LOG BOOK OF

LABORATORY
JOOD ALASQAH
CHAPTER 01

Sample
Collection
PRE-ANALYTICAL
 Types of Samples
Blood samples: whole
blood, plasma, and
serum
Phlebotomy Urine sample: 24
hours urine, and
Blood Sample routine urine
Collection Procedure Stool Sample
Sputum
Swabs
01 Identify patient

Wash hands and

02 sterilize them,
and wear gloves

Patient
What test
03 preparation by
fasting
needs fasting?
Assemble
04
equipment
Fasting blood glucose,
need 8 hours.
Lipid profile which is
05 Position patient
recommended to fast
for 12 hours.
06 Apply tourniquet Order of
Draw
Select vein site
07 median cubital vein
Varies
is the best, if not found Blood Cultures
basilic or cephalic vein # of inversions 8 - 10
Light Blue
Clean site with Sodium Citrate
08
Alcohol # of inversions 3-4
Red
Clot Activator
09 Venipuncture # of inversions 4-5

Gold
SST (Serum Sperator Tube)
Fill the tubes in # of inversions 4-5
10 the right order Light Green
mix well with the Lithium Heparin
additives # of inversions 8 - 10

Dark Green
11 Remove tourniquet Sodium Heparin
with the last tube filled # of inversions 8 - 10

Lavender
EDTA
12 Place gauze # of inversions 8 - 10

Gray
Sodium Fluoride/Potassium
Oxalate
# of inversions 8 - 10
13 Remove needle

14 Dispose the device NOTE: If there is no blood culture, SST

tube (with no additives) collected
before sodium citrate tube for more
accurate result in coagulation
studies
 Apply pressure
15 and bandage the
arm

16 Label Tubes

Venipuncture
Equipment
Gauze
Sponge
Gloves Alcohol
Wipes

Sharps
Needles Container Syringes
Bandages

Collection Tourniquet Requisition

Tubes Form
 Sample Handling
and Transporting
m ple s must be
All sa s in fectious
re d a
conside autions
by takin
and wea
g p
ring
re c
g lo v es while
pecial
Possible Errors
handlin
tests mo
g.
s
s
t
o
b
m
e
e
p
s
ut in ice during sample
immedia
te
monia
ly , l ik e ACTH
collection and their
and Am
effect to test
results
01
Contamination
It is caused by proceeding the wrong
order of draw, like doing EDTA tube
draw before heparin tube draw.
It will cause hypocalcaemia and
hyperkalaemia.

02
Hemolysis
caused by incorrect needle size,
improper tube mixing, incorrect filling of
tubes, excessive suction, prolonged
tourniquet, or may be by patient having
disease causes his red cells to breakdown
like hemolytic anemia.
 Effect of hemolysis on blood test
Increased: potassium, lactate dehydrogenase,
SGOT/AST, SGPT/ALT, creatine kinase, iron, phosphate,
total protein, albumin, calcium, alkaline phosphatase
Decreased: troponin T, haptoglobin, bilirubin,
amylase, bicarbonate

03
Lipemia
caused by inadequate time of blood
sampling after the meal. It can corrected
by ultracentrifugation.

04
Effect of lipemia on
blood test Clotted Sample
Increased: bile acids, caused by improper or delayed tube
direct bilirubin, TIBC, mixing. it can have effect on Prolong
magnesium
Decreased: sodium,
in coagulation study, and cause low
potassium, chloride, platelets.
bicarbonate, lactate
dehydrogenase

NOTE: icteric can happen if the patient has high level of bilirubin
CHAPTER 02

Blood
Bank
 What is Blood bank?
It is the place where blood is
collected from donors , typed ,
separated into components
,stored ,and prepared for
transfusion to recipients .

Types of Samples
EDTA blood sample
(RBC/plasma)
Donor's blood bag used for blood grouping
,phenotyping ,cross matching
and antibody screen and
identification

Rejecting Criteria for Rejecting Criteria for

EDTA blood sample Blood bags
Wrong tube Hemolyzed unit
Wrong label Clotted sample Lipemic unit
Incorrect or incomplete High/low volume
request Broken unit
Clotted sample Incorrect or incomplete
Hemolyzed sample information
Lipemic sample
 Donation area
One unit can produce:
Packed RBCs
Fresh frozen plasma (FFP)
Cryoprecipitate (CRYO)
Platelet concentrate

Can be donated once

every 90 days

Whole blood donation

SAGM : Saline-Adenine-
Shelf-life : 35 days ( whole Glucose-Mannitol
blood with CPDA), 42 days CPDA : Citrate-
for Packed RBCs with Phosphate-Dextrose-
SAGM Adenine

Volume required :
450-500 ml collected
in 10-15 minutes

it is an automated donation
when donor can only donate
one of the blood component
( mostly platelets )

Can donate platelets

Apheresis once every 14
days and plasma every 28
days

Shelf-life
platelets - 5 days
plasma - 1 year
Criteria of acceptance and rejection of the donor

Age: 18-65 years

Weight: above 50 kg
Temperature
37 degrees Celsius Blood pressure
systolic
(maximum 180 – minimum 90)
Hemoglobin
diastolic
for men 14-17 g/dl
(maximum 100 – minimum 50)
for women 12.5-14 g/dl
platelets count
Pulse ratio at least 150,000 (for
50-100 per minute platelets apheresis)
Food
Sleep
donor most take a meal
at least 6 hours
during the last 3 hours.
before donation
Surgery or hijamah or
tattooing Vaccination
donation is acceptable after 2 weeks for Measles (rubeola),
only after one year of mumps, polio, typhoid, yellow fever
the surgery or hijamah vaccines , 4 weeks for Rubella, chicken
or tattooing pox (varicella-zoster) vaccine
Haemoglobinopathies
only trait can accepted
( in sickle cell History of recent
trait , whole blood only infection
accepted ) defer for 14 days
after recovery

Pregnancy , menstruation and

lactation
can't donate (up to 6
months after delivery for
pregnant)

The period time between each donation

Whole blood donation: every 90 days (12 weeks)

Platelet donation: once in a 14-days period
(2 weeks)
Plasma donation: every 28 days
Red cell donation in conjunction with another
component: every 56 days (8 weeks)
 Blood bags separation

Whole
blood

Test for :
HIV
HTLV
Hepatitis C
Hepatitis B
Malaria
Syphilis Packed Platelet
ABO + RhD RBCS rich
plasma

35 days
at 4

Platelet
concentrate FFP

5 days 1 year
at 22 at -30

ABO Blood grouping and Rhesus factor (RH)

To detect blood group it can

Red blood cells done by slide method, tube
have many method or by gel card .
antigens (mostly
p
their surface . roteins) in
clinically importantthe most
are ABO and RH. antigens
Method of detection can be forward (To
antigens A and B a detect antigen using patient's packed red
important becaus re the most cell and anti-A,anti-B and anti-D reagent )
e
of theses antigens presence or reverse (to detect antibody using
if the blood group will decide patient's plasma and A1 and B cell
O, the most signA, B, AB, or reagnet)
antigen is the D ant ificant Rh
igen.
 Group A Group B Group AB Group O Rh + Rh -

Red Blood
cell type

Antibodies None None

in plasma Anti-A and
Anti-B Anti-A Anti-B Anti-D

Antigens
in red None None
A and B
blood cells A antigen B antigen antigens Antigen D

Slide
method

Tube
method

Gel card
A+

Agglutination interpretation

4+ 3+ 2+ 1+ 0 Mixed
field
 Blood Grouping

Newborn Blood Grouping

Only a forward
ABO typing is
performed

DCT will run in

the same card
with ABO
typing

Discrepancies
Blood group Discrepancies

Typing discrepancies exist when:

The forward and reverse reactions do not match,
There are unexpected/weak reactions,
And/or the current results do not match those
obtained on a previous specimen from the same donor
unit or patient specimen.
Serologic Typing Discrepancies
 Du test method

It is method to detect weak D antigen , which is the normal

D antigen but with weak expression so in routine blood
grouping it will show as Rh negative while it is not. This
method done by using tube or gel card.
Because of the weak reaction of weak D antigen we need
to incubate red blood cells with anti-human globulin
(AHG) and anti-D at 37 C . if D antigen is present, anti-D
will sensitize the cells and agglutination occur and patient
will report as Rh positive ,

Rh phenotyping

There are 5 principle Rh antigens

that may be found in most
individuals, C,c,E,e,D and d .
known donor and patient
phenotype will help to reduce
transfusion reactions ccee K-

D>c>E>C>e
D is highly immunogenic and e rarely immunogenic .

