Presented by

Al Shaimaa M. Taha
Assistant Lecturer of Biochemistry
 Collection of Blood

Capillary Blood

The heel of an infant up to

The ‘ring’
one year old
finger of a child
Venous Blood
 Anticoagulants

tri-Sodium Citrate
•
EDTA
EDTA.K
(10g%)
salt
recommended
Heparin
in
is
than
(3.2g%)
EDTA.Na (more soluble).
• 25μl EDTA • +for1mlclinical
blood; chemistry
• ESRtests and Sod. Citrate
(400μL
never immunophenotyping.
exceed EDTA are completed to 2ml with
volume because • not it recommended
causes for routine
blood)
shrinkage and hematological
degenerative tests • PTbecause
and APTTit (200μL Sod.
changes to cells.causes cells to clumpCitrate
and heparin
is completed to
• CBC and gives a blue background
reticulocyte to blood
2ml with blood).
count. films.
• It is not suitable for
Stability of Anticoagulated Blood
Storage Anticoadgulated Blood Type
Citrated-Blood
Storage -1Anticoadgulated Blood Type
 Room
EDTA-Blood Temp
- 1 • ESR Determination
Immediately.
Room
4°C up Temp.
to 4 hrs • Within 2 hrs.
(ESR )
4°C • Within 24 hrs.
- 20 4°C
°C •PT and•APTT
RBC’sDetermination
Count
Avoid Storage at
Room25°C
Temp • Platelet Count
2-3 days
Avoid at 4°C
Storage at •Hb Determination
4°C
Avoid Freezing
 Citrate anticoagulated blood
• Even when citrated blood is stored at 4–8˚C, there is a decrease in the
ESR due to changes in erythrocyte shape affecting rouleaux.
• The ESR should be measured within 4 hours of collecting the blood.
• Coagulation tests should be carried out as soon as possible after blood
is collected into citrate anticoagulant.
 }
Laboratories Hematological Tests
1. WBC’s count
2. Differential WBC count
3. RBC’s count
4. HCT measurement
5. Hemoglobin measurement
6. Blood indices calculation
CBC
7. Reporting blood films
8. Platelet count
9. Tests to screen for a bleeding disorder (PT & APTT)
10. ESR
Improved Neubauer Ruled Counting Chamber
WBC’s
Count
WBC’s Count (thousands/ mm3) =
Cells counted x 50
 WBC’s
Differential
Reagents
Absolute Methanol (Fixative Solution)

Eosin (red dye, Soln. I)

has a negative charge and stains positively
charged (acidophilic) substances
(hemoglobin)

Methylene blue (blue dye, Sol. II)

has a positive charge and stains negatively
charged (basophilic ) substances (DNA,
RNA and nucleoproteins)
Blood Film Preparation

Blood Film Staining

 IDEAL BLOOD FILM

Well fixed thin blood film.

 Optimally stained smears have the following
characteristics:
1. RBC’s should be pink or yellowish red
2. Platelets are violet to purple granules
3. Neutrophil: Nucleus is dark blue, cytoplasm is pale pink, granules is
reddish lilac
4. Eosinophil: Nucleus and cytoplasm are blue, granules are red to red
orange
5. Basophil: Nucleus is purple or dark blue, granules are dark purple
(almost black)
6. Monocyte: Nucleus is violet, cytoplasm is sky blue
7. Bacteria are blue
8. Area between cells are clear
EXAMPLES OF UNACCEPTABLE SMEARS
too acidic suitable too basic
Count tail body head
Observing Direction
Normal MORPHOLOGY OF WBC IN
PERIPHERAL BLOOD
Staff Neutrophil Segmented Neutrophil
(3-5%) (50-70%)
Basophil Eosinophil
(0-1%) (2-4%)
Lymphocyte Monocyte
(25-40%) (2-8%)
ABNORMAL CHANGES
OF WBC MORPHOLOGY
Toxic Granulation
Dohle body
Hypersegmentation Degeneration of nucleus
Anisocytosis of vacuolization
neutrophil
Auer Bodies(Auer Rod)
RBC’s
Count
RBC’s Count (million/ mm3) = Cells
counted x 10000
 Hematocrite (HCT) or Packed Cell
Volume (PCV)
Defi niti on: It is a measure of the volume of packed RBC’s
expressed as percentage of whole blood.

