Page 1 of 11

بسم الله الرحمن الرحيم

2لاب فسيو

بالاعتماد على سلايدات الدكتور و كلامه و مصادر خارجية

السلايدات رح تكون داخل صندوق

Differential WBC Count

اته و صفاته الاساسية ن ن اعداد خلايا الدم البيضاء ... احنا اخذنا بالتفصيل الممل كل نوع و ايش ممي الاختلاف بي

ن المعدل الطبيعي لكل نوع الهدف من هذا الفحص نعرف اعدا د خلايا الدم البيضاء بانواعها المختلفة ولازم نكون عارفي

ي جسم الانسان ني اشارة لوجود خلل ف

... طبعا اي زيادة او نقصان بتعطينن

ي اشارة ل neutrocyteمثال اخذناه بالباثو لو زاد acute bacteria infection and necrose tissueفهذا بعطينن

الاساس الي رح نعتمد عليه بالفحص نوخذ عينة دم و نصبغ بصبغة خاصة سريعة التحضي و يدوية و كل نوع بعطي

ه ه عن غي ن استجابة معينة بمي

clean slide 2بتشبه الجهاز الي منستخدمه بالسكري مشان يطلع دم ..... lancet 1المكونات الي رح نحتاجهم

وري جدا تكون نظيفة حنى يطلع الفحص صحيح .... نوع من انواع microscope ......diff quick stain 3ضن

الصبغات

Aim:

To determine the percentage of each type of leukocytes in the peripheral blood.

Certain percentages can change during certain diseases so this test is valuable for

the diagnosis of these diseases.

Principle:

The test depends on making a blood film from the peripheral blood and staining it with Diff

Quick stain using manual method. Different types of WBCs are differentiated by their

morphological and staining characteristics.

Material:1 Lancets 2 Clean slides 3 Microscope 4 Diff Quick Stain

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I. Preparation of blood smear Is made on an ordinary slide

وه ح نفس الخطوات و كثي مرتب بفضل تحضن شفت فيديو بشر

رابط الفيديو

https://www.youtube.com/watch?v=nbRUiWl2Qrs

ح الارب ع خطواتركزوا عالصورة الي تح ت لانه بتشر

1. Slides must be spotlessly clean.

Hold them always by the edges.

Never put your fingers on the surface

of the slide. Prepare two clean slides.

2. Obtain a drop of blood from the tip

of the middle finger and place it at the

end of one slide. Put the slide on the

surface of your bench. Use the second

slide as a spreader by placing its

smooth edge at an angle of 45º to the

first slide.

3. Move the spreading slide backward

until it touches the drop of the blood

as in the diagram. The drop of the

blood will immediately spread across

the edge of the spreading slide

4. With the spreading slide at the angle

of 45º, push it slowly forward and

steadily across the horizontally placed

first slide

Page 3 of 11

سمك العينة بعتمد عالزاوية الي مسحت فيها الدم

كان السمك اكير 45كل ما كانت الزاية اكير من

ما بحتاج نعرضه للحراة نهائيا

Dلازم تكون لون العينة زي

بكون جهزت عينة الدم مشان اصبغها 8بنهاية خطوة

5. The thickness of the film may be

regulated by increasing or

decreasing the angle between the

slides or by varying the speed of

spreading. A quick spreading gives

a thick film; the wider the angle

the thicker the film.

6. Allow the blood smear to dry in

air at room temperature; it must

not be heated

7. A satisfactory film is very evenly

distributed and yellowish orange

in colour ; the red cells should be

close together but not overlapping

each other. Check that by looking

at the fill under the microscope

8. The “zone of morphology”

should be at least 2 cm in length.

The smear should occupy the

central area of the slide.

Page 4 of 11

تشوفوا الفيديو بالاول بعدين رح تصي الخطوات سهلة و ما رح تتغلبوا بالحفظقبل ما نحكي بالمرحلة الثانية بفضل

رابط الفيديو

https://youtu.be/Y27TvaJMKRE

II. Staining of blood smear

Do not stain a film until you have made a satisfactory one.

The stain used is the Diff Quick stain. This stain contains;

1 Diff Quick fixative reagent ( Triarylmethane dye, Methanol)

2 Diff Quick solution 1 (Xanthene dye (Eosin Y) in phosphate buffer)

3 Diff Quick solution 2 (Thiazine dye in methylene blue and azure A

phosphate buffer

1. Air dry the smear

2. Dip slide for 30 seconds into Fixative. Allow excess to drain after each

dip.