To detect the phenotyping we will use gel card .

because of its clinical significant,kell antigen will be
test with Rh phenotype.

Coombs test
Direct coombs test (DCT)
To detect patient red blood cells that already
Coombs test is used to detect the sensitized with antibody or complement in their
presence of antibody IgG or surface . DCT is used to assist in the diagnosis of
hemolytic disease of the newborn (HDN) and
complement that already attach autoimmune hemolytic anemia.
(sensitized) to red blood cell antigen or
can attach to it and cause hemolysis.
There is 2 types of this test , direct and
indirect Indirect coombs test (ICT)
To detect unexpected antibodies in patient's
plasma that can bind to their antigen in donor
red blood cell and cause hemolysis . antibody
screen test use ICT principle .
 DCT

ICT

Interpretation of coombs test

DCT ICT

+ - + -
There are There are no There are There are no
incomplete incomplete incomplete incomplete
antibodies (IgG antibodies (IgG antibodies (IgG antibodies (IgG
or C3d) attached or C3d) attached or C3d) present in or C3d) present in
to RBCS to RBCS patient's serum patient's serum

Antibody screening test Antibody identification test

It is done by using 3 cells and Antibody identification panel is

patient's plasma to detect done following a positive
unexpected antibodies that have screening test to
the ability to destroy donor's red cell identify unexpected antibodies in
after transfusion . Antibody the patient's plasma that cause
identification needed if antibody this positive result by using 11 O red
screening is positive cells
. .
 Cross matching

It is done b
transfusion as fina efore
to l step
determine the
compatibility between
donor and recipie
check ABO compant by
and tibility
detect clinically
significant antib
that missed in ICT. odies

No agglutination
Major cross match

Compatible
.

Agglutination
Donor's red cells Patient's plasma Not Compatible
. .

No agglutination
Minor cross match

Compatible
.

Agglutination
Patient's red cells Donor's plasma Not Compatible
. .
 Blood Issuance

1 Blood issuance slip

4 Observation
checklist paper
2 Blood issuance report
paper
Take a Segment
5 from Blood bag
Check The
3 information Loudly

Blood Units Receiving

Receiving Blood Units

Write the Receiving Date and Time
Check the Temperature of the log tag
Temperature Should be between 1-10 C
Check the bags for any clots or hemolysis
Check the certificate of screening Tests

Donor Blood Group Confirmation

Confirm Rh- by Du test (weak D)

Write Blood Units numbers in Tracking system or

Blood inventory
 Example of Machine used in
blood bank
ORTHO VISION® Swift Analyzer
for ID-Cards
Principle : designed for automated
ABO, RhD , Rh Phenotyping , detection
and identification of clinically relevant
antibodies, crossmatch, and DCT
using Gel Cards, utilizing
hemagglutination and gel filtration as
the principle of operation.

Quality Control

“Ready-to-use” human matrix

derived with known antigen or
antibody to run the Dialy
control material
CHAPTER 03

Hema-
tology
 What is Hematology?
It is the study of blood components
which includes: red blood cells,
white blood cells, and platelets in
order to diagnose the diseases
related to it.

Types of Samples
Whole Blood Sample
obtained from EDTA tube for
Body Fluids CBC, electrophoresis, G6PD
Plasma Sample
test, blood film, ESR and
obtained from sodium
malaria screen
citrate tube for
coagulation studies
EDTA tube
and special tests

Sodium
citrate tube
1:9

Rejecting Criteria
Wrong tube Hemolyzed sample
Underfilled or overfilled, especially Lipemic sample
with sodium citrate tube (if more or Clotted sample
less than 10% it is accepted) Incorrect or incomplete
Wrong label request
 Complete Blood Count
CBC
Parameter Normal Range

White Blood Cell Count

4.5 – 11.0 X 10 3/µL
WBC

Red Blood Cell Count Male: 4.5 – 5.5 X 10 6/µL

RBC Female: 4.0 – 5.0 X 10 6/µL

WBC differential count

includes: lymphocyte, Lymphocyte 20 - 40% of WBC
monocyte, Monocyte 2 - 8% of WBC
neutrophil,basophil and Neutrophil 40 - 60% of WBC
Basophil 0.5 - 1% of WBC
eosinophil
Eosinophil 1 - 4% of WBC
Hematocrit
Male: 42 - 52%
HCT
Female: 36 - 46%
which means The percentage of total
blood volume that consists of RBCs

Hemoglobin
Male: 14.0 – 17.4 g/dL
HGB
Female: 12.0 – 16.0 g/dL
total amount of the oxygen carrying
protein in the red blood cells

Mean Corpuscular Volume

MCV 80 – 100 fl
average size of red blood cell

Mean Corpuscular
Hemoglobin
28 - 34 pg
MCH
is the average amount of hemoglobin
(HGB) for each red blood cell
 CBC cont.
Parameter Normal Range

Mean Corpuscular
Hemoglobin Concentration
32 – 36 g/dL or %
MCHC
average concentration of hemoglobin
in each red blood cells

Red Cell Distribution Width

RDW 12.0 – 14.6%
variation in the size of RBCs

Platelet count
150 - 450 X 10 3/µL
PLT

Mean Platelet Volume

6.8 – 10.2 fl
MPV

Reticulocyte Count less than 2%

pre-mature red blood cell

Principles of CBC Analyzers

Electrical Impedance Method
A stream of cells in suspension passes
through a small aperture across which
an electrical current is applied. Each cell
pass will alters the electrical impedance
and can thus be counted and sized.
Particles such as blood cells are
nonconductive but these particles will
suspend in an electrically conductive
diluent. As a dilute cells suspension
drawn through the aperture, the passage
of each individual cell will increases the
impedance (resistance) of the electrical
path between two electrodes that are
located on each side of the aperture.
 Flow Cytometry
A laser beam passes through cells
suspension so that different optical
properties can be measured.

Fluorescent Detection
A cells labeled with fluorescence, and laser
beam will excite these molecule, then light will
emit at different wavelength and the amount of
fluorescence will indicate the percentage of cell.

Optical Light Scatter

A laser beam passes through each cell
flows, and cell will scatter the light in
different directions depend on its
granularity (side scatter) and its size
(forward scatter ).

