the VDRL

test
97
RPR.
Materials
and reagents provided in

the RPR test kit
Antigen delivery needle to deliver 60 drops/ml of
the antigen suspension
Control sera, positive and negative

Disposable droppers to deliver 50ml of
serum or plasma
Plastic-coated RPR test cards, each with
two rows of five wells
Prepared RPR antigen suspension
98
RPR.
RPR qualitative test
1.
Remove the reagent kit from the refrigerator and
allow the reagents to warm to room temperature

2. Reconstitute the control serum by adding the
recommended volume of distilled water
3. Label each well on the RPR card with the
laboratory number of a sample to be tested,
including wells for positive, weakly positive and
negative control sera
99
RPR.
4. add 50ml of unheated serum or plasma to the corresponding

well.
5. Gently shake the antigen suspension and add one free-falling
drop to each well using the antigen delivery needle provided.
Carefully mix the antigen suspension and serum. Use a new stirrer
for each sample. Spread to cover the area of the well.
6. Place the card on the mechanical rotator under a humidity cover
and rotate for 8 minutes. If a mechanical rotator is not available
rotate the card by hand with a steady circular motion for 2
minutes, then place it in a moist chamber containing wet tissue or
filter-paper for 6 minutes. Remove the card and rotate briefly to
obtain the final reading. Take care to avoid crosscontamination of
samples
10
RPR..
7. Remove the card from the rotator and examine it
macroscopically in a good light. The positive control
serum should show clearly visible agglutination. The
negative control serum should show no agglutination.
8. Record the test results:
Small to large flocculated clumps: reactive
Even turbidity of the particle suspension: nonreactive
9. Prepare serial dilutions of any reactive sera to
estimate the antibody titre
10
10
10
Treponemal tests
use
Treponema pallidum antigens to detect
specific antibodies that have developed in

serum in response to syphilis infection
The
procedures
specificity
of
are
used
positive
to
verify
the
reactions
in
nontreponemal tests
highly
specific and sensitive but they cannot
differentiate between active and past syphilis

infections; neither can they be used for
evaluating therapeutic response
10
Treponemal.
Includes:
1. Fluorescent
Treponnema
antibody
Absorption test (FTA-ABS-test)

2. T. pallidum immobilization test (TPI)
3. Treponemal pallidum complement fixation
test
4. Treponomal pallidum particle agglutination
test
10
Fluorescent treponemal antibody absorption test

(FTA-Abs)
The
antigen used for the FTA-Abs test consists
of Treponema pallidum (Nichols strain) which

is fixed with acetone on a slide
Inactivated
patient serum is incubated with a
sorbent consisting of Reiter treponemes for

absorption of nonspecific treponemal group
antibodies
After
absorption, patient serum is added to the
slide
10
FTA-Abs
Specific
antibodies in the serum bind to the
surface of the treponemal cells

After
rinsing, a conjugate of antihuman
antibodies with a fluorescent stain (fluorescein

isothiocyanate) is added to the treponemes
The
conjugate will bind to the antibodies that
have bound to the treponemes and can be

visualized by fluorescence microscopy
10
FTA-Abs
Reactivity
can be observed three weeks after
infection and is permanent in untreated patients

A
positive reaction in the FTA-Abs test indicates a
high probability of syphilitic infection

False-negative
results are exceptional and may be
due to poor quality antigen

False-positive
results have also been reported in
patients with hepatic cirrhosis

Cross-reactivity
between T. pallidum and Borrelia
burgdorferi (Lyme disease) has been demonstrated

10
FTA-Abs
The
main advantages of the FTA-Abs test is its high
specificity and sensitivity as well as the early onset of

reactivity
Materials
and reagents provided in the FTA-Abs
test kit
Buffer solution
Control sera, positive and negative
Fluorescein-labelled antihuman immunoglobulin
(conjugate)
Lyophilized extract of Reiter treponemes (sorbent)
T. pallidum smears fixed to slides
10
procedure
1.
Remove the reagent kit from the refrigerator and
allow the reagents to warm to room temperature.

2. Allow the required number of slides to warm to
room temperature for 15 minutes.
3. Dilute 50ml of serum with 0.2ml of sorbent and mix.
Dilute positive control and negative control sera in
parallel
4. Cover the smear on one slide with 10ml of buffer
solution (conjugate control) and the smear on a
second slide with 10ml of sorbent (sorbent control).
11
FTA-Abs
5.
On the remaining slides cover the smears with 10ml
each of serum dilution and control serum dilutions

6. Place the slides in a moist chamber for 30 minutes at
37 Co. Wash the slides for 5 minutes in Tween-PBS
solution. Repeat each wash. Rinse in distilled water,
then drain and leave to dry in a slide box.
7. Cover the smears on all slides with 10ml of
antihuman immunoglobulin diluted in buffer solution
according to the manufacturers instructions.
11
FTA-Abs
8. Place the slides in a moist chamber for 30

minutes at 37 C. Wash the slides for 5 minutes
in Tween-PBS. Repeat each wash. Rinse in distilled
water, then drain and leave to dry in a slide box.
9. Cover each smear with 2 drops of mounting
medium and a coverslip.
10. Check that the conjugate, sorbent and negative
serum controls do not fluoresce:
11
FTA-Abs
The
degree of fluorescence can be graded
as:
Non-reactive 0
Borderline 1+, 2+, 3+
Reactive 4+
A
reaction of 2+ or greater is regarded as
indicative of T. pallidum infection
11
Antistreptolysin O (ASO) test

Streptococcal
infections are very common in
all populations, and a high percentage of

people
will
have
antibodies
against
streptococci
The
B-haemolytic group A streptococci
produce two haemolysins:

Oxygen-labile streptolysin O and
Oxygen-stable haemolysin S
Only
reduced (non-oxidized) streptolysin O is
immunogenic and is used for

the test
11
ASO.
The
antistreptolysin O test is based on the fact
that
patients
with
Streptococcus
pyogenes
infections develop antibodies that inhibit the

haemolytic activity of streptolysin O
The
antibodies are usually long-lasting and a
single increased titer is not an indication of a

current infection
Only
a fourfold or greater rise in titer on
successive serum samples taken 1014 days

apart should be considered indicative of recent
infection
11
ASO.
This
test is mainly used in the diagnosis of
acute
rheumatic
glomerulonephritis
fever,
and
other
acute
post-
streptococcal diseases
There are two types of commercial antistreptolysin O

test kits:
The ASO latex slide agglutination test is used to
screen sera to identify those with raised ASO titres
The ASO tube test is a haemolysis inhibition test
that is used to determine ASO antibody titer in
serum samples that are positive in the ASO latex
11
ASO latex slide agglutination

Materials
and reagents
Disposable cards, with 6 wells each

Disposable dropper
Positive control serum
Sensitized latex reagent (with streptolysin
O)
11
procedure
1.
Dilute the serum 1: 20.
2.
Place 1 drop of the serum solution in a well on

the disposable card
3. Use a new dropper to add 1 drop of sensitized

latex reagent
4. Use an applicator stick to mix the two drops
and spread them over the entire well.
5. Examine for agglutination within 2 minutes
A
positive reaction appears as a fine
flocculation (agglutination) yitayewb2011@gmail.com

within 2 minutes
11
ASO tube test

Materials
and reagents
Positive control serum

Reduced streptolysin O antigen (dried
preparation)
Sheep red blood cells
Standard antistreptolysin O antibody (dried
preparation)
Streptolysin O buffer (25\ concentrated
solution)
11
procedure
1. Reconstitute the reduced streptolysin O antigen