Procedure
Calculati on
Interpretati on of HCT

HCT values are reduced in anemia.

Increased values are found when there is loss of plasma as in

severe burns and dehydration. Also in all forms of
Polycythemia.
 Determination of
Hemoglobin Concentration
 Reagents
Procedure
1. 10μl R1 (Concentrated Reagent)
EDTA-anticoagulant in Dark
blood is added to Bottle
a test tube containing 2.5ml
working solution. Potassium ferricyanide
2. Shake well to avoid clumping of erythrocytes.
Potassium cyanide
3. Stand for 5mins. Potassium phosphate
4. Read *R1
againstisworking
storedsolution
at room at (520-560nm).
temperature.
Calculati on Solution in Dark Bottle
Working Reagent
The contents of one bottle of R1 is diluted to 2500ml
Hemoglobin Conc.
with deionized (g/ dl) =
water.
*Working reagent is stable up to expiry date when store
Specimen
at room temperature.absorbance x 36.77
Calculation of CBC
Indices
 )%( HCT
10 X = MCV
RBC (million/mm3) )fL 78-95(

Hb (g%)
10 X = MCH
RBC (million/mm3) )pg 27-33(

Hb (g%)
100 X = MCHC
)%( HCT )% 30-40(
 Step-by-Step Diagnostic
Sequence
1. RBC and platelets counts, Hb, HCT and indices.
2. A careful evaluation of the differential blood count

3. Analysis of the cell composition of the bone

marrow
Reporting blood films
 Platelet Count
1-
2- Procedure
Using Hemocytometer
Differential Blood Film
Reagents blood is diluted 20 times with 1% ammonium oxalate in a
1.EDTA-anticoagulant
1.Use
Ammonium the oil
clean eppindorf andimmersion
stand
oxalate, (1%lens
for 30mins
10g/l for complete lysis of RBC’s.
w/v)
Toestimate
2.Make sure
make theml:
the
100 number
central gridof platelets
areas of the chamber and the special hemocytometer
per field.
cover glass areoxalate.
completely
Ammonium . . .clean
. . . .and
. . .dry.
. . . . . . . . . . 1.0 g
2.Look
3.Slide
at 5-6 fields and take an
the cover glass into position over the grid areas.
Distilled
average.water. . . . . . . . . . . . . . . . . . . . . . . . 100 ml
storeone
4.fill at of
3.Multiply 2–8theC.
thegrids of the chamber with the sample, taking care not to overfill
average by 20,000.
the area.
For use: Filter immediately before use to ensure the reagent is free
5.Leave the chamber undisturbed for 20mins. to allow time for platelets to settle.
from particles which could be mistaken for platelets.
6.Count the platelets in the five of the 25 groups of 16 small squares of the
chamber marked P in fig using the 40x objective.

Calculati on
1) <8 platelets/OIF = decreased
1) 8 to 20 platelets/OIF = adequate
Platelets2)Count (thousands/ mm
>20 platelets/OIF ) = Cells counted x 1000
= increased
3