Radio Frequency conductivity

measurements depend on cell interior
density (granules and nucleus)

Spectrophotometry
measurement of the reflection or
transmission properties of the light at
different wavelength
 Examples of Automated CBC
Analyzers
Sysmex ™ XN-1000 Automated
Hematology Analyzers
Principle: Fluorescent Flow Cytometry
(WBC Diff and IG, NRBC, RET),and
cyanide-free sodium lauryl sulphate
detection method ( absorption
photometric methods ) for HGB , DC XN-1000
SHEATH FLOW DETECTION METHOD or
electrical impedance for
platelets,HCT and red cells

XE-2100 Sysmex ™ XE-2100 Automated

Hematology Analyzers
Principle: Fluorescent Flow Cytometry
(for WBC diff ,NRBC,RET,optically
measured PLT) . RF/DC detection
method (RBC)

Abbott Alinity H-series

Principle: WBC Diff, retic, RBC –
Optical Scatter and Fluorescence
/Platelet – Optical Scatter for
platelets, HCT and red cells.

CELL-DYN RUBY Abbott

Principle: Multi-Angle Polarized
Scatter Separation (MAPSS™)
technology
 UniCel® DxH 800 Coulter®
Cellular Analysis System
Principle: Electrical impedance

Anemia Workup
Low HCT and HGB

Examination of CBC and peripheral blood smear MVC > 100

MVC < 80 MVC 80 – 100 Macrocytic Anemia

Microcytic Anemia Normocytic Anemia Megalocytes&

segmented neutrophils
on peripheral smear
Serum iron studies Reticulocyte count

Low iron and < 2% > 2%

ferritin (hypoproliferative) (hyperproliferative)
with high TIBC

Low/normal iron
and Leukemias Hemorrhage Absent:
Iron deficiency low/normal ferritin aplastic Hemolytic non-
anemia with low TIBC anemia anemias megaloblastic
Pure red cell
aplasia
Other marrow Alchohol abuse
Mentzer index failure syndromes Myelodysplastic
(MCV/RBC<13) suggests a syndrome
Thalassemia component Liver disease
of anemia of Present: Congential bone
chronic disease megaloblastic marrow failure
with iron syndromes
deficiency
anemia
Vitamin B12
and/or
Folate deficiency
Drug-induced
 Peripheral Blood Smear
Done to study the Thin Smear preparation
morphology of blood
that help in diagnos cells
various primary andis of Slide most be rapidly air dried by
secondary blood a waving the slide or using a fan, then
nd slide should be labeled with patient
blood related disea
Smear has 2 types, ses. name and ID. After that put it in
th
and thick. Thin bloo in fixation (methanol) for 5-10 minutes
then stain it after it has dried with
smear is mostly usedd
counter stain (giemsa, leishman and
study cell morpholo to wright stain )
gy

Blood cells morphology

 RBCs Abnormalities
Abnormal RBC morphology and
their most associated diseases

Spherocyte
hereditary spherocytosis
Stomatocyte
Microcytic RBC hereditary stomatocytosis
iron deficiency anemia
Target Cell RBC
Macrocytic RBC thalassemia
vitamin B12/folate
deficiency
Sickle Cell RBC
Spurr Cell RBC sickle cell disease
(Acanthocyte)
liver disease Teardrop
Blurr Cell RBC bone marrow fibrosis
(Echinocyte) Hemoglobin C Crystals
uremia & liver disease hemoglobin C disease
Schistocyte
microangiopathic hemolytic
anemia Red Cell Agglutinate
Bite Cell RBC multiple myeloma
G6PD deficiency
Elliptocyte Rouleaux
hereditary elliptocytosis multiple myeloma
 RBCs abnormalities

RBC SIZE & HG ABNORMALITY RBC SHAPE ABNORMALITY RBC INCLUSIONS

MICROCYTES HYPOCHROMIC
(SMALL IN SIZE) SEEN IN IRON
DEFICIENCY ANEMIA AND
THALASSEMIA.
MACROCYTES, (LARGE IN SIZE)
SEEN IN VIT B12 OR FOLIC ACID
DEFICIENCY.

WBCs abnormalities

QUANTITATIVE ABNORMALITIES QUALITATIVE ABNORMALITIES

LEUKOCYTOSIS – HIGH NUMBER PRESENCE OF IMMATURE CELLS

OF LEUKOCYTES ABNORMAL MORPHOLOGY
LEUKOPENIA –LOW NUMBER OF
LEUKOCYTES
 Platelets
abnormalities

QUANTITATIVE ABNORMALITIES QUALITATIVE ABNORMALITIES

THROMBOCYTOSIS –HIGH NUMBER PLATELET SATELLISM (PLATELETS

OF PLATELETS CLUMPED AROUND NEUTROPHILS)-
THROMBOCYTOPENIA –LOW CAUSED BY EDTA GIVES A FALSE LOW
NUMBER OF Platelets PLT COUNT
PLT CLUMPING - CAUSED BY EDTA
GIVES FALSE :
LOW PLT COUNT
GIANT PLATELETS
SMALL PLATELETS
GRAY PLATELETS (DEGRANULATED)

GIANT PLATELETS Clumps

GRAY PLATELETS
SATELLISM

CBC abnormal results

Low Platelets

Request Na citrate Low

Thrombocytopenia
tube
Normal
Platelets

Pseudothrombocytopenia
 MCHC > 37

Incubate sample At 37
High Hb Cold agglutinin
degree (In Waterbath) corrected

Not
Corrected

Plasma Exchange
with normal saline
Corrected Not
Corrected

Sample was Need Further

lipemic/Icteric with investigations
normal Hb

Cold agglutinin disease (CAD) is a

rare type of autoimmune hemolytic
anemia. Cold temperatures activate
your immune system to destroy red
blood cells>

Hematopoiesis
Multipotential hematopoietic
stem cell
(Hemocytoblast)

Common myeloid progenitor Common lymphoid progenitor

Erythrocyte Mast cell Small lymphocyte

Natural killer cell
Myeloblast (Large granular lymphocyte)

T lymphocyte B lymphocyte

Megakaryocyte
Basophil Eosinophil
Neutrophil Monocyte
Plasma cell
Thrombocytes

Macrophage
 Erythrocyte Sedimentation Rate
ESR
Manual Method
ESR test is to detect inflammation by
Modified Westergren Method is used.
measuring the speed at which red Sample will be in special sedimentation test
blood cells settle to the bottom of a tubes containing sodium citrate.
straight glass test tube. ESR is not
recommended as a screening test to 1 3
detect inflammation in asymptomatic
individuals because it has low
specificity and sensitivity. RBCs have
2
draw blood blood cells fall cells stick
a net negative surface charge and into tubes to bottom in together and
tend to repel from each other. If an hour sink
positively charged plasma proteins Automated Method
increase due to inflammation, the ROLLER 20LC automated
repulsive forces are partially or totally ESR analyzer will read cell
counteracted resulting in aggregation aggregation by capillary
photometry technology.
of RBCs. The faster the settling of
cells is, the higher the ESR.

Sickling SolubilityTest
A Sickling Test is a m A clear appearance seen with hemoglobins
of blood & and solu ixture that are more soluble in the solution
tio
a test tube that give n in
turbid appearance s a
Turbid appearance is seen with insoluble hgs
differences in lysis due to that form liquid crystals and give a cloudy
of
insoluble hemoglob the appearance.
and soluble form of in S
Hemoglobin A.
 Malaria Screening
Rapid test for the qualitative
detection of four kinds of circulating
plasmodium (P. falciparum , P. vivax ,
P. ovale, and P. malarae) antigen in
human whole blood specimens. The
test is done by using
Immunochromatographic technology
based on the capture of the parasite
antigens in which parasite lactates
dehydrogenase of P.falciparum from
blood using antibodies against this
antigen.

Principle

If the test is positive we confirm it by

staining thick and thin blood smear.
Screening kit

Malaria Morphology
Falciparum Vivax Malariae Oval

Ring Stage

Trophozoite

Schizont

Gametocyte
 Glucose 6 Phosphate Dehydrogenase
G6PD

G6PD is intracellular enzyme to

Fluorescent Spot Test
protect hemoglobin from oxidative
denaturation by producing NADPH.
G6PD-deficient RBCs cannot generate
sufficient NADPH to reduce Normal
glutathione and oxidative damage
will result. Because of oxidative
Intermediate
damage, Heinz bodies will appear control
and RBCs are removed from the
circulation by hemolysis. We use
Deficient
fluorescent spot test to diagnose
G6PD deficiency by using UV light.

Body Fluid Analysis

Manual Automated
If sample is too turbid, we dilute it
Done by CBC analyzers
Neubauer Hemocytometer Chamber
Neubauer Calculation
chamber

Neubauer Hemocytometer Chamber

if WBC number is high
Counting

Cytospin
ABCD is It is a cytology
for RBC technique
count done to
concentrate
cells then stain
1-5 is for with counter
WBC stain to examine
count the slide.
 Hemoglobin Electrophoresis
Qualitative Disorders
This technique is done to measure
Abnormal hemoglobin
and identify the different types of
hemoglobin in the blood. Normal Hemoglobin S: Indicates sickle cell anemia
if patient have no symptoms and
adult without any hemoglobin percentage of hemoglobin S is less than
synthesis defect would have 3 types 40% it is sickle cell trait. If patient has
symptoms and percentage of hemoglobin S
of hemoglobin: Hb A (98%-95%), Hb A2 more than 40% it is sickle cell disease.
(2%-3%) and Hb F ( 1%-2%). Hemoglobin C, E,M and D
Presence of any abnormal
hemoglobin that results from genetic Quantitative Disorders
mutations. β-Thalassemia ( major and minor )
α-Thalassemia

principle

Coagulation Studies
Screening Test for Coagulation Function
Hemostasis in human PT /INR: it is done to evaluate extrinsic and common
pathway and monitor warfarin therapy
body have 2 parts, APTT: it is done to evaluate intrinsic and common pathway
primary (platelets and monitor heparin therapy
and blood vessel ) Fibrinogen: it is done to evaluate fibrinolysis system and
monitor disseminated intravascular coagulation
and secondary Thrombin Time: it is done to evaluate common pathway
(coagulation factor). D-dimer: it is done to evaluate fibrinolysis system
In the lab we mostly Special Tests
test and measure the
Factor Deficiency Evaluation.
parameters related VII / IX / vWF. etc.
to secondary Mixing study
Circulating Inhibitor screening.
hemostasis. Lupus / ATIII / protein S/C
 coagulation cascade

Normal & Critical Values

Test Normal Critical

Value Value
PT 10-14 sec above 50
sec
INR 0.2-1.2 Above 5

APTT 26-38 Above 100

D-dimer less than 0.5 Above 0.5

fibrinogen 180-350 Less than

mg/dl 100

Examples of Coagulation
Studies Machines
STAR Max, STAGO
coagulation analyzer
Principle : clotting method Sysmex® CS-2500
using movements of
magnetics bets and Principle: Reaction-kinetics
measure when its stopped analysis and four
measurement principles
(clotting, chromogenic,
immunoturbidimetric, and
aggregation**) are
combined on one analyzer.
 Mixing Study
Patient plasma Normal plasma
PT prolong (Pooled)
PT normal
factor level 100%

50% patient: 50% Normal

Done if we have prolong PT or

APTT to determine factors
Result : PT prolong Result : PT normal
deficiency or presence of
No Correction Correction
inhibitors.

Inhibitors Factor
Deficiency
Lupus
anticoagulant

Critical Values and

How to Deal With it
Hemoglobin (less than 6g/dl or
more than 22 g/dl)
Low: rerun sample in other device, if
same result come, send result Platelets (less than 30,000/ul)
High: check for lipemia and icterus if Check for clots, if present reject if not
present reject, otherwise rerun sample rerun sample on other device, if still low
in other device, if same result come, we do blood film to decide if it platelets
send result, if not should check aggregation (clumping) or confirmed
machine quality thrombocytopenia. If under microscope
there is platelets aggregation (because
patient have EDTA allergy) we order new
sample with sodium citrate tube if
platelets are low under microscope so it
WBC ( less than 2000/ul or more is confirmed thrombocytopenia.
than 30,000/ul )
Rerun sample in other device (with
differential count ) and do blood film, if
you get the same result, send, otherwise
check machine quality and linearity. Coagulation studies (PT,PTT,INR)
prolonged
Check for clots, if present reject sample,
otherwise, rerun sample if we get same
MCHC (more than 40 g/dl) result we will inform doctor and do
mixing study
Cold agglutinin suspected, RBCs will be
low, MCH and MCV high. To correct, we
incubate sample in water bath at 37 C
for 30 min then rerun sample. If it is still
above 40 , re-incubate.
CHAPTER 04

Clinical
Biochemistry
 What is Clinical
biochemistry?
Clinical biochemistry lab will
provide the measurements of
biochemical parameters to
diagnose diseases and monitor
treatments .

Types of Samples heparin

tube
Plasma sample
obtained from Lithium Heparin
Tube "green tube" for routine
analysis
Serum sample Plain
obtained from red or yellow tubes
plain tubes for iron and TIBC
analysis
EDTA tube
Whole blood sample
obtained from EDTA tube
"lavender" for HbA1c sodium flouride
Plasma sample tube
obtained from sodium fluoride
tube"gray" for glucose analysis
Body fluids
for electrolytes, protein, and
glucose measurements.
urine container
Urine ( 24 hour and routine )
for electrolytes measurement
and Creatinine clearance

Rejecting Criteria
Wrong tube Hemolyzed sample
Leakage sample Lipemic sample
Wrong label
Incorrect or incomplete request
 Liver function tests

Bilirubin metabolism

Evaluation of synthetic function

Total protein, Albumin and globulin

Protein in the plasma contain albumin,
globulin and clotting proteins.
Albumin is the main protein in the blood, Evaluation of hepatocellular function
which has a lot of functions like transport
hormones and maintain osmotic pressure. Alanine aminotransferase (ALT)
Whereas globulin will be used mainly in Also called serum glutamate pyruvate
immune system . transaminase ( SGPT)
Albumin/globulin ratio ALT enzyme is mainly synthesized in liver
A/G ratio is to represent how many cell, so it is more specific indicator for liver
remaining fraction of proteins there is. inflammation.
Aspartate aminotransferase (AST)
Also called serum glutamate
Evaluation of cholestasis oxaloacetate transaminase (SGOT)
&bile flow blockade AST enzyme is found in many organs
ike liver, heart, RBCs and skeletal muscles.
Alkaline phosphatase (ALP) If only AST increase, it'll indicate acute
ALP enzyme is presence mainly in the cells myocardial infarction .
lining the biliary ducts, but also present in If both ALT and AST increase, it'll indicate
bone, intestine and placental tissue . ALP liver disease
often increases if bile duct blocked, but
can increase in case of hepatitis and
cholestasis.
Gamma-glutamyl transferase (GGT)
GGT enzyme is mainly present in liver and
biliary system, and small amount can be
found in kidney and pancreas. In case of
alcoholism, GGT will be high.
Bilirubin (total, direct and indirect)
Bilirubin results from breakdown of RBCs.
Measurement of bilirubin will help in
diagnose bile block, liver disease, blood
hemolysis and helps to differentiate
between jaundice types.
 How to diagnose jaundice ?

LDL

Lipid profile HDL Triglycerides

Done to determine the risk of

heart diseases
Total cholesterol

Anemia profile
Transferrin
Iron

Serum Iron
Measurement of amount of circulating iron that bound to
transferrin . iron test is always ordered after abnormal result
in CBC.

Total iron binding capacity (TIBC)

Serum iron bound to transferrin TIBC
It is to measure the blood's capacity to bind iron with
transferrin . TIBC value calculated from iron and UIBC value,
UIBC [µg/dl] + Iron [µg/dl]

Unsaturated iron binding capacity (UIBC)

UIBC
It is to measure the amount of free binding sites in transferrin.
 Diabetic profile
Done to diagnose diabetes and
to determine treatment response

Fasting blood glucose :

8 hours fasting is recommended.
normal 70-110 mg/dl
Random blood glucose:
Normal: 80- 120 mg/dl
Hemoglobin A1c (Hb A1c) :
the average glucose concentration
in red blood cells (RBCs) during
their 120-day lifespan

Cardiac function test

Done to evaluate heart function and it is done
to help diagnose chest pain or other signs
and symptoms of a heart attack .

Electrolytes test
Done to measure electrolytes to diagnose the
conditions that result from imbalance in
electrolytes quantities .

Sodium It is the most abundant cation in extracellular fluids.

+ Because of sodium function in water balance and metabolic
Na functions, sodium analysis is done to monitor kidney diseases
and treatment of hypertension .
Potassium
+ It is the most abundant cation in intracellular fluids. Potassium
K test is done to monitor acute or chronic kidney failure .

Calcium
+2 It is the most abundant mineral in the body, mostly in the
Ca skeleton. Calcium test is done to evaluate diseases in kidney,
parathyroid glands, and bone .

Phosphate
-4 Phosphate test is done to check phosphate levels in kidney
PO disease and bone disease patients .

Magnesium
+2 Magnesium test is done to evaluate of the severity of kidney
Mg problems and diagnosis of gastrointestinal disorders .

Chloride
- It is the most abundant anion in extracellular fluids. Chloride test
Cl is done to diagnose and monitor kidney disease, heart failure
, liver disease, and high blood pressure.
 Trace elements
Zinc
+2 Measurement of zinc in the blood help to diagnose deficiency or
Zn monitor response to supplementation .

Copper
Measurement of copper in the blood help to help diagnose and
Cu monitor Wilson disease .

Kidney function tests

BLood test
Urine test
Urea
After breakdown of amino acid ammonia will be produced, Electrolytes measurements
ammonia will then convert to urea in the liver and be
excreted in urine.
Blood urea nitrogen (BUN)
It measures the amount of nitrogen contained in urea. BUN 24 hour urine
provides measurement of glomerular filtration rate .
Creatinine
It is a waste product that results from muscle metabolism after It is mostly done to estimate
breakdown of creatine. the kidney will filter the most of creatinine glomerular filtration rate (GFR )
Uric acid and how the kidney effectively
It is the final product from purine breakdown. Uric acid remove the creatinine from the
measurement is mostly done to diagnose gout . blood.
Ammonia Creatinine clearance (CC)
Done to measure the high level of ammonia that result from it is the amount of creatinine in the
kidney or liver diseases . plasma that is removed from
the body by the kidney. CC will
estimate GFR by calculating:

24hour Urine protein

It is to measure how many protein
is spilled in the urine to diagnose
proteinuria .

Examples of Machines used in

clinical biochemistry lab
SIEMENS Dimension

Principle: spectrophotometer by
measuring transmitted light at
specific wavelength .
Dimension
 Roche COBAS 6000 C501

Principle: ion-selective electrode for

sodium, potassium, and chloride .
spectrophotometer for other tests.
6000 C501

Abbott Alinity c chemistry

analyzer
Principle: Spectrophotometer

Alinity c

Critical value for clinical

biochemistry lab
CHAPTER 05

Hormone
 What is Hormone?
it is a chemical massages send
from endocrine glands to its target
in the body . Each gland release a
various types of hormones that do
a various functions .

Types of Samples
Plasma sample
Serum Sample obtained from EDTA tube
obtained from red or "lavender" for ACTH test.
yellow tube for routine
analysis and tumor EDTA tube
markers.

Plain tubes

Rejecting Criteria
Wrong tube Hemolyzed sample
EDTA tube without ice Lipemic sample
Wrong label
Incorrect or incomplete request
Thyroid function test

Anemia profile

Cardiac Markers
 Fertility evaluation

Bone health evaluation

Diabetic profile
 Adrenal gland function

Tumor marker

Examples of Machines used in

hormone lab
Roche Cobas E411 analyzer

Principle: Electro-Chemiluminescence
technology

Cobas E411
 Roche Cobas 6000 E601
analyzer
Principle: Electro-Chemiluminescence
technology

Cobas E602

ADVIA Centaur XPT

Immunoassay System
Principle: Chemiluminescence
technology

ADVIA

Principle
CHAPTER 06

Serology &
Immunology
 What is Serology &
Immunology?
Serology: It is the study of antibodies in
the serum that formed in response to an
antigen, so it is all about antigen
antibody reaction.
Immunology: is the study of the immune
system, which protects us from infection,
including study of auto-antibodies.

Types of Samples
Plasma sample
Serum Sample obtained from EDTA tube
obtained from red or "lavender" for PCR.
yellow tube for routine
analysis and ELISA test EDTA tube

Plain tubes

Rejecting Criteria
Wrong tube Hemolyzed sample
Wrong label Lipemic sample
Incorrect or incomplete request
 Routine tests
Routine tests in serology lab depends on different
principles including: Agglutination, Precipitation,
Flocculation, and Nephlometry, Hemagglutination.

Principles of Routine tests

Agglutination
Agglutination will
appear if the
Sample containing Agglutination antigen and
Antibody coated with target analyte
latex antibody in the
(antigen) zone of
Antigens or antibodies equivalence .
(in the kit) should
coated with latex or
RBCs to visualize the
reaction by naked eye .

C-reactive protein is a protein appears in the blood in response to inflammation but it is not
specific indicator of inflammation . CRP test can be done manually using latex agglutination test
CRP
Tests that based on

or automatically by nephlometry .
Rheumatoid Factor is an abnormal protein occurs in the serum in case of rheumatoid
agglutination

arthritis, But absence of RF does not exclude the diagnosis or existence of RF. RF test can be
RF done manually using latex agglutination test or automatically by nephlometry.
Antistreptolysin O ASO is the antibody made against streptolysin O, an oxygen-labile
hemolytic toxin produced by streptococci , so its aids in the diagnosis of several conditions
ASO associated with streptococcal infections . ASO test can be done manually using latex
agglutination test or automatically by nephlometry .
Salmonella possesses two major antigens namely somatic antigen (O) and a flagellar
Widal antigen (H). Widal test is used to diagnose enteric fever . This test helps to detect presence of
salmonella antibodies in a patient’s serum .
Brucella abortus can cause an infection in human that may be acute, relapsing, chronic, or
Brucella subclinical. Brucella melitensis can cause Malta fever.Direct agglutination assay is a
commonly used serologic test for Brucella infection.

Prozone phenomenon
Prozone phenomena it is when there is high antibody concentrations
but no reaction occurs. As the antibody concentration is lowered
below the prozone by doing dilution, the reaction occurs. The cause
of prozone phenomenon may because antibody excess or blocking
antibody or presence of nonspecific inhibitors in serum .
Flocculation

Sample containing Flocculation

Antigen coated with target analyte
carbon (antibody)
Tests that based on
Flocculation

Syphilis is sexually transmitted disease caused by bacteria called Treponema pallidum . If

the result of RPR is positive its confirmed by TPHA. The antigen used in RPR is a Cardiolipin, in
RPR which micro particulate charcoal particles are used to enhance the visual difference
between positive and negative results .

Indirect Hemagglutination immunoassay (IHA)

Sample containing Agglutination

Antigen sensitized target analyte
RBCs (antibody) Positive result
RBCs settled as
a fine carpet

Antigen sensitized No Agglutination

RBCs
Negative result
Intact button
shape in the
Sample with no bottom of the
antibodies well
Tests that based on

Treponema pallidum haemagglutination is done to detects specific

Treponema pallidum antibodies in serum to confirm of active syphilis
infection.
If antibodies against T.pallidum are present in the diluted serum,
IHA

TPHA sensitized red blood cells will agglutinate, resulting in a cloudy

red/brown deposit coating the well like a carpet. If there is no
antibodies, sensitized red blood cells will not agglutinate, resulting in
a ring-like deposit at the bottom of the well. The reaction is carried
out in a U-microplate
 Dilution method
For any positive result for routine manual test we need to make a
dilution for patient serum with normal saline to know antibodies titer
(except RPR test ). Each test have different dilution titer .
Dilution can be done on slide or tube .

Same as tubes , just put

50ul from saline in all
wells and put 50ul from
serum in first will and mix Just make sure that serum and
and take 50ul to the next normal saline have the same
and so on .. then put quantity.
drop from reagent in all Add drop from the reagnt on
wells and see the result. slide with drop from each tube
and see result .
if the agglutination for example
stop on third dilution we can say
that the antibody titer is 8 and so
on.

Procedures for routine tests

with dilution titer

we put
different serum
concentrations
to prevent
prozone
phenomenon

Nephlometry
It is an automated machine that involves immunochemical
determination of protein in serum. In this method, the light,
which is scattered by the antigen-antibody complexes, is
measure. The intensity of the measured light scatter is
proportional to the amount of the antigen- antibody
complex in the sample. A reference curve by calibration is
created with known antigen levels. The scattered light
signal of the samples can then be read from this curve as
an antigen concentration.

Automated
Tests that besed on

IgG - use as indication for chronic infection

IgM - Use as indication for acute infection
Nephlometry

IgA - protect against microorganism invasion and present in secretions

C3 and C4 Complement - done when suspected that individual
complement component concentrations are abnormally reduced.
Ceruloplasmin - done to diagnose copper metabolism disorders, that is, BN ProSpec®
Wilson’s disease "ceruplasmin will be low" System from
Alpha1-antitrypsin - done to diagnose neonatal respiratory distress
syndrome, severe protein- losing disorders, and pulmonary emphysema. Siemens
CRP
RF
ASO
 Enzyme-linked
immunosorbent assays
(ELISA)
ELISA is the most common immunoassay Component
(antigen-antibody reaction) type and used to 1.Coated well- made of polystyrene (immobilize
detect the presence and measure the antigens or antibodies)
amount/concentration of specific analytes (e.g. 2.Sample diluent - kits dependent .
antibodies ) in a sample for diagnosis and 3.Conjugate- enzyme labeled antigens or
vaccination. ELISA techniques use antibodies antibodies e.g horse radish peroxidase or
linked to an enzyme as horse radish peroxidase alkaline phosphatase
or alkaline phosphatase. Ag-Ab reactions are 4.Substrate-it’s peroxidase conjugate, it’s react
detected by the enzyme-substrate reaction, with enzyme of conjugate to produce color
color change indicates an antigen-antibody 5.Wash concentrate- buffer solution,
reaction has occurred. containing detergent to remove excess
In general, ELISAs can be grouped into: direct antigen or antibody .
(sandwich) to detect antigens, indirect to detect 6.Stop solution- stop the enzyme-substrate
specific antibodies. reaction

Direct ELISA
Sample Incubate Incubate Incubate Read

wash wash stop

solution

Target antigen Conjugate Substrate

Indirect ELISA
Sample Incubate Incubate Incubate Incubate Read

wash wash wash stop

solution

Target antigen Primary antibody Conjugate Substrate

secondary antibody

Sandwich ELISA
Sample Incubate Incubate Incubate Read

wash wash stop

solution

Target antigen Conjugate

Primary antibody Substrate
secondary antibody
Competitive ELISA
Sample Incubate Incubate Read

wash wash stop

solution

Target antigen Antibody Conjugate Substrate

 The Recombinant ImmunoBlot
Assay (RIBA) Test
Principle

Polymerase chain reaction

PCR
Polymerase chain reaction test is done to
amplify many copies of DNA sample to study it
and help in diagnose diseases .
 Exmple of Machines used in
Serology and Immunology lab

DSX - 4-plate ELISA Processing

system from Dynex
Principle: ELISA colorimettric.
Used to measure virus, bacteria and
parasite antigens or antibody against
these antigens .
DSX

DiaSorin ETI-MAX 3000

Principle: ELISA colorimettric .
Used to measure virus, bacteria and
parasite antigens or antibody against
these antigens .
ETI-MAX

Alegria® by ORGENTEC
Diagnostika
Principle: ELISA colorimettric.
Used to diagnose AutoImmune
Diseases .
common tests done :
ANA Alegria®
ANTI ds DNA
ANTI SSA , ANTI SSB
ANTI PHOSPHOLIPID SCREEN

Phadia 250 by THERMO

scientific
Principle: ELISA colorimettric .
Used for Sensitivity tests

Phadia 250
CHAPTER 07

Micro-
biology
 What is Microbiology?
it is a study of microorganisms that includes :
virus, bacteria, parasite and fungi

Types of Samples
Stain
Culture
Gram stain
Acid fast bacilli
Blood, urine, stool,
semen, CSF, Body
fluids
Swabs
Tissue Hair, skin scrabs and
nail

Fungal

Rejecting Criteria

Wrong Label
Incorrect/ Sample Leaakge
Incomplete
specimen
Information

Wrong Sample
 Culturing
Culture Media
Enriched media (have 5% sheep blood)
Most of the bacteria can grow on BAP
Blood Agar Plate

Use to see the type of hemolysis

(streptococcus especially)
Incubate in 5% CO2 container
plates

Enriched media ( heated RBCs )

Rich with factors V & X
Allow some fastidious organisms to grow, like Haemophilus influenzae
Chocolate Agar

and Neisseria spp.

Incubate in 5% CO2 container
plate

Selective & differential and sometimes indicator media

Differential > lactose fermentation, E.coli > pink dry colony,
Klebsiella > pink mucoid colony
Other Enterobacteriaceae are non-lactose fermenters ( yellow or
colorless colony )
Selective > have bile salt and crystal violet that inhibit gram-positive
Macconkey Agar

and allow gram-negative to grow

Growth on blood & McConkey > gram negative
growth only on blood > gram-positive
Indicator > have neutral red ( pink colour)

plate

a non-selective chromogenic culture medium

Chrom Agar

used to differentiate Candida types

plate
Agar
 Selective and differential media

Macconkey
Selective for E. coli 0157:H7

Sorbitol
Bile salts and crystal violet > inhibit gram-positive bacteria
Potassium tellurite and Cefixime > allow E. coli 0157:H7 to grow

Agar
Differential > sorbitol fermentation
E. coli 0157:H7 > non sorbitol fermenter

Selective media for fungal growth

Dextrose Agar

Low ph (acidic ) will inhibit bacterial growth

Sabouraud

plate

Selective and differential media

Used to Isolate all Staphylococcus
Selective > high concentration Salts, only gram-positive especially
Mannitol Salt Agar

Staphylococcus Aureus can grow

Differential > mannitol fermentation staphylococcus aureus ( yellow
color)
Use for Methicillin-resistant Staphylococcus aureus (MRSA) screen
Phenol red used as an indicator
Mannitol agar with Oxicillin used for MRSA as a specific media
plate

Cysteine, lactose, and electrolyte-deficient agar

Differential media > lactose fermentation (yellow color)
for urine culture
Yeast shows white colony > Do germ tube test to see if it is candida
albican (positive for germ tube) or not.
Bromothymol blue used as an indicator
Proteus can't do swarming
Look for colony forming unit (CFU), 100 colonies or with single type
colony is significant, for catheter patient 10-15 colony is significant .
CLED Agar

plate

Simple media
Mueller Hinton

Used for antibiotic sensitivity test

plate
Agar
 Xylose Lysine Deoxycholate agar
Selective media: some references consider it as differential (because
of different colony color and shape will appear)
Selective for salmonella (black center colony, H2S production) and
shigella ( colourless colony)
Used for stool culture
Before culture on XLD agar, put stool in Selenite F broth (enrichment
media) that have high salts concentration to enhance salmonella
and shigella growth and inhibit other.
E.coli (lactose fermenter) show yellow colony > report as flora
Proteus same as salmonella , but show swarming and fishy smell
plate
XLD Agar

Selenite F
broth

Thiosulfate-citrate-bile salts-sucrose agar

Selective media for vibrio cholera ( golden yellow colony )
Have bile salts to inhibit gram +ve
Bromothymol blue use as indicator
Before culture on TCBS, put sample in alkaline peptone water ( have
electrolytes and nutrients ) to enhance V.cholera growth
TCBS Agar

plate
alkaline
peptone
water

Culturing Technique
Culturing is done
inside class II
biohazard safety
cabinet to protect
Culture Plate Methods specialist and the
air from ant
Semi-quantitative Quantitative contamination
Point of specimen Point of application
application of calibrated volume
of specimen
1 streak area
1 streak area
2 streak area

for urine culturing

3 streak area
22 cross-streak
cross streak
 All culture media will
incubate inside general

Incubation purpose incubator at 37

C. Chocolate agar will
incubate in CO2
incubator.
Bacterial need 24h to
Urine
48h to grow Fungal
CLED agar
need 1-4 weeks to grow
Stool macConkey XLD agar
agar
macConkey Chocolate
Body fluids Blood agar
agar agar
Pus macConkey
Blood agar
agar
Sputum/tracheal macConkey Chocolate
Blood agar
aspiration agar agar
macConkey Mannitol Sorbitol
Food poisoning Blood agar macConkey XLD agar
agar salt agar agar
Nasal swab Mannitol
(MRSA screen) salt agar
Tissue macConkey Chocolate
Blood agar
agar agar
Swabs (throat, macConkey Chocolate
Blood agar
eye, ear) agar agar

Blood Culturing
When patient have signs and symptoms of sepsis, blood
culture is ordered

3 6ºC Indicators of Sepsis

t e m p e rature: < (chills
B o d y (10 0 º F )
r >3 8ºC
(9 7 º F ) o
) s/L
or fever nt < 4 x 109 cell
u n)
WBC co r fungal infectio
o
(in viral 9 cells/L (i
n
2 x 1 0
or > 1 n)
c t e r ia l infectio > 90 beats
b a rt b e a t :
id h e a
Ra p Sample Collection
ute 0
per min athing rate: >2
re
Rapid b er minute Blood sample on
p
b re a t h s bottles
(at least 2 set
'aerobic (blue) and
anaerobic (purple) '
from different site
in the body)
 Required Sample Amount

Aerobic Anaerobic
bottle bottle
Syringe amount amount of
needed of blood. blood.

Adult 20 ml 10 ml 10 ml

Bottle contains:
20 ml 2.5 – 10 2.5 – 10
Pediatric ml ml SPS (sodium
polyantha
0.5 – 1 sulfonate) that act
Infant 3 ml 0.5 – 1 ml
ml as anticoagulant
Resin: acts as
neutralization of
antibiotic.
A gel sensor (on
Any positive blood culture will the bottom) to
subculture on Blood, Chocolate and detect bacterial
MacConkey agar growth

If results are needed as soon as possible, we will do

gram stain to give antibiotic to patient.

It is an automated
rapid microbial BACT/ALERT® 3D
detection that capable
to detect ant microbial 1. CO2 is released
2. CO2 reacts with dye
growth in blood culture 3. LED activates fluorescence
bottles by using 4. Detector reads fluorescence
5. Data is analyzed
colorimetric sensor that 6. Performing positivity analysis
changes from gray to 7. positive vial is announced by
alert
yellow in the presence
of CO2 produced by
growing
microorganisms.
sensors will scan every
ten minutes, if growth is
detected alarm will
start

Normally bottles will take 1-5 days inside the

machine to give the result. If brucella is
suspected, 14 days are needed to get the
working principle
result. Fungi need 14 days and anaerobic
bacteria will incubate for 5 days.
 Identification and Antibiotic
Sensitivity
Biochemical Tests
Catalase test

It is used to identify bacteria that

produce catalase enzymes.
Catalase test is always used to
distinguish between
staphylococcus (catalase positive)
and streptococcus (catalase
negative) Catalase
2HO
2 2 2HO+O
Oxidase test Hydrogen
2 2
Peroxide Water
Oxygen

It is used to identify bacteria that

produce cytochrome oxidase
enzymes. Oxidase test done
to distinguish between
Enterobacteriaceae (oxidase
negative) and Pseudomonas
(oxidase Positive)

Coagulase test

It is used to identify bacteria that

produce coagulase enzymes and
make clots in plasma. coagulase
test is done to distinguish between
staphylococcus aureus (coagulase
positive) and other staphylococcus
(coagulase negative).
 Microscopic Examination
Gram Stain

Gram Staining Procedure

Acid fast stain

It is also called Ziehl-Neelsen stain, by

using sputum sample to diagnose
tuberculosis infection. Acid fast stain is
used also to diagnose other mycobaectria.

KOH test

KOH Preperation for microscopic

analysis of scale for fungal forms
KOH test is used as primary and first
method for screening fungal infections by
detecting fungal elements

microscopic
view

Germ tube test microscopic

view

Used for identifying candida albicans.

If we have a white colony on CLED agar
with urine culture we suspect candida
albicans Infection so we will do this test
 Cultural Characteristics
Pigment Production Colony Morphology

Odor Production Colony Size

Manual Antibiotic Susceptibility Testing

The tube is incubated
for the bacteria to grow

n
h e m o s t commo t
T to tes
The inoculum density
is standardized using Mcfraiand

method
standard A cotton swab dipped
in the inoculum suspension is swabbed over
2-3 identical colonies are picked from the plate and the entire surface of agar to give a

antibiotic ility
transformed the the broth lawn culture

is
sus ce p tib
diffusion
disc by using
method Hinton
M u elle r
dia
agar me
Incubate under 37C for 24h

Disc added

Example of Automated Antibiotic

Susceptibility Testing and Biochemical
Tests
Vitek 2
It is an automated machine
that use Minimum Inhibitory
Concentration (MIC)
principle to test antibiotic
susceptibility and
colorimetric technique to
detect biochemical tests
 MRSA Screen
Nasal swab

Mannitol salt agar PCR

mecA gene
Yellow colonies Colorless colonies
MRSA
Antibiotic Susceptibility Other
Testing staphylococcus

Cefoxitin screen positive Cefoxitin screen negative

Oxacillin resistant Oxacillin sensitive

MSSA
MRSA

Critical Lab Results

Positive AFB smear
Positive blood culture
Positive CSF culture
Positive result in any
sterile body fluids
Positive MRSA
 Bacteria identification charts
GRAM +VE COCCI

Catalase Catalase

Staphylococcus Streptococcus

Hemolysis Hemolysis Hemolysis

Coagulase Coagulase
Bile esculin
S.aureus
Optochin Optochin Enterococcus
Beta hemolysis Novobiocin Novobiocin Resistant
Golden Yellow
Sensitive
colonies Sensitive Resistant
S.pneuomonia S.viridans
S.epidermidis S.saprophyticus
White colonies
White or yellow Bactiracin Bactiracin
colonies Sensitive Resistant
S.pyogrnes S.agalactiae
Wide zone Narrow zone

GRAM -VE BACILLI

Lactose fermentation Lactose fermentation

Slow Rapid
Oxidase Oxidase
Citrobacter
Pseudomonas
Serratia
Indole Indole
E.coli Klebsiella
H2S production H2S production
dry colonies mucoid colonies
Enterobacter
Shigella Salmonella
Yersinia Proteus
swarming growth
fishy smell
Acinetobacter
Catalase +ve
 GRAM +VE BACILLI

Spore formers Non-spore formers

Aerobic Anaerobic Acid fast Not acid fast

Bacillus Clostridium Mycobacterium Listeria

Nocardia Corynebacterium
Lactobacillus

GRAM -VE COMMA-SHAPED

Oxidase

Grow in 42 Grow in alkaline media Urease producing

Campylobacter jejuni Vibrio cholerae Helicobacter pylori

yellow colonies on TCBS

GRAM -VE COCCI

Maltose fermentation Maltose fermentation

Neisseria meningitidis Neisseria gonorrhoeae

GRAM -VE COCCOBACILLI

Brucella
Bordetella pertussis
Haemophilus influenzae
Francisella tularensis
Pasteurella
CHAPTER 08

Parasit-
ology
 What is Parasitology?
Parasitology lab is all about
examination of urine, stool, semen
and body fluids to mainly diagnose
parasitic infection and other
abnormalities related with kidney and
intestinal tract etc.

Types of Samples
Urine sample
first morning mid-stream
Semen sample urine collected in urine
for semen analysis Stool sample provides the best results for
collected in stool
urine analysis
container for Routine
Examination and Occult
Semen Blood. urine container
container

Stool container

Rejecting Criteria
Wrong container. Leakage of the sample
Wrong Label
Incorrect/Incomplete specimen
Information
 Urine examination
Urine color
Visual examination Colorless : sign of over hydration . If
accompanied by excessive thirst , Diabetes
Color – normally pale yellow insipidus is suspected .
Appearance (turbidity) –normally clear
Odor - no abnormal odor
Amount : normal is 1.5-2 ml Pale yellow : this is the optimal color of urine.

Dark yellow : patient needs to drink more water

Dipstick
Orange : sign of dehydration or liver disease

Red : sign of kidney disease or due to certain

medications .

Dark brown : sign of urinary tract infection or

due to antibiotics

Blue or green : in some rare genetic diseases

Purple : in klebsiella pneumoniae infection

Normal results
Specific Gravity : 1.010-1.030
pH : 4.5-8
Blood , protein , Leukocyte esterase ,
ketones , glucose and nitrites :
negative
 Microscopic examination
By doing wet mount : First take an amount
from urine in a tube for centrifuge, then
take the sediment (one drop) on a slide
then put coverslip and observe with 10x
lense then 40x.

What can be seen and what it can be

indicate ?

1. White blood cells – infection or

inflammation.
2. Red blood cells - kidney disease
3. Bacteria - infection.
4. Epithelial cells - infection or kidney
disease.
5. Parasites - parasitic disease
6. Crystals - sign of kidney stones.
7. Casts - sign of a kidney disorder.

Example of Automated urine

analysis
LabUMat 2 & Urised 3 Pro
machines
PLabUMat 2 is for chemistry analysis
Urised 3 Pro is for microscopic
examination
 Stool analysis
stool
Visual examination
small amount small amount
Color
Volume
Consistency
Odor
Presence of blood, worms and pus. saline Iodine

Microscopic examination one drop one drop

by saline or iodine wet mount

mix and put coverslip

Occult blood test

It is a rapid test to check any hidden
blood in stool.
Blood can be present in case of ulcers,
inflammation of intestine, benign 1ox lense
or malignant polyps . 40x lense

Occult blood kits

 Semen analysis
Semen analysis Involves analysis of chemical and
physical characteristics of semen, shape, motility,
and count of sperms.
Semen must be delivered after
collection within 30-60 minutes

Chemical and physical characteristics

with normal results
ph – alkaline
Color – white
Viscosity – viscus (< or = 2 cm thread after liquefaction
Liquefaction time – 15-30 minutes
Volume – 1.5-2 ml

Microscopic examination
By wet mount
CHAPTER 09

Histopath-
ology &
Cytology
 What is Histopathology
and cytology?
Histopathology : it is study of tissue sections
under microscope to diagnose a various
diseases .

Cytology : it is study of cells in body fluids

to help in diagnosis various diseases .

Types of Samples
Histopathology sample
all biological tissues either
whole organ or pieces that
Cytology sample
filled with 10 % formalin for
Bone marrow.
fixation
PAP slides.
FNA (fine needle
aspiration)
Body Fluids.

Rejecting Criteria
Incorrect or incomplete request Tissue come without
When tissue section is not fixative
immersed in 10% formalin.
Wrong label
 Histopathology workflow
01 Receiving Check if request form is complete :
Patient name.
Unit record number.
Type of the specimen.
Date of collection.
Make sure that sample filled with
10% formalin

Grossing and Grossing is to describe any abnormalities in

the tissue by pathologist and then cut the
02 cassette parts of interest that will help in diagnosis
and put them in the cassette with label
preparation

Fixation in 10% formalin for preserve the

tissue
Dehydration in a series of ethanol (alcohol)
solutions of increasing concentration
Tissue until 100% alcohol to remove water from
03 tissue.
processing Clearing in xylene as intermediate
solvent to remove alcohol
Infiltration with liquid paraffin wax

A cassette is placed on top of the mould

and filled with paraffin wax and allow it
to solidify to make block .
04 Embedding

05 Trimming
By using rotary microtome , block will be
cutted into very thin sections.
section will float on a water bath then
picked up and placed on microscope
slides.
06 Cutting The slides are then dried on a hot plate
to help the tissue adhere to the slide.

There is 2 type of stains , routine and special

stain.
07 Staining Routine stain : Hematoxylin and eosin (H&E)
Hematoxylin (stain purple)
Eosin (stain pink )
 Special stain

By using DPX substance .

08 Mounting

Microscopic
09
examination

Immunohistochemistry
Method of detecting the presence
of specific proteins in cells or
tissues by using antigen-antibody
reaction .

Cytology
Body fluids, centrifuge and we work
with the sediment or using cytospin

In cytology there 3 types of Swabs obtained by brushing or

specimens scraping, to study epithelial cells

Aspiration , put on slide as smear

All types of sample we put it on a slide and fix

it with alcohol , then stain it with pap stain and
H & E stain and mount with DPX and examine
under microscope.