3. Dip slide for 30 seconds into Stain 1. Allow excess to drain after each dip.

4. Dip slide for 30 seconds into Stain 2. Allow excess to drain after each dip.

5. Rinse slide in distilled water or Weise's buffer, pH 7.2 to remove excess

stain.

6. Allow to dry in air or rapidly dehydrate in absolute alcohol.

7. Examine at low power to identify structures and then under oil

immersion

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ي بنصح تشوفوا ة و نفس الاشر ن الفيديو هذولة المرحلة الثالثة و الاخي هي

https://www.youtube.com/watch?v=ihSnTKLUazQ&t=132s

https://youtu.be/DFzndODp86s

III. Examination of blood smear:

WBC counterخلية بتطلع معي و من خلال 100مشان احسب عدد خلايا الدم البيضاء لازم اعد اول

100او 50قليل و احدد احسن منطقة بعدهيك برفع لحد microscopeبالبداية لازم تكون عيار ال

• The monolayer is the area where the cells are examined and a 100 cell

differential leukocyte counts performed.

• The monolayer section is usually a distance of one 10x field behind the

feathered edge of the smear and is the optimal area for examination of

cells. White blood cells are uniformly distributed in this section.

• The student should scan the smear at low magnification first to locate

the optimal area for differential leukocyte count.

• Once the optimal area has been located, switch to a higher

magnification ( 50 or 100x oil immersion objective ) and begin your

differential count and morphology assessment.

• Begin the count by moving back and forth across the smear in a pattern

that avoids covering the same territory . This can be done by moving in

a zigzag fashion across the slide (as in the diagram

• Identify each leukocyte that is encountered until 100 cells have been

counted and sorted by type, giving you a percentage of each cell type or

a relative differential leukocyte count.

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زي بالصورة zig-zagببلش العد عن طريق

)6.300/cu mm-e Granulocytes (70%Th

Basophils Eosinophils Neutrophils

(0%-1%) (1%-4%) (50%-70)% Percentage

liberate histamine and heparin during allergic

reactions.

phagocytize and destroy the antigen-antibody complex

highly phagocytic (often called macrophages).

function

7μ 13μ 10-12μ size

-Scanty or abundant cytoplasm.

-Large deep blue cytoplasmic granules,

blue-black nucleus. -Lobulated nucleus (2 or

3 lobules). -They liberate heparin

into the blood, a substance that can

prevent blood coagulation.

-Large sized cells with abundant cytoplasm

-Large red-orange cytoplasmic granules, blue-purple nucleus Mutilobulatednucleus (2 or 3

lobules)

*abundant cytoplasm *Small pink granules in the

cytoplasm. *Multilobulatedpurple nucleus (three lobes or

more).

Character

after allergic reaction (such as asthma, parasitic infestation,

and Hay fever .)

acute pyogenic infection. Increase in NO

Page 7 of 11

2.700/cu mm)-The Agranulocytes (30%

Monocytes Lymphocytes

(2%-8%) (20%-40%) percentage phagocytic and help in tissue

repair after injury. -They are the scavenger cells and they are transferred into

tissue macrophages to engulf the dead tissues and

bacteria.

* specialized in production of immunity such as the synthesis of antibodies

function

-The Largest WBC with irregular blue or purple nucleus (Kidney shaped

nucleus).

Variable -7μ in diameter (small) to 10μ in diameter

(large)

Size(shape)

-Abundant blue-gray cytoplasm (nongranular).

with no cytoplasmic granules, light blue cytoplasm

-The scanty cytoplasm is pushed to the periphery of the cell by the large deep blue

or purple nucleus -Small lymphocytes have very large spherical nucleus, large lymphocytes have large oval

indented nucleus

Character

*innate immune * chronic inflammation.

-They perform a major role in immune system and increase in number during

chronic and viral infections

Increase NO

Page 8 of 11

Bleeding Time Test

(Duke Method)

يف ...هاي مسؤول ن ي اشارة لفاعلية الصفائح الفحص هذا اخذنا قبل هيك و بقيس الوقت اللازم حنى يتوقف الينية الصفائح و بالتالي بعطينن

ي طريقة ثانية باستخدام جهاز الضغط بس بتوخذ الدموية ... بحتاج اعمل جرح بالجلد ن دقايق و مش عملية ( 10... )ف

قبل ما نحكي عن الخطوات بفضل تشوفوا الفيديو

https://youtu.be/3UNK7w-Upss

Definition:

A bleeding time test determines how quickly the bleeding stopes. The test involves making small

punctures in the skin. The test is a basic assessment of the platelets function (i.e. platelet plug

formation).