*OIF= oil immersion field

Interpretati on of platelet count
I. Thrombocytopenia
I. REDUCED PRODUCTION OF PLATELETS
● Infections, e.g. typhoid, brucellosis
● Deficiency of folate or vitamin B12
● Aplastic anaemia
●Drugs (aspirin), chemicals (benzene), alcoholism
● Leukaemias, lymphoma, myeloma, myelofibrosis, carcinoma
● Hereditary thrombocytopenia (rare condition).
II. INCREASED DESTRUCTION OR CONSUMPTION OF
PLATELETS
● Disseminated intravascular coagulation (DIC); activated procoagulants are
released into the circulation. Platelets and coagulation factors are
consumed and fibrin is deposited in small vessels, activating the
fibrinolytic system.
● Infections (malaria)
● Hypersplenism
● Immune destruction of platelets, e.g. systemic lupus erythematosus (SLE),
chronic lymphatic leukaemia, lymphomas and HIV/AIDS. Also, exposure
to drugs, e.g. quinine, mefloquine and penicillin.
 II. Thrombocytosis
●Chronic myeloproliferative diseases, e.g. essential
thrombocythaemia, Polycythemia Vera, chronic myeloid
leukemia, myelofibrosis.
● Carcinoma (disseminated)
● Chronic inflammatory disease, e.g. tuberculosis
● Hemorrhage
●Sickle cell disease associated with a nonfunctioning spleen or after
splenectomy.
● Iron deficiency anemia, associated with active bleeding.
Diagrammatic representation of
blood coagulation.
Coagulation Factors
I. Fibrinogen.
II. Prothrombin.
III. Thromboplastin.
IV. Calcium.
V. Proaccelerin (Labile factor).
VII. Proconvertin (Stable factor).
VIII. Antihemophilic globulin A.
IX. Christmas factor.
X. Stuart- Prower factor.
XI. Plasma thromboplastin antecedent.
XII. Hageman factor.
XIII. Fibrin Stabilizing factor.
Tests to Screen for a Bleeding Disorder
PT
PT APTT
APTT
a. Centrifugate
Calculation:
Is a method of choicethe for:
citrate-anticoagulant Calculation:
a. Centrifugate
Is a method the for:
of choice citrate-anticoagulant
1- bloodPT
Patient
1.Monitoring at 1500rpmSec. for 15mins. therapy 1-
= ?anticoagulation blood
The at 1500rpm
results
1.Monitoring may be for 15mins.
reported
anticoagulation directly in
therapy
b.
2- Incubate
Mean
(Coumadin Normal
and 100μl
PT PT reagent= in
“MNPT”
dicumarol). W.B. at b.
? Sec. Incubate
units of ?100μl
(heparin). Sec. APPT reagent in W.B.
3- 37˚C(R)
Ratio
2.Screening for=the
10mins.
Patient PT / MNPT
extrinsic at 37˚C for
clotting system 2.Screening the10mins.
Or
intrinsic clotting system
c.
4- Incubate
INR VII).
(factor
ISI120μl plasma in W.B. at c.
= (R) , where ISI for this kit = 1.5 2- Incubate
VIII, IX, XI, XII).2 W.B. at 37˚C
as ratio:
(factors 120μl CaCl in
37˚C for the
*INR=international
3.Screening 5mins. normalized
clotting ratio.in the 3.Screening
factors R for
= APTT
5mins.of
thepatient plasma
clotting (Sec.)/
factors in the
d. Transfer
*ISI=international100μl
common pathways sensitivityfrom patient
(factors II,index.plasma
V, X). to d. APTT
Transfer
common of FNP
100μl
pathways (Sec.)
from
(factorspatient
II, V, plasma
X). to
*N.R.tube
4. LiverINR (b) andtest.
in healthy
function immediately
persons = 0.6 start– 1.2
the tube (b), shake well&incubate at 37˚C
stop watch.
(differs from lab to lab). for 5mins.
e.
N.R.Switch
*Oral 10-14 off the
anticoagulant
Sec stopfrom
(differs watch
therapeutic when
labrange
to lab).the e.
INR N.R.Transfer
22-34 Sec.100μl fromfrom
(differs tubelab (c)totolab).
tube
first
= 2 –fibrin strand from
3.5 (differs is visible
lab toand the gel
lab). (d)&start the stop watch immediately.
In a graduated
clot formation Wassermann
begins. tube, 200μl 3.2%f.sodium Shake citrate
tube are
(e) completed
well&incubate to 2mlin with
W.B.
venous blood (in a ratio of
f. Repeat the above steps to normal 1 citrate:9 blood when HCT<55% for
at 37˚C for 20Sec. PT only).
healthy persons. blood
Citrate-anticoagulated g. Following 20Sec. blood
Citrate-anticoagulated Incubation, remove
Centrifugation the tube; gently tilt back&forth until a
Centrifugation
Poor Platelet Plasma (PPP) Poorgel clot Plasma
Platelet forms; (PPP)
switch off the stop
PT reagent {thrompaplastin (III)+excess APPT watch&record
reagent{Kaolin the(surface
time. activator)+
CaCl2 (IV)} h. Repeat (platelet
phospholipid the above steps +toexcess
substitute)} fresh
CaCl2normal
(IV)} plasma (FNP) of normal
Clot is formed Clot is formed
persons.
 Sources of error when performing PT&APTT
tests
1. Difficulty in obtaining a venous sample, resulting in hemolysis or
small clots in the sample.
2. Delay in testing the plasma and leaving the sample at room
temperature.
3. Using tubes or pipettes which are not clean and dry or are
contaminated with detergent. Whenever possible use disposable
tubes.
4. Not timing accurately the different stages of the test (a stop-watch
must be used).
5. Not pipetting correct volumes of plasma and reagents.
6. Using unsatisfactory reagents (will be detected when testing
control plasma).
 Causes of Acquired Hypercoagurable State (INR<0.5)
• Advancing age • Antiphospholipid antibody
• Prior Thrombosis syndrome
• Malignancy • Myeloproliferative Disorders
• Estrogens • Heparin-induced
thrombocytopenia (HIT)

Causes of Inherited Hypercoagurable State (INR<0.5)

• Antithrombin deficiency
• Protein C deficiency
• Protein S deficiency
• Factor V Leiden mutation (Factor V-Arg506Gln)
• Prothrombin gene mutation (G A transition at position 20210)
• Dysfibrinogenemias (rare)
 ESR
Reagents
Procedure
1.
3.2g%
Pipette
tri-sodium
0.4 ml citrate
of sodium citrate anticoagulant into a small
container.
Principle
2. Add 1.6 ml of venous blood or EDTA anticoagulated blood and
mix well.
When Removeblood
citrated the cap in
of the
a container and place the sample in
the ESRpositioned
vertically stand. Insert a Westergren pipette and ensure it is
Westergren
positioned
pipette vertically.
is left undisturbed, red
cells
3- aggregate,
draw the bloodstack
to the together
0 mark oftothe Westergren pipette.
form rouleaux,
4- Set the timer forand
1 hour.sediment
Ensure the ESR stand and pipette will
through
not be the plasma.
exposed to direct sunlight.
5- After exactly 1 hour, read the level at which the plasma meets the
red cells in mm.
Factors Aff ecti ng ESR
● Fibrinogen, immunoglobulins, and C reactive protein increase
red cell aggregation.
● Sedimentation is increased when the ratio of red cells to plasma
is altered(in anemia).
● Sedimentation is reduced when the red cells are abnormally
shaped (sickle cells). High temperatures (over 25˚C) increase
sedimentation.
 Source of Error
● Using the wrong volume of blood to anticoagulant.
● Blood not sufficiently mixed with anticoagulant.
● Clots in the blood. Even the smallest fibrin clot in the sample will
invalidate the test result.
● Air bubbles at the top of the column.
● Testing blood samples at the hottest time of the day, or leaving tests in
direct sunlight. Temperatures over 25˚C increase sedimentation.
● Using a pipette which is not clean or not dry.
● Pipette not positioned vertically. Even slight variations from the
upright increase sedimentation.
● Not checking whether the ESR stand is level on the bench.
● Placing an ESR stand on the same bench as a centrifuge where
vibration will interfere with sedimentation.
● Measuring the ESR when a patient is dehydrated.
Interpretation of ESR test results
Reference range
Men . . . . . . . . . . . . . . . . . . . . . Up to 10 mm/hour*
Women . . . . . . . . . . . . . . . . . . .Up to 15 mm/hour*
Elderly . . . . . . . . . . . . . . . . . . . Up to 20 mm/hour*
*These figures should be checked locally. Higher values may need to
be applied in tropical countries.
Note: Higher values are obtained during menstruation and pregnancy.
Causes of a significantly raised ESR
• Most types of anemia
• Acute and chronic inflammatory conditions and infections (HIV
disease, tuberculosis, acute viral hepatitis, arthritis, bacterial
endocarditis, pelvic inflammatory disease, systemic lupus
erythematosus
Causes of a significantly lowered ESR
• Sickle cell anemia
• Polycythemia
• DIC (fibrinogen is consumed)