with the appropriate volume of distilled water
2. Reconstitute the standard antistreptolysin O
antibody with 10 ml of streptolysin O buffer
3. Dilute the streptolysin O buffer with 480ml of
distilled water before use
4. Wash and centrifuge 1 ml of sheep red blood
cells three times in streptolysin O buffer and
pipette the supernatant fluid off. Add streptolysin
O buffer to give an 8% cell suspension
12
Procedure
5. Allow the reagents and serum samples to
reach room temperature.
6. Make a 1: 10 dilution of patient serum in a
test-tube (0.1 ml serum + 0.9 ml Streptolysin
O buffer). Prepare 2 master dilutions from the
1 : 10 dilution as shown in the table below:
12
Serum
Buffer
SDilution
0.2 ml (1 : 10)
1.8ml
1:100
1.0 ml (1 : 100)
0.5ml
1:150
7. Prepare the following dilution series with

streptolysin O buffer for each of these master
dilutions
12
Tub Serum
e
Buffer
Dilution
Reduced
streptolysin O
(1 :
0.8ml
1:50
0.5ml
(1 :
0.5ml
1:200
0.5ml
(1 :
0.5ml
1:400
0.5ml
1:800
0.5ml
0.2 ml
10)
0.5 ml
100)
0.5 ml
200)
0.5 ml
400)
0.5 ml
150)
0.5 ml
300)
0
2
3
4
5
6
(1 :
0.5ml
(1 :
0.5ml
1:300
0.5ml
(1 :
0.5ml
1:600
0.5ml
1.5ml
1.0ml
0.5ml
12
Procedure
8. Rearrange the tubes in rising dilutions: 1 : 50, 1 : 200, 1 :
300, 1 : 400, 1 : 600, 1 : 800.

9. Add 1.5ml of buffer to control tube 7 and 1ml of buffer to
control tube 8.
10. Add 0.5ml of reduced streptolysin O to all test-tubes,
except control tube7.
11. Mix and refrigerate at 4 C for two hours to allow the
antibodyantigen reaction to take place.
12. Add 0.5 ml of the 8% cell suspension to each tube,
including control tubes 7 and 8, mix and incubate in a waterbath at 37 C for 30 minutes.
12
Procedure
13. Centrifuge the tubes at 1000 g for 2 minutes and observe
for haemolysis. Control tube 7 should show no haemolysis and
control tube 8 should be completely haemolysed.
14. The ASO titre is determined as the highest dilution showing
no sign of haemolysis:
If there is haemolysis in all tubes, report the result as ASO
titre
less than 200 IU.
If there is no haemolysis in the tubes with a higher serum
dilution, report the result as ASO reactive with the titre.
12
WIDAL TEST
The
Widal test is one of the most utilized
diagnostic tests for typhoid fever in developing

countries
This
test demonstrates the presence of somatic
(O) and flagellar (H) agglutinins to Salmonella

typhi in the patient's serum using suspensions of
O and H antigens
Antigens
of S. paratyphi A, S. paratyphi B, S.
paratyphi C are included in most commercial kits

12
Widal
The
recommended method of performing the
Widal test is by the tube agglutination technique

where serial two-fold dilutions of the subject's
serum are tested
The
over reliance on the Widal test in the
diagnosis of typhoid fever has led to the

underutilization of blood culture
The
unavailability of microbiologic facilities and
the long waiting time for culture results have been

identified as reasons for the preference for the
Widal test
12
Widal
Traditionally,
a "'positive" Widal test is based
on a fourfold rise in O agglutinins in repeated

tests or a titer of 1:80 or greater in a single
test
12
Procedures
Slide method
Specimen: serum/plasma
Take
Add
clean slide
a drop of serum, which is obtained, from
non-hemolyzed blood
Add
a drop of antigen suspension, which is non-
expired,
Mix
well antigen suspension and serum.
Look
for agglutination.
12
Procedures.
Tube method
Used to confirm slide test method
Sample: serum/plasma
Procedure
1. For each antigen arrange 10 small test
tubes in a rack.
2. Place 0.9ml of saline in the 1 st tube and
0.5ml in the remaining 9 tubes
3. Add 0.1ml of fresh cell-free serum to the 1 st
tube
13
Procedures.
4.
Mix and transfer 0.5ml to tube 2,3,4,5,6,7,8,and

tube 9. From tube 9 discard 0.5ml.
Tube 10 will contain only saline and will

serve as a negative control (antigen control)
5.
Mix antigens well and add 0.5ml to each tube. Mix

by gently shaking the tubes.
6.
The final dilutions are 1:20, 1:40, 1:80, 1:160, etc.
7.
Incubate the tubes at 37oC for 2-4 hrs
8.
Report the highest dilution with definite clumps

13
Procedures.
Tube dilution
0.1
0.5
0.1 0.5
0.5
0.5
0.5
0.5
0.9
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
initial
Tube
10
initial
Tube
1
10
21
3 2
1:2560
Dilution 1:20
1:10
1:10
1:1280
1:5120
1:2560
Dilution
Dilution
Dilution
factor
5120
factor
10
2560
20
10
0.5
1:20
1:40
1:40
1:80
1:5120
1:80
1:160
160
80
0.5
0.5
0.5
65
80 40
0.5
0.5
40 20
5120
0.5
0.5
0.5
67
0.5
0.5
1:320
1:640
640320
0.5
0.5
0.5
0.5
87
1:160
1:320
320
160
0.5
0.5
1:640
1:1280
1280
640
2560
1280
13
Factors need to be considered for interpretation.
For
example:
Endemicity of enteric fever in an area of investigation

Administration of antibiotics
Immunization with any typhoid vaccine or a previous
infection or exposure
The stage of the disease at the time of collection of

sample.
Early in the disease low antibody titers are

found
The antibodies start rising after 1st week of illness
and do so until 3-4 week
13
Reporting widal test

If no agglutination occurs the test should be reported as:
S.
typhi O titer less than in 20 (O 1:20)

nonreactive
S. typhi H titer less than in 20 (H 1:20)

Interpretation of the Widal Reaction
A
Negative test does not necessarily mean the patient is
not infected. Reaction occurs in infected patients about
50% during the 1st week
80% in the 2nd week,
90-95% in the 3rd or 4th week

13
Causes of positive widal result

The patient being tested has typhoid fever
Chronic salmonellosis
Vaccination with typhoid vaccine
cross-reaction with non-typhoidal Salmonella
variability
and
poorly
standardized
commercial antigen preparation

infection
with
malaria
or
other
enterobacteriaceae
13
Causes
of negative Widal agglutination tests
absence of infection by S typhi

the carrier state
an inadequate inoculums of bacterial antigen
in the host to induce antibody production
technical difficulty or errors in the
performance of the test
previous antibiotic treatment
13
Agglutination test for

rikettsianceae
Rickettsiaceae
The
genus rickettsia contains several species and sub
species.
Although
classified as bacteria, rickettsia resemble
viruses in that they are mostly obligate parasites and

are unable to survive as free living organisms
They
are about the size of the largest viruses and can
just be seen with the light microscope.

Rickettsia
resembles bacteria also by virtue of their
morphology and microscopic visibility
13
Rikettsianceae
Unlike viruses, however, rickettsia contains both

RNA and DNA, multiply by binary fission, have cell
wells that contain muramic acid, possess enzymes,
and show sensitivity to antiseptic and antibiotics.
Species in the genus rickettsia have been sub

divided into three groups of antigenically related
microorganisms:
typhus group
scrub group
spotted group
13
Rikettsianceae
Serologic diagnosis
Weil-Felix
is
an
agglutination
rickettsial
infections
using
test
particular
for
various
strains
of
bacteria of the genus Proteus that have antigens in

common with the rickettsiae to be identified
Indirect fluorescent antibody (IFA) test. It is of

value not only in diagnosing acute infections but also
in serological epidemiological studies
Complement fixation (CFT) test, which is not

sensitive as IFA test
13
Weil-Felix reaction test
Principle; agglutination test based on the crossreactions occur between antibodies produced in
acute rickettsial infections and the OX-19 and OX-2
strains of Proteus vulgaris and the OX-K strains
of Proteus mirabilis
Three strains of Proteus species have been found

to be useful in diagnosing rickettsial diseases;
these have been labeled OX-2, OX-19 and OX-K.
Antibodies produced at day 6 and high at 2nd week

14
Proteus group used and

Organism
Disease
degree of reaction
OX-19
OX-2
OX-K
Typhus group
Endemic typhus,Brills
R. prowazeki
disease
++++
+/-
R. typhi
Murine typhus
++++
+/-
++++/-
Scrub typhus group

R. tsutsugamushi
Scrub typhus
(R.orientalis )
Spotted fever group:
R. conori
R. conoripeiperi
R. siberica
R. rickettsia
------Asian tick fever

Rocky
mountain
+/++++ +/++++ 0
+/++++ +/++++ 0
+/++++ +/++++ 0
spotted +/++++ +/++++ 0
fever
14
Weil-Felix
False negative- common especially with R.

tsutsugamushi infections
False positive- Proteus infections, relapsing

fever, brucellosis, infectious mononucleosis,
and other acute febrile illness.
14
HIV Serology
Several laboratory methods are available to screen

blood, diagnose infection, and monitor disease
progression in individuals infected by HIV.
HIV tests can be classified into:
detect antibody
identify antigen
detect or monitor viral nucleic acids, and
estimate of T-lymphocyte numbers (cell
phenotyping)
14
HIV Serology..
The
isolation of HIV, its nucleic acid and
detect HIV antigen are mainly used to

detect
early
HIV
infection
before
antibodies develop
HIV Antibody Tests
Based on a multi test algorithm for
detecting antibodies to HIV by using
screening and confirmatory tests.
14
HIV Serology..
Common
HIV antibody tests are:

ELISA
Rapid Tests
Western Blot
14
Enzyme Linked Immunosorbent Assays

(ELISA)
Performing an ELISA
ELISA
can
be
performed
with
serum,
plasma, urine, oral fluids, or dried blood

spots
Can take from 2 to 4 hours to perform
(including
specimen
preparation
and
dilution) and an additional 3 to 4 hours if a

screening result has to be confirmed
Manufacturers instructions provided with
14
ELISA..
General
1.
steps for performing an ELISA
Dilute the specimen in the specimen buffer and put it in a

micro well plate containing HIV antigen already bound to the
plate.
2.
Incubate the plate as per protocol and then wash as

indicated.
3.
Add antihuman immunoglobulin-enzyme
conjugate,
which will react with the HIV specific antibody if present

4.
Incubate
5.
Wash the plate, add the substrate and incubate as prescribed
6.
Add a stopping solution to terminate the enzyme reaction and

read the absorbance of the solution using spectrophotometer.
14
ELISA..
A
positive reaction has occurred if the

specimen in the specimen well changes color
or becomes colored
Positive reaction indicates the presence of
HIV-specific antibody in the specimen.
The reaction is best read quantitatively with
an ELISA plate using spectrophotometer
(ELISA reader).
14
Rapid Tests
General Description
Most HIV rapid tests contain antigens to
HIV-1 and HIV-2 and detect antibodies to
both
A positive test result is indicated by
clumping, a spot dot or line depending on
the test format
The sensitivity and specificity of the latest
generation of rapid tests are similar to
14
Rapid Tests
Sensitivity approaches 100%; specificity is
>99%
Negative tests can be reported as negatives
Positive results should be confirmed
Useful in situations where immediate results
are important to manage decisions
15
Rapid Tests..
Simple
rapid assays are
Capillus HIV-1 HIV-2 agglutination assay

Determine HIV1 and 2
immunochromatographic test
Uni-gold HIV1 and 2 immunochromatographic
test
15
Rapid Tests..
Determine HIV1/2
Is an immuochromatographic test
Detects antibody of HIV1/2 antigens on serum, whole blood,
plasma
Uses recombinant antigen
Test
method
Sample added to sample application pad

Samples are serum, plasma, whole blood
Result read after 15 minute
Sensitivity 99.4-100%
15
Capillus is
Simple
Rapid
Agglutination test
Available as a 20 test or 100 test kit requires storage at 2-8 oC
The test method

uses recombinanat antigen derived from HIV 1 and 2 invitro to
detect antibody
Uses serum, plasma whole blood or
No agglutination indicate that it is negative
Has high degree of accuracy

Sensitivity reported are 100%
Specificity 98-99%
15
15
Uni-gold
HIV tes
Is an immunochromatic test
Detects antibodies
Samples are whole blood, serum, plasma
Uses recombinant HIV 1/2antien
do not require refrigeration
Test method
2 drops of whole blood, serum, plasma is
added to sample port
Then add a buffer
Read the result after 10 minutes (pink
color will appear)
Has sensitivity of 100%
Specificity 99.7-100%
15
HIV
C
T
HIV
HIV
15
Previous test algorithm
15
Current test algorithm
15
Pregnancy Test (HCG)
Pregnancy begins with conception-that

is, the fertilization of an egg by a
sperm
During
pregnancy,
the
chorionic
membrane of the placenta produces a

hormone
called
gonadotrophin
stimulates
the
human
chorionic
(hCG),
which
secretion
of
15
HCG
Human chorionic gonadotrophin (hCG) is

synthesized in large amounts by the
placenta, and it appears in urine, blood,
amniotic fluid and serum relatively soon
after implantation
of the
developing
embryo.
The presence of this hormone serves as

basis for pregnancy testing
16
HCG
Production of hCG increases steadily during

the first trimester, peaking around the 10th
week of gestation
Levels then fall to less than 10% of the first

trimester levels during the reminder of the
pregnancy
The
urine and serum of pregnant women
contain
high
concentrations
of
human
chorionic gonadotrophin (hCG)

16
HCG
Specific
and
sensitive
analytical
methods for the -chain sub unit of

hCG permit the detection of pregnancy
as early as 8 days after ovulation (1
day after implantation)
HCG concentrations climb early in

pregnancy, reaching a maximum by 8
to 10 weeks of gestation
16
Application of pregnancy
tests
Pregnancy tests are important in investigation
of suspected:
To detect early pregnancy
To determine the adequacy of hormone
production
In
threatened abortion: to conform the
abortion is complete or not. In this case the

quantity of hGC decreases gradually
16
Detection of hCG in urine
A number of serologic tests have been used

in pregnancy testing; each designed to
detect minute amounts of hCG when it
appears in the urine during the first few
weeks of pregnancy.
The methods most commonly used now are

based on the agglutination inhibition test
16
The amount of hormone excreted in the urine is

almost the same as that found in the blood.
By about the 8th
day after the first missed
period during the pregnancy, the hCG level is

about 1000IU/L (1IU/ml), and by the 12 th day it
is about 2500IU/L(2.5 IU/ml).
Laboratory
pregnancy
tests
are
based
on
detection of rapidly rising levels of hCG in urine .
16
Specimens
Urine specimen
An early morning specimen is preferable because

this is the most concentrated and will therefore
contain the highest level of hCG
The urine must be collected in
a clean container, which is free from the all traces

of detergent.
If the specimen cannot be tested immediately it

should be refrigerated at 4c, but for not longer than
48 hrs.
16
If the urine is cloudy it should be flittered or

centrifuged and the supernatant fluid used.
Specimens that are heavily contaminated, or

contain large amounts of protein or blood, are
not usually suitable for testing.
There are different types of commercially

produced kits for pregnancy tests and each of
this is with the technique and precaution with
instructions.
16
Types of urine testing kits

1. Rapid
I.
latex slide test

Direct agglutination test
II. Indirect or inhibition test

2. Heamagglutination
technique (tube tests of
the inhibition type)
16
Rapid latex slide test of the inhibition

I.
Direct slide agglutination test

(Direct latex slide tests).
recently introduced techniques,
more sensitive than the inhibition
tests
16
I. Direct slide agglutination test

Principle
In the direct slide test, the latex reagent consists of
particles coated with anti hCG antibodies. This reagent is
mixed directly with the urine there will be agglutination,
which is visible to the naked eye if the urine contains the
hormone hCG. If not there is no visible agglutination.
In the direct slide test, therefore, agglutination of the
particles indicates positive tests and no agglutination
indicates a negative test.
17
Procedure
Prepare clean, dry, detergent free slides.
Take one drop of early morning urine in
Pasteur pipettes and drop on slide.
Take 1 drop of reagent (anti-hCG antibody).
Mix gently; and see for agglutination.
Nowadays people use random urine for pregnancy
test. Because they consider as the hCG hormone may
present at any time in the urine.
But the morning
urine is more preferable due to content of high

concentrated hCG.
17
If
hCG is present in the urine, it will combine

with the antibodies and causes agglutination
of the latex particles.
Urine
latex reagent
Result
(hCG antibody
agglutination
Coated particles)
hCG
Antigen
is
present
hCG
antigen
combines
with hCG
antibody on
latex
particles
17
If
no hCG is present in the urine, there will be

no agglutination of the latex particles.
Urine
latex reagent
Result
(hCG antibody
No
agglutination
Coated particles)
No hCG
Antigen
No hCG antigen
combines with hCG
antibody on latex
particles
17
ii. Indirect latex slide test

(inhibition latex slide test)
Principle:
Colored latex or other visible particles coated with hCG

antibodies to hCG and urine are mixed with particles.
Negative urine results in visible agglutination; and

positive urine results, presence of hCG in urine inhibits
agglutination (or protein flocculation).
Reagents:
Antiserum containing anti-hCG-antibody
Latex reagent containing polystyrene particles coated

(sensitized) with the hCG antigen
17
Procedure:
Take clean slides which is free from detergent
Mix a drop of urine with one drop of antiserum on slide
Add latex reagent
See for positive or negative test.
If HCG is present in the urine it will combine with the

anti-HCG antibody.
This will leave no antibody free to combine with the

latex HCG and therefore there will be no agglutination
of the latex particles.
17
Factors that affect pregnancy tests
The time in the pregnancy when the test is

carried out
The presence of excessive amounts of protein

or blood in the urine may cause false positive
results
Detergent contaminated urine
Bacterial contamination of the urine may

cause unreliable results.
Drugs, which may cause false-positive results

17
Queez
1.
In ASO test for B-haemolytic group A streptococci
infection we are targeted to Streptolysen O, why

not we use the other hemolysin?
A. Because it is easily produced by the bacteria in

artificial culture media
B. The other hemolysins are highly immunologic
C. Since Streptolysin O is highly immunologic
D. We can use either of hemolysin for diagnosis
purpose
E. Non
17
2. In two fold serial dilution method of

widal test the dilution factor of tube 6 is (
in the first tube 0.1ml serum and 0.9ml
saline are mixed)_________
A. 1:160
B. 1:320
D.1:1280
E.1:640
F.1:80
17
3.
Identify incorrect statement about the interpretation
of widal test among the following alternatives

A. Positive widal test result indicates the patient being
tested has typhoid fever
B. Positive widal test indicate the individual is in carrier
state
C. Positive widal test can be due to infection with other
enterobacteriaceae
D. Negative widal test can indicates the bacterial dose
is small and cant induce the immune system of the
individual suspected for salmonella infection
E. Non
17
4. You are invited by Deberberhan university students clinic to

give one day service in your profession. You observed that most
of the students were complaining fever, headache, and malaise
and skin rash. You suspect that the cause in common is
recketicial infection and the weilfex test shows positive result,
which of the following is true about weilflex test?
A. OX-19 and OX-2 antigens of weilfex test are derived from
Proteus mirabilis strains
B. OX-K antigens of weilfex test are derived from Proteus vulgaris
strains
C. The positive weilfex test may be due to Proteus infection
D. For additional confirmation widal test must be done
E. non
18
5. A serological test which is requested

for thyphus suspected patient
is/are___
A. Widal
B. Welflex
C. RPR
D. all
18
Immunohematology
tests
is more commonly known as "blood banking

deals with the concepts and clinical techniques
related to modern transfusion therapy
an application of the principles of immunology
to the study of:
red cell antigens and
their corresponding antibodies on blood for
resolving the problems of blood transfusions
18
Blood Group Genetics

The
ABO phenotypes and their

corresponding genotypes
Phenotype
Genotype
AA, AO
BB,BO
AB
AB
OO
18
Genotype versus phenotype

Phenotype
Physical expression of inherited traits,

Determined by reacting red cells with known
antisera
Genotype
Actual genes inherited from each parent

Can only be inferred from the phenotype
Family studies are required to determine the
actual genotype .
18
Inheritance pattern of blood group

antigens
In
most cases blood group antigens are
inherited with co dominant expression.

The
product of each allele can be identified
when inherited as a co dominant trait

If one parent passed on an A gene the other
parent passed on a B gene, both the A and B
antigens would be expressed equally on the red
blood cells
18
Recessive or dominant inheritance patterns
recessive
inheritance would require that the same alleles from
both parents be inherited to demonstrate the trait
Dominant
expression would require only one form of the allele
to express the trait.
A
and B genes are dominant
gene is recessive, since it is expressed only when
both parents contribute the O allele.

18
ABO inheritance: Punnet squares
Two group A parents can have a group O

child
The parents of an AB child can be A, B or
AB, but not group O
A
AA
AO
AO
OO
18
Chromosomal
assignment
Chromosomal
assignment of genes of blood group system
Blood group system

chromosome
Rh---------------------------------------------------------1
Duffy------------------------------------------------------1
Gerbich--------------------------------------------------2
MNS------------------------------------------------------4
Kell--------------------------------------------------------7
ABO-------------------------------------------------------9
Kidd-------------------------------------------------------18
Lewis-----------------------------------------------------19
Landsteiner-Wiener-----------------------------------19
Lutheran-------------------------------------------------19
Hh---------------------------------------------------------19
P-----------------------------------------------------------22
18
Blood cell antigens

Red blood cell antigens
A
unique set of red blood cell Ag is
determined through genetic

inheritance.
These
antigens project from the
surface of the RBC in three

dimensional configurations
As a result, they are accessible to Ab
18
In
biochemical terms these antigens
may take the form of:

proteins,
Glycoprotein,
Glycolipids
Some
of the red blood cell antigens are more
immunogenic than the others

Example
The D antigen within the Rh group system
19
Human leukocyte antigens (HLA)

Is
possessed by nucleated cells such as
leukocytes and tissues

Can
readily provoke an immune response if
transferred in to an allergenic (genetically differ)

individual.
Encoded
by genes which are parts of Major
Histocompatibility Complex (MHC) gene system.
19
Human Leukocyte antigens

The
MHC system is important in the:
recognition of non self ,

coordination of cellular and humoral
immunity , and
graft rejection
19
Blood group Abs & their

stimulation
Blood group antibodies are classified into:
Natural and
Immune antibodies
Natural / non red cell immune Abs
Are RBC Abs in the serum of an individual that

are not provoked by previous RBC sensitization
Do
not react above the body temperature

19
They
are high molecular weight
cannot cross the placenta

They
are mainly IgM type
Exhibit
optimum in vitro agglutination in
saline media so we call them;

complete antibodies.
Optimum
reaction at room temperature or
lower
cold agglutinins
19
Immune antibodies
Produced
due to previous antigenic
stimulation either by transfusion or pregnancy
Characteristics
Mainly IgG
Optimally
react at 370C
warm agglutinins.
Causes
Can
more serious transfusion reactions
cross the placental barrier
Detected
after 3-6months of birth

19
Frequency of ABO blood groups in different

population
A%
B%
AB%
O%
Asian
28
27
40
African
26
21
49
Nepalese
33
27
12
28
Ethiopian
31
23
40
19
In-vitro detection of blood group Ag

and Ab reaction
The blood group Anti-serum
A
substance used to determine a persons blood

type, by identifying antigens present on the red cell.
Is highly purified solution of antibody.
named on the basis of the antibody it contains
For Example:
Solution of Anti-B antibodies is called anti B
antiserum
Preparation
inoculating animals with an antigen or
collecting serum from humans who have been sensitized
with corresponding antigens
19
The
presence of In-vitro antigen and antibody

interaction can be detected by:
Hemolysis
Precipitation
Agglutination (Most commonly used)
Agglutination:
Visible
clumping of particulate Ags caused by

interaction with a specific Ab
Occurs in two stages:
Sensitization (physical attachment of Ab
molecules to Ags on the RBC membrane)
lattice formation (Is the establishment of
cross links between sensitized particles and
Abs resulting in clumping
19
19
Routine ABO phenotyping