Indication:

The bleeding time test is a common test to screen patients having bleeding tendency (i.e.

prolonged bleeding times). The bleeding time is used to measure the primary phase of

hemostasis.

Procedure:

•Clean the puncture site with an spirit to minimize the risk of infection.

•Make small cuts with the lancet on the tip of the middle finger (or the earlobe). The puncture

should be 3-4 mm deep to cause slight bleeding.

•Start the stopwatch immediately, swab the cut with filter paper every 30 secondsuntil no

more blood is absorbed (i.e. no more blood spots on the filter paper).

•Record the time it takes for the bleeding to stop.

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Clotting Time Test

(Capillary Tube Method or Wright’s Method)

extrinsicو هذا بحفز ال tissue thromboplastin (factor III)بفرز damageهسا الدم اول ما يتعرض

pathway ة حنى تتكون الخير

platelet aldheranceبدون intrinsicلانه انا بدي احسب فقط aPTTبستخدم intrinsicمشان اقيس ال

Interpretation:

Normal bleeding time is between 2-5 minutes. Prolonged bleeding time could indicate low

platelet count or the platelets are dysfunctional, and further testing will be required.

Abnormal results could indicate one of the following conditions

•Thrombocytopenia انخفاض عدد الصفائح الدموية

•Medications (Example aspirin and other cyclooxygenase inhibitors, & antibiotics such as

penicillin and cephalosporins)

Anti plateletزي

•Uremia (uremic platelets synthesizeless thromboxane A2, and the blood vessels in patients

with uremia produce greater quantities of platelet-inhibitory prostaglandin)

•Liver failure

•Leukemias

•Von Willebrand’sdisease

Indication:

•Clotting time is used as a screening test to measure all stages in the intrinsic coagulation system

and to monitor heparin therapy.

•This test is sensitive only in extreme factor deficiencies(such as in hemophilia). Therefore, it is of

limited use in today’s medical practice and its use is confined to demonstration.

Page 10 of 11

بناء عالفيديو

Definition:

•Is the time required for a fresh sample of blood to form a clot in vitro. The basis for the test is that

whole blood will form a solid clot when exposed to a foreign surface such as a glass tube.

•Clotting time test is a rough measure of all intrinsic clotting factors in the absence of tissue factors.

•Variations are wide and the test sensitivity is limited. The more recent sensitive test for the intrinsic

pathway is the partial thromboplastin time (PTT). Normal value of clotting time is 3-8 minutes.

Material Required:

•Sterile disposable pricking

needle or lancet

•Stop-watch

•Dry glass capillary tube

(narrow diameter top 2 mm,

minimum 10 cm long)

•Cotton Swab of absorbent

cotton

•Spirit wetted, cotton swab

•70% v/v ethyl alcohol

Procedure:

1.Apply alcohol 70% v/v to clean the fingertip with

cotton swab. Allow it to dry naturally.

2.Prick the finger. Remove the first drop.

3.Dip one end of the capillary into blood drop gently

without pressure (better to use more than one capillary

tube).

4.The timer is started when the first blood start to enter

the first capillary tube.

5.Allow to fill the capillary with blood by lowering the

end of fitted capillary. (Do not suck the blood).

6.After every 30 seconds, using stop-watch, break a

small piece of capillary.

7.Repeat breaking at regular time intervals, till fibrin

thread appearsat the broken end of capillary tube. Do

not pull away the cut pieces long apart and bristly.

8.Record time interval between pricking finger and first

appearance of fibrin thread between the broken ends

of capillary tube. That is clotting time of blood.

9.Don't forget to dispose of the broken capillary tube in

the SHARPS CONTAINER

Page 11 of 11

ي حال كان الوقت اكير ندقايق بكون السبب : 8ف

THE END

• Factors V, VII, VIII, IX, XI, XII Deficiencies

• Hemorrhagic disease of Newborn

• Vitamin K deficiency

• Heparin Therapy

• Presence of Circulating antibodies

(inhibitors)

• Anemia and leukemia

• Afibrinogenemