Lab Work 1 Anticoagulants, ESR and PCV - PowerPoint Presentation

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Lab Work 1Anticoagulants, ESR and PCV

Dr Than

Kyaw

27 February 2012

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COAGULATIONComplex process of blood clottingImportant part of hemostasisCessation of blood loss from damaged vessel(s)Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). (Refer to theory) 2

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ANTICOAGULANTSDrugs that prevent the clotting (coagulation) of blood by inhibiting the formation of one or more clotting factorsThe reagents employed should not bring about alteration of blood components. 3

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USES OF ANTICOAGULANTSIn vitro - experiments - blood tests

In vivo - usually given as treatments

- to prevent thrombus extension and embolic complications by reducing the rate of fibrin formation

- Deep vein thrombosis and pulmonary embolism.

-

Myocardial

infarction

-

Cerebrovascular

disease - Blood transfusion

4

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5Types of anticoagulants

Ammonium and Potassium

Oxalate

Ammonium oxalate – expand the RBCs

Sodium oxalate – shrink the RBCs

Also k/s double oxalate, balanced oxalate

Mixture of ammonium oxalate and potassium oxalate (3:2)

1

mg of double

oxalate - sufficient

to prevent clotting of 1 ml of blood

action

: precipitate

ionic calcium and

form insoluble

salts

.

safely

used for

haematocrit

,

Hb

estimation

, RBC and WBC counts,

prothrombin

time and

bilirubin

concentration etc.

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6Types of anticoagulants

2. Sodium Citrate (

trisodium

citrate)

- 3.8% aqueous solution is used.

- 9 parts of blood: 1 part of sodium citrate solution

-

action

: Chelating

effect on calcium ion

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7Types of anticoagulants

3. E.D.T.A (

Versene

or

Sequestrene

)

-

di

-sodium salt of ethylene

diamine

tetra acetic

acid (EDTA); powerful

anti-coagulant

-

action

: stable

chelating effect on calcium

- 1 to 2 mg per ml of blood

- can be used in the blood transfusion

- prevents blood platelets clumping in vitro; so can be employed for

platelets count

- Blood from some birds and reptiles

hemolyzes

when collected

into

EDTA

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8Types of anticoagulants

4. Dicoumarol

(

Bishydroxycoumarin

)

- O

btained from spoiled sweet

clover;

inactive in vitro

-

A

ffect

the transport of vitamin K to its site of action.

-

Vit

K is required for normal synthesis of

prothrombin

and

other clotting factors.

-

Interferes

with factor VII and factor X in the liver

- R

educes the Christmas factor (IX)

- Reduces the adhesiveness of platelets

- I

ncreases

antithrombin

concentration in plasma

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9Types of anticoagulants5.

Heparin

- a natural anti-coagulant which aids in maintaining blood in a fluid

state.

- main source in the body - mast cells (liver, lungs etc)

- inhibits the reaction between thrombin and fibrinogen

- also interferes with the formation of

thromboplastin

- Heparin retards agglutination and lyses of platelets

- used in the ratio of 0.1 to 0.2 mg per ml of blood

- should not use for blood films; it gives faint blue back-ground on

staining.

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10Types of anticoagulants

6. Hirudin

:

- P

resent in the

buccal

glands of leech

- A

powerful anti-coagulant

- I

nhibits the conversion of

prothrombin

to thrombin.

7.

Venoms

- V

enoms of certain snakes contain a powerful anticoagulant effect

- I

nterferes with the action of

thromboplastin

or

- D

eplete the blood fibrinogen.

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118.

Contact and Smoothness of Surface

-

The clotting of the blood can be prevented when it comes in contact with the tissue fluid or with the substance which is wet or has a smooth surface.

-

When

the vessel in which the blood is collected is coated with thin layer of paraffin wax, blood remains in fluid state for half an hour.

Types of anticoagulants

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12Lab 1. (b)

Erythrocyte sedimentation rate (ESR)

Aim

:

To determine the ESR of the blood sample using

Westergren

/

Wintrobe

tube

Principle

A simple non-specific screening test

I

ndirectly measures the presence of inflammation in the body

A

column of blood taken in a narrow tube

K

eep vertically undisturbed

RBCs tend to settle down due to

higher specific gravity

than plasma

T

endency of RBCs to settle more rapidly in the face of some disease states

U

sually

due to increase

in plasma fibrinogen,

immunoglo-bulins

, and other

acute-phase

reaction proteins

-

Changes in red cell shape or numbers may also affect the ESR.

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Aggregate or Rouleaux formation

Single rouleaux formation

Medium density aggregate

Small high density aggregate

Small

rouleaux

network

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14Method 1

Method 2

Westergren

tube

300 mm in length (graduated 0 – 200 mm)

Internal diameter 2.5 mm

Both ends open

3.8% sodium citrate solution

Blood

Stand (rack)

Wintrobe

tube

110 mm long (graduated 0 – 100 mm)

Internal diameter 3 mm

One end closed

Pasteur pipette

EDTA

Blood

Stand

(rack)

Requirements

:

ESR

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Species

Site of collection and permitted conditions

Dog, Cat

Cephalic

,

saphenous

, femoral and jugular veins

Ruminants

Jugular

vein, tail vein

Swine

Jugular

vein, anterior vena cava, ear veins

Chicken

Brachial

wing vein, right jugular vein

Blood sample collection

ESR

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16ESR

Procedure

(

Westergren

)

Collect 2 ml of blood into a syringe by venue puncture

transfer into a vial containing 0.5 ml of 3.8% sodium citrate solution

(

blood to citrate ratio of 4:1

)

Mix gently the contents of the vial by repeated inversion

Suck the blood mixture carefully into the

Westergren

pipette up to the 0 mark

without introducing any air bubbles.

Fix the pipette vertically in the

Westergren

rack

Allow it to remain for 2 hours

Screw clamp at the top as well as pit at the bottom provided by rubber pads

ensuring right fixing without allowing leakage.

It also avoids the effect of atmospheric pressure.

Observe the pipette for the column of clear plasma at the end of 1 h

and the 2

nd

hour.

Note the readings

in mm per 1

st

hour and 2

nd

hour.

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ESR

Species

Westergren

tube

Wintrobe

tube

mm at end of 1 hour

mm at end of 2 hour

Record your observed values

after 1 hour and then 2 hour

.

Precautions

The pipettes should be kept perfectly vertical.

Blood column should be without

bubles

.

Use the blood within 2 hours of collection (6 hours if kept 4



C).

The test should be carried out at the room temperature.

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19ESR

Procedure

(

Wintrobe

)

- Performed similarly

as

Westergren

method

- EDTA (or double oxalate)

anticoagulated

blood without extra

diluent

is drawn into the tube.

- The value is usually higher than

Westergren

method.

- The method also is exposed to atmospheric pressure.

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ESR

Some interferences which increase ESR:

increased level of fibrinogen, gamma globulins.

technical factors: tilted ESR tube, high room temperature.

Some interferences which decrease ESR:

abnormally shaped RBC (sickle cells).

technical factors: short ESR tubes, low room temperature, delay in test performance (>2 hours), clotted blood sample, excess anticoagulant, bubbles in tube.

Chronic inflammatory disease (collagen and vascular diseases) increases ESR.

Polycythemia

decreases ESR.

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Red blood cells have settled, leaving plasma at the top of the tube. Reading: 18 mm/hourB

A

A at “0” hr; B after 1 hour

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Lab 1. (c) Hematocrit

or Packed Cell Volume (PCV)

Aim

: to estimate the packed cell volume of the ------------------- blood

Principle

- PCV

(

hematocrit

) - one of the most common blood test performed

-

PCV is the percentage of RBC in the blood.

- E

asy, inexpensive, reliable, and provides valuable information.

-

The term “packed cell volume” describes the principle behind the test.

- B

lood is drawn into a capillary tube,

- Take care not to overfill the tube.

- By c

entrifugation cellular components are ”packed” into the bottom of

the capillary tube.

- RBCs

at the bottom of the tube,

B

uffy coat (leukocytes

a

nd platelets)

C

lear to yellowish top layer is plasma.

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Hematocrit

Procedure

Fill two

hematocrit

tubed

¾ full

with blood containing anticoagulant

(by capillary action).

Blood may be obtained directly from the patient. Use a hematocrit tube containing an anticoagulant. - Wipe the tube with a

kimwipe

or blotting paper

-

Place finger over the “non-blood” end of the tube and push the

opposite end into a clay sealant 3-4 times

- Place the

hematocrit

tube in a centrifuge with the

clay end toward

the periphery

- Place centrifuged

hematocrit

tube on a reader with the top of the clay sealant at the 0% mark and the top of the plasma layer at 100%

Centrifuge at 10000 rpm for 5 minutes.

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Hematocrit

Procedure (continued)

Read the % of RBC which is read at the top of the RBC layer,

do not include the

buffy

coat.

Note and record the color of the plasma.

(

eg

. Clear and transparent, white and cloudy, etc)

Note an increase or decrease in the size of the

buffy

coat.

The

buffy

coat is usually less than 1 mm wide.

Also, note the color of the

buffy

coat which is normally white.

Record your findings carefully and write a lab report.

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Hematocrit

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Format of the report

DVT 1073

Fisiologi

Haiwan

II

Lab work no. ____

Name: ____________ No.

Matrik

: ______________ Date: ________

Title of the laboratory work.

Aim

Requirements

Procedures

Observation and findings

Discussion/Comments/conclusion

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The End