Principle
When
an antigen is mixed with its

corresponding antibody under the right
conditions it causes agglutination or
haemolysis of the red cells.
The ABO phenotype is determined when the
red blood cells are directly tested for the
presence or absence of either A or B antigen.
Serum testing provides a control for red
blood cells testing
Clinical significance
For safe blood transfusion
Prevention of Hemolytic Disease of the Fetus
and Newborn (HDFN)
20
In vitro serologic reaction

ABO phenotyping has two components:
1. Forward grouping- test RBC for ABO
antigens
2. Reverse grouping- test serum for ABO
antibodies
Donor and recipient red blood cells must be
tested using anti- A and anti-B
Donor and recipient serum or plasma must be
tested for the expected ABO antibodiesusing
reagent A1 and B red blood cells.
Cord blood and samples from infants less than
4 months old should be tested by forward ABO
grouping .
20
The methods for grouping

1. Tube method
2. Emergency tile or slide grouping
3. Tile or slide grouping of blood donors
1.Tube grouping method

Specimens
-Patients serum
-Patients cells
Equipments and reagents
Anti- A serum and anti- B serum

Antigen A and antigen B ( Red cell suspension)
test tube
Centrifuge
wash bottles
normal saline
20
Method
1. Take 4 tubes and label them 1-4.
2. Place each of the following in its numbered tube.
Tube 1: 1 volume of anti- A sera.
1 volume of 3-5% suspension of
patients washed cells
Tube 2: 1 volume of anti- B serum
1 volume of 3-5% suspension of patients

washed cells.
Tube 3: 1 volume of patients serum.
1 volume of 3-5% suspension of washed
RBC (reagent RBC) containing
antigen A
20
Tube 4: 1 volume of patients serum.

1 volume of 3-5% suspension of washed
RBC (reagent RBC) containing antigen B
3. Mix the contents of each by gently taping the tube
with the finger and leave for five minutes at room temp.
4.
Centrifuge the tube for one minute at 1000 RPM for
one minute.
5.
Replace the tube in the rack in the same position as

before centrifugation, and read the result by gently
tapping
each
tube,
looking
for
agglutination
or
haemolysis
20
6.
Check the tube which shows no visible
agglutination by examining the contents

microscopically on a slide using a 10x objective.
7.
Decide what a blood group of a patient is and
record the result in the book provided and one

the patients form.
Controls
Anti- A and Anti- B should be controlled using
known A cells and B cells.
20
Table
2.2. ABO phenotyping Reaction
Phenotype
RBC reaction
Anti-A
Serum or plasma reaction
Anti-B
A-cells
B-cells
AB
+=
agglutination,
O=
no
20
The RH blood group system

D antigen
Is
the most immunogenic antigen in the Rh
system
More
than 80% of D-negative people receiving a
D-positive red blood cell transfusion produce an

antibody with anti- D specificity.
For
that reason ,a D-negative patient should
receive D- negative red blood cells.

20
Can
cause a severe hemolytic transfusion reaction in a
recipient
if
transfused
with
blood
possessing
the
offending antigen
Being
IgG, are capable of crossing the placenta and are
associated with hemolytic disease of the new born (HDN)
For safe blood transfusion
Prevention of Hemolytic Disease of the Fetus and
Newborn (HDFN)
20
Slide Test
Specimen
Washed
RBC(40-50%)
Equipments and reagents

Anti-
D serum
Microscopic slide
Microscope
Applicator stick
Albumin (Control)
20
Slide Test.
1.
Place a drop of anti-D on a labeled slide.
2.
Place a drop of Rh control (albumin or other

control medium) on another labeled slide.
3.
Add two drops of 40-50% Red cell

suspension to each slide.
4.
Mix the mixture on each slide using an

applicator stick, spreading the mixture
evenly over on most of the slide.
21
Interpretation of the test result

-Agglutination of red cells ----------Rh positive.
-No agglutination of red cells------Rh negative.
A smooth suspension of cell must be observed
in the control.
Note: Check negative reactions
microscopically.
21
THE ANTI-GLOBULIN (COOMB`S)

TEST
Is
a sensitive technique to detect incomplete

Abs(produced by previous exposure)
Abs that are sensitized but which fail to
agglutinate RBCs suspended in saline at room

temperature, mainly IgG
are agglutinated by the anti-IgG in antiglobulin
serum through the linking of the IgG molecules on

neighboring red cells.
21
Types
of AHG tests
the direct antiglobulin test (DAT)

the indirect antiglobulin test (IAT).
Direct antiglobulin test (DAT)
It is the detection of in vivo sensitized RBC.
Clinical significance:
Diagnosis
of autoimmune hemolytic anemia
Investigation
of drug sensitized RBCs
Investigation
of transfusion reactions
21
Principle
Washed red blood cells from the patient are
directly tested with the antiglobulin serum
Specimen
2-5% RBC suspension
Procedure
1.
Place one drop of a 2% to 5% saline
suspension of cells to be tested in a labeled
tube.
2.
Wash 3 or 4 times with saline. After last
wash decant completely. Add one or two
drops of antiglobulin serum and mix.
3.
Centrifuge: examine for agglutination with
an optical aid; record results.
21
Indirect antiglobulin test (IAT)
It is the detection of Ab that may cause RBC

sensitization invitro.
Clinical Significance:
Detection
and identification of unexpected
Antibodies
Cross
matching
Detecting
RBC Antigens not demonstrable by
other techniques
21
Principle
The
Antibody-containing serum is incubated with specific
RBCs which, following washing, are reacted with antiglobulin

serum to see whether RBC sensitization has occurred.
Equipments and Reagents
Test
tubes
Centrifuge
Microscope
Microscopic
slides
AHG
Physiological
saline
Anti-D
21
Procedure
1.
Place two to six drops of the serum under test (patients serum) in a test
tube.
2.
Add one drop of washed 5% suspension of the test cells (donors RBC,
screening RBC, etc) optional: Two drops of 20-22% bovine albumin may be
added to the mixture
3.
Mix well
4.
Incubate at 370C for 15 to 30 minutes.
5.
Centrifuge immediately on removal from the incubator for 15 seconds at

3400 rpm: examine for hemolysis and agglutination using an optical aid,
and record results
6.
Wash 3 or 4 times in large amounts of saline.
7.
Add one or two drops of antiglobulin reagent.
8.
Mix well.
21
9.
Centrifuge at 3400 rpm for 15 sec.
10.
Examine for agglutination using an optical

aid.
11.
Record results
12.
Add one drop of known sensitized red cells to

all negative test. Centrifuge at 3400 rpm for
15 sec: examine for agglutination. If no
agglutination is seen, the test result is invalid
and must be repeated.
21
THE CROSS-MATCH (COMPATIBILITY TESTING)

It
is a procedure performed before transfusion
to select donors blood that will not cause any

adverse reaction, (hemolysis /agglutination)
It
helps the patient to receive maximum
benefit from the transfusion of red cells

This is done by ensuring compatibility
21
Principle
Serum
of the recipient / donor is tested against
the red cells of the donor/ recipient under

different conditions in order to establish their
compatibility
It
Will:
prevent immunization of the patient

guarantee normal survival of transfused
erythrocytes
detect all unexpected antibodies in a patients
serum
22
Types of Cross Match

Two types
Major cross-match:
involves
mixing recipients serum with the
donors red cells

is
much more critical for assuring safe
transfusion than the minor compatibility test

called
major b/c the Abs in the recipients serum
are most likely to destroy the donors RBC
22
Minor cross match:

Involves
mixing the donors serum with
patients red cells

Called
minor because
any Ab in the donors serum will be diluted

by the large volume of the recipients blood
the destructed RBCs of the patient may be
compensated by the transfused RBC of the
donors
22
Choice of Blood for cross-match

The
blood selected for cross-match should be of the
same ABO and Rh (D) group as that of the recipient

However,
Rh positive recipients may receive either
Rh positive or Rh negative blood

Whenever
possible blood of the patients own blood
group should be given

Otherwise
the following rules should be applied
Group A patient.
- Should receive group A blood, if not available group O
Group B patient
- Should receive group B blood, if not available group O
22
Group O patient
Can only receive group O blood
Group AB patient
Should receive from group AB, if not possible
can receive blood from group A,B, and O
22
THE DONATION OF BLOOD

Selection of blood donors
Criteria:
Age
Between 17 65 years
Hemoglobin
Females
Males
In
-not less than 12.5 g/dl (PCV 38 %)
-not less than 13.5 g/dl (PCV 41%)
both sexes Hgb above 19g% (Hct above
57%) are not acceptable

22
Pulse, Blood pressure and

Temperature
Pulse - between 60 100 per minute
B.P
Systolic between 90 and 180 mmHg
Diastolic between 50 and 100 mmHg
Temperature
should not exceed 370C
22
Weight
A
person
between 45-50 kgs can donate 350 ml of

blood
above 50 kg can donate 450ml of blood
Donors
with unexplained weight loss of a
significant degree are not acceptable to

donate
22
Pregnancy
Are
excluded from donating for 1 year after
the conclusion of their pregnancy

Medication
Deferral
of donors depends on the nature of
the disease for which the drug was ordered

Surgery
If
the surgery is minor (such as tooth
extraction) a donor is excluded until healing is

complete and full activity has been resumed
22
Illness
Prospective donors with:
disease of the heart, liver, lungs, or
history of cancer, or
bleeding problems should be excluded
Donors
who have had leukemia must never be
accepted
Donors
with a previous history of tuberculosis
are acceptable after completion of therapy

and if the disease is no longer active
22
Infectious diseases
A
donor must be free from transfusion
transmissible infections
Donors having had close contact with an individual
with viral hepatitis must be deferred for 1 year
Donors with history of malaria, previously resident in
an endemic area, should be deferred for 3 years after
becoming symptomatic or after leaving the endemic
area
Persons at high risk for acquiring or transmitting AIDS
should not donate blood
23
Selection
Previous donation
An
interval of at least
four months for men and

six months for women, is required before
the next donation
23
Vaccinations
Persons recently immunized with
toxoids and
killed viral, bacterial and rickettsial vaccines are

acceptable, if they are symptom free and not febrile
A donor who has received an attenuated live virus

vaccine such as mumps or yellow fever is deferred for
2 weeks after the last immunization
If rabies vaccination has been given following a bite

by a rabid animal, the donor must be deferred for 1
year after the bite
23
Transfusion reactions
Also
Are
called adverse reactions to transfusions
any unfavorable responses by a patient
following transfusion of blood, blood

components or derivatives
Example:
Facial flushing
Chest/back pain
Chills
Fever
23
Types of Transfusion Reaction

A .Hemolytic reactions:
-
abnormal destruction of RBCs of either the donor or

recipient
B. Non hemolytic reactions
-not usually associated with RBC hemolysis

a. Febrile reactions (pyogenic reactions)
b. Allergic reactions
c. Bacteriogenic reactions
d .Circulatory overload
23
Types of Transfusion..
Cause of haemolytic transfusion

reactions (HTR)
incompatible blood
blood under great pressure
transfusion of hemolyzed ,overheated or

frozen blood
HTR can be
1. immediate or
2. delayed
23
1. Immediate Type
hemolysis
evidence is obtained during or
immediately after blood is infused due to

destruction of donor cells by the recipients
Abs
Two
types:
a. intravascular
b. extravascular
23
a. IntravascularIt is due to:
ABO incompatibilities due to clinical errors

(Usually)
Antibodies to other blood group antigen systems
may also be involved in immediate HTR (Rarely)
b. Extravascular
It is due to:
-Coating of red cell antigen by incompatible Abs
which
results in the removal of the red cells in liver &
spleen
-Any IgG, non agglutinating, non-complement
binding
form of antibody
23
2. Delayed HTR
Are
not so dramatic and are usually
noticed a week or two after a transfusion

Abs
to Ags other than ABO ( Rh, Kell,
Duffy, Kidd) Ag systems are commonly

responsible)
They
are subtle in their manifestations
and present
Only as a mild jaundiceyitayewb2011@gmail.com
23
2.Non-Hemolytic transfusion reactions

Characteristics of non hemolytic reactions:
shortened post transfusion survival of
RBCs
febrile reactions
allergic response
23
a. Febrile reactions (pyogenic reactions)

The patient develops fever and chills during or
after transfusion
The rise in body temperature may be due to
leukocyte Abs , platlet Abs or Pyrogens
Prevented by giving leukocyte and platlet poor
RBCs
24
b. Allergic reactions
Most common type is urticaria (an itchy
rash)
Characterized by flushing of the skin
Commonly caused by the interaction b/n
transfused IgA and class specific anti-IgA in
the recipients plasma
Controlled by giving anti-histamines
24
c. Bacteriogenic reactions
Caused
by bacteria that may contaminate
solutions or equipments before sterilization

Transfusion of bacterially contaminated
blood components
unusual color or
the presence of hemolysis, both of which
may suggest bacterial contamination
24
Signs of bacteriogenic
reactions
Rapid onset of chills & fever

Vomiting
Diarrhea
Shock
24
Microbiological Tests
A. Gram stain
Most
bacteria are differentiated by their gram
reaction due to difference on their cell wall

structure.
Gram-
positive bacteria are bacteria that stain
purple with crystal violet after decolorizing with

acetone alcohol.
Gram
- negative bacteria are bacteria that stain
pink with the counter stain (safrainin) after losing

the primary stain (crystal violet) when treated
with acetone alcohol.
24
Micr
Procedure
1. Make the smear

2. Allow to dry
3. Rapidly pass the slide (upper) three times through the flame
4. Cover the fixed smear- with crystal violet for 1 minute and wash
5. Add Gram's Iodine for 1 minute and wash
6. Add acetone alcohol until it decolorized and wash
7 Cover the smear with safranin for 1 minute
8. Wash and dry
Result
Gram - positive bacteria . . . . . . Purple /dark red/

Gram - negative bacteria . . . . . pink
24
Micr
B. ziehl- Neelson staining Method

Ziehl
Neelson stain (Acid fast stain) in used
stain mycobacterium and other acid fast

organisms which cannot be stained with gram
stain.
Principle-
once the mycobacterium is stained
the primary stain it can not be decolorized

with acid. So named as Acid fast bacteria
24
Micr
Procedure for zielhl- Neelson staining method
1.Prepare
the smear from the specimen and fix it by passing
through the flame

2.Place
fixed slide on a staining rack and cover each slide with
concentrated carbol fuchsin

3.Heat
the slide from underneath with sprit lamp until vapour
rises(don't boil it) and wait for 3-5min

4.Washed
5.Cover
off stain with clean water
the smear with 3% acid alcohol soln until all color is
removed (2 minutes)
6.Wash
off the stain with clean water and cover the slide with 1%
methylene blue for one minute.

7.Wash
Result
and dry and Examine with microscope.

Acid fast bacilli . . . . Red
Back ground . . . . Blue
24
Micr
CULTURE
Culture
Media -It is the media containing the
required nutrient for bacterial growth

Use
of culture media
1-for isolation and identification of

microorganism
2-to perform anti microbial sensitivity test
24
Micr
Type of culture media
1-Basic /simple/media: It is a media that support
the growth of micro organism that do not
require special nutrient
Eg. Nutrient broth and Nutrient agar
2-Enriched media :Media that are enriched with
whole blood ,serum ,special extract or vitamins
to support growth of pathogenic bacteria
Eg. Blood Agar and Chocolate agar
24
Micr
3- Enrichment media
Fluid
media that increase number of pathogen by
containing substance that discourage the multiplication

of unwanted bacteria
Eg. Selenite F broth media
4- Selective media
Media which contain substance (antibiotics) that prevent

or slow down the growth of bacteria other than pathogen
from which the media are intended
Eg Modified thayer martin agar and Macconky
agar
25
Micr
5- Differential (Indicator) media
Media
to which indicator substance are added
to differentiate bacteria
Eg. Macconky agar and TIBS agar
(Thiosulphate Citrate
Bile salt Sucrose)
6-Transport media:-Media containing ingredients

to prevent the over growth of commensal and
ensure the survival of pathogenic bacteria,
when specimen can not be cultured soon after
collection
25
URINALYSIS
Urine
analysis is the way of looking for :
Color
Composition
Concentration
25
The Composition of Urine

Urine
A fluid extracted by the kidneys, pass through

the
ureters,
stored
in
the
bladder,
and
discharge through the urethra
in
the
presence
of
disease
conditions,
depending on the abnormality, the urine will

have abnormal constituents
Freshly voided urine from healthy individuals
is clear and pale yellow, It is slightly acidic (pH

5.0 to 6.0)
25
Normal Composition of Urine
Water- 95%
Ions:
sodium
potassium
sulfate
phosphate
Nitrogenous waste:
urea
uric acid
Creatinine
25
Abnormal constituents include ;

-keton bodies
- protein
-bacteria
-blood
-glucose
- pus
-certain crystals
-casts
-nitrite
25
Physical examination of urine

The
first part of routine urinalysis
Simple and It provides hint for the
subsequent chemical and microscopic

urinalysis
It includes
measurement of urine volume
Color
Foam
Measuring the specific gravity
25
Volume
Reported
The
as ml per 24hr
24-hour urine voided by a healthy
adult range 600-2000ml

Children (6 to 12 years) about 1000ml
infants about 600ml
Factors
that affect the urine output
fluid intake
Diet
physiological and environmental factors of the
body
25
Anuria :urine
formation<100ml per 24 hours
Complete urinary tract obstruction

Hemolytic transfusion reaction , Acute renal
failure, Acute glomerulonephritis
Polyuria:
urine volume is greater than normal
>2000ml per 24 hours for prolonged period

Diabetic mellitus
Tubular necrosis [improper function of urine
tubules]
Intravenous fluid intake
25
Oliguria
:urine volume is less normal
<400ml per 24 hours for prolonged period

Dehydration or poor blood supply to the
kidneys
Mechanical obstruction of the urinary
system [e.g. due to renal calculi or tumors]
Excessive salt intake
Diuresis:
temporal increment of urine due
to excessive fluid intake

25
color
Urine color is recorded within 30 minute after collection. Normal color ranges
from deep yellow to light yellow

Colorless urine may indicate
Large fluid intake
Diabetic mellitus
Alcohol consumption
Dark yellow or brown red urine may indicate
Concentrated urine
Decreased fluid intake
Fever
Dehydration
Yellow brown or beer brown color may indicate the presence of bilirubin
Clear red color may indicate presence of hemoglobin
Cloudy red or smoky red color indicates hematuria
26
Foam
Normally
fresh urine produces small amount of white foam,
But during certain abnormal conditions, it may be changed

Yellowish foam indicates the presence of bilirubin in the urine
large amount of white foam indicate high concentrations of
protein
Transparency
Freshly
voided urine specimen is normally clear and transparent
Occasionally
turbidity of urine may result from
White blood cells [pus cells]

Kidney stones, Yeast cells
High number of bacteria cells
26
pH
pH is the unit that describes the acidity or

alkalinity of a solution
It can be measured using litmus paper or

reagent strip
Alkalinity
of freshly voided urine, indicate a
urinary tract infection

Persistently
acidic urine in diabetic acidosis
resulting from an accumulation of ketone

bodies
26
Persistent alkaline urine(>6)
may be caused by:

UTI
Renal failure
Vomiting
Alkalizing drugs such as streptomycin,
kanamycin etc
26
Persistent acid urine (pH < 6)

Malabsorption syndromes
Diabetic ketoacidosis
Dehydration
Starvation
And also certain drugs such as Phenacetic
26
Chemical examination of urine
Determination of Glucose
Presence
of detectable amount of glucose in
urine is Glycosuria
it
could be physiological(high carbohydrate
diet, pregnancy)
Pathological Glycosuria
A. Diabetes mellitus
B.
Glycosuria due to other endocrine
disorders
26
Measured
When
by urine dipstick method
the test strip is
dipped in urine the

reagents are activated
and
chemical
reaction occurs
The
chemical reaction
results in a specific
color change
26
Positive
Glucose
(Normal for comparison)

26
Ketones
are
a group of three related substances
Acetone
acetoacetate (acetoacetic acid) and
-hydroxybutyrate ( -hydroxybutyric
acid)
Formed
in liver from aceto acitate (oxidation
product of amino acid, fatty acid)

26
Positive
urine Ketones
(Normal for comparison)

26
cases :
starvation and diabetes mellitus
during prolonged vomiting
severe diarrhea
high fat intake and low carbohydrate diet
For
diagnosis of the severity of
diabetes
27
Determination of Urinary Protein

Test
for urinary protein is one of the most
important and valuable parts of the routine

urinalysis
Albumin
is one of the important proteins,
which appears in urine during a pathological

condition
27
Causes of Proteinuria
1. Increased permeability of the glomerulus
2. A decrease in normal reabsorption in the
tubules
Types of Proteinuria
classified according to the relationship of

its etiology to the kidney and also the
mechanism involved
Functional Proteinuria
Systemic disease or renal pathology
Proteinuria
27
A.
Functional Proteinuria: not associated with renal

damage
1. Accidental or false proteinuria:
occurs when mixing of urine with a proteinous fluid

such as pus, blood or vaginal discharge
2. Physiological proteinuria:
It is usually less than 0.5 gm/24 hr.

associated with exposure to heat or cold, emotional
stress, and later stage of pregnancy
3. Postural (orthostatic) proteinuria:
associated only with the upright position or sitting for

a long time
27
B. Systemic disease or renal pathology Proteinuria.
1. Pre renal protein urea: not due to primary

renal disease
Variety of toxic condition
Renal hypoxia
Hypertension
2.Renal proteinuria: primarily kidney disease
Glomerulonephritis
Nephritic syndrome
Destructive parenchymal lesions (tumor,
infection )
27
Determination of Hemoglobin
Hemoglobin appears in the urine when there is extensive or

rapid destruction of circulating RBCs
Reticuloendothelial system
cannot
metabolize or store the
excessive amounts of free hemoglobin

Normal
Can
range 1-1.4g/l
be measured by strip test
Principle of strip test

The
hemoglobin test spot of the reagent strip contains a
buffered mixture of organic peroxide and a chromogen

The
color of the chromogen changes in presence of hemoglobin
turns
blue in case of Hemoglobinuria, which is often associated
with hematuria
27
Hemoglobinuria
may occur with severe
intravascular hemolysis
hematuria-
a condition when intact red
blood cells are present in the urine

Hematuria is used to indicate bleeding
somewhere in the urinary tract
Usually both red blood cells and hemoglobin
mark this disorder
27
MICROSCOPIC URINALYSIS
Is direct visualization of urine sediments under

microscope
It
is more of quantitative test
One
can do microscopic examination to see
organized and non-organized sediments
27
Organized (Formed )
elements
RBCs/HPF
WBCs/HPF
Epithelial cells / LPF
Casts / LPF
Parasites/LPF
Bacteria / HPF
Yeast Cells /LPF

Spermatozoa
27
Non-organized
Triple phosphates
Amorphous phosphate
Calcium carbonate
Calcium phosphate
Calcium Oxalate crystals
Alkaline Urine Crystals
27
Organized Urinary Sediments

RED
BLOOD CELLS
Red blood cells are not usually present in
normal urine
Appearance:
Normally RBCs appear in the fresh sample
as intact, small and faint yellowish discs,
darker at the edges
28
When
the number of RBCs is found more than
their normal range, usually greater than 5

RBCs/HPF it may indicate:
Presence of disease conditions in the urinary tract, such
as:
Acute and chronic glomerulonephritis
Tumor that erode any part of the urinary tract
Renal stone
Cystitis
Trauma of the kidney
traumatic catheterization
28
Interfering factors
Factors
that may result falsely in high number
of RBCs, i.e. without the presence of actual

renal or other normal physiological
disturbances included:
Menstruation
Vaginal bleeding
Some
drugs:
Aspirin ingestion or over dose

Anticoagulant therapy over dose
28
LEUKOCYTES (WBCs)
Normal
range:
Appearance:
0-5WBC/HPF
normally, clear granular disc
shaped
White
blood cells
a few are normal

high numbers indicate inflammation or infection
some where along the urinary or genital tract
Reporting
of of WBCs
When 0-5 leukocytes / HPF are seen-- normal
5-10 leukocytes / HPF are seen-- few leukocytes / HPF
28
10-20 leukocytes/HPF are seen--->moderate leukocytes/ HPF

20-30 leukocytes /HPF are seen ----> many leukocytes / HPF
Above 30 leukocytes / HPF / are seen - full/field
Increased
number of leukocyte in urine are seen in case
of:
Urinary tract infection such as renal tuberculosis
All renal disease
Bladder tumor
Cystitis
28
EPITHELIAL CELLS
Epithelial
cells
cells are large and flat

normal cells that line the urinary and
genital tract or renal tubules
28
Presence
of epithelial cells in large number
may indicate:
Acute tubular damage
Acute glomerulonephritis
Silicate over dose
Note: The presence of large number of

epithelial cells with large number of
Leukocytes and mucus trades (filaments) may
indicate Urinary Tract Infections (UTI)
28
Reporting of epithelial
cells
Epithelial
cells distribution reported after looking
under 10x objective of the microscope

Usually
they are reported semi quantitatively by
saying
1-3 epithelial cells /LPF
2-4 epithelial / LPF
6-14 epithelial / LPF
15-25 epithelial/ LPF
Full of epithelial cells / LPF when the whole field

of 10 x objective covered by epithelial cells
28
Casts
Casts
are long cylindrical structures that result

from the solidification of material within the
lumen of the kidney tubules
Formed
by precipitation of proteins, and

aggregation of cells within the renal tubules
Most of them dissociate in alkaline urine, and

diluted urine even in the presence of Proteinuria
Pathological
Conditions that favors for the

creation of casts include
The presence of protein constituents in the
tubular urine
Increase acidification
28
Most urinary casts are formed either in the

distal convoluted tubules or in the
collecting ducts, because urine more
concentrated and maximally acidified here
Casts formed in the collecting tubules tends
to be very broad, and usually indicates the
significant reduction in the functional capacity
of the nephron and indicate severe renal
damage
Major casts are:
hyaline
epithelial
white blood cell, and. red blood cell casts
granular (coarse and fine), waxes, Fatty
28
Hyaline Casts
All
hyaline cast have a precipitated protein
matrix, so there has to be renal

Proteinuria for these to be formed
29
Presence
of large number of hyaline casts may show
possible damage of glomerular capillary

membrane
This
damage permits leakage of protein through
glomerulus and result in precipitate and gel

formation (i.e. hyaline casts) in the tubule. Thus
this may indicate:
Nephritis
Chronic renal disease
Congenital heart failure
29
Cellular Casts
As
the protein concentrates in the distal
tubule & becomes stickier, cells can become

entrapped
These
become Hyaline Casts with Inclusions
& while the formal name would be for

example
Hyaline-WBC
Cast,
they
are
frequently simply referred to as WBC Cast
29
White blood cell casts

formed by aggregates of white blood cells that
trapped in protein matrix in the renal tubular
lumen
An excess of white blood cells, singly or in
clamps, in the urine may indicate inflammation
white blood cell casts definitely are renal origin
They characteristically seen in acute
pyelonephrities and occasionally in
glomerulonephirites
29
Red blood cell casts

- Usually, they found in hematuria. Red blood
cell casts are usually diagnostic of glomerular
diseases
- Normal range: normally not seen in normal
individual
- Appearance
- Formed usually after accumulation of
cellular element
in the renal tubules
29
Epithelial Casts
Epithelial Casts are composed largely of tubular
epithelial cell desquamated within the tubule
Inflammation
of the kidney may cause greater
sloughing of renal epithelial cells, so large

number of epithelial casts is indicative of renal
parenchymal disease with tubular damage
29
Reporting of casts
Casts
are examined under 10x objective of
the microscope
Casts
are reported quantitatively by saying:
Few casts / LPF

Moderate casts / LPF and
Many casts / LPF
During
the report the, type of cast that is
seen should also be mentioned

29
PARASITES
Parasites
that can be seen in urine microscopy
are:
Trichomonas
vaginalis
Schistosoma
haematobium
* Other parasites such as Entrobious

vermicularies also may occur due to
contamination of the urine with stool
29
YEAST CELL
Yeast
cells are fungi that are not normally

seen in health individuals
They are usually of Candida species
(Candida Albicans) and are common in
patients with
Urinary tract infection

Vaginites
Diabetic mellitus
Intensive antibiotic or immunosuppressive
therapy
29
BACTERIA
Bacteria
are commonly found in urine
specimen because of abundant normal

microbial flora of the vagina
Most common cause of UTI
cell
can be identified by bacteriological
culture
Presence
of bacteria may indicate the
presence of UTI or contamination by genital or

intestinal microflora
common
bacteria???
29
THANKS !
!!
30

Basic Clinical Tests Guide

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Basic Clinical Tests Guide

Uploaded by

Copyright:

Available Formats

Basic Clinical Chemistry Tests

Chemical analysis of body fluids

LIVER FUNCTION TEST

Liver is the 2nd largest

The heaviest gland

Located just below the diaphragm

Site of intermediary metabolism and

If liver is diseased it loses partially or totally

Lipid and proteins)

of bile salt (800-100ml of bile/day)

disorder of unknown cause

involves children's /some adults/

after the recovery from viral infection

liver function tests

Bilirubin /total and direct/

Comes from the degradation of Hgb as well as a

The human erythrocytes have life span of

erythrocytes is hydrolyzed in the reteculo

dissociate to iron and porphrin

bound with albumin go to the liver

up by hepatocytes(albumin detached from

non albumin proteins (Y and Z) bind and

conjugation takes place

the ER of hepatocyte the enzyme Uridine

transfers glucuronic acid molecules to bilirubin

Secreted in to bile canaliculi

Bacterial enzyme act on bilirubin

enters the high pH of

returns to the liver

All re-excreted into

bile except 2-5% via

Most (85%) UBG is

symptom of yellow staining of

connective tissue from excess

- sclera of the eyes

describes the dark

yellow-brown color of serum

is commonly caused by hemolytic of red blood

cell like hemolytic anemia and acute malaria

increased destruction of red cell result in large

load of bilirubin- albumin complex to the liver than

As a result of this the predominating plasma

It is a type of jaundice associated with liver

The liver has some problem and cant metabolize

In this case liver is unable to conjugate

Post hepatic jaundice

This type of jaundice is mainly due to

Since conjugated bilirubin is normally excreted

In post hepatic jaundice the predominating

Laboratory Diagnosis Of Jaundice

Icterous Index method

2. Chemical Methods (for direct & indirect bilirubin)

Icterous Index method

yelowish color of serum is mainly due to

intensity of the serum is directly read on a

spectrophotometer compared with a standard

:Bilirubin present in the sample reacts with diazotized sulfanilic

acid in the presence of caffein benzoit forming a purple colored

reaction is stopped by addition of acetic acid which destroys the

purple colour azobilirubin converted to a blue color azobilirubin by

the addition of alkaline tartarate solution

intensity of the blue color is directly proportional to the amount of

total bilirubin present in the sample .The absorbance read at 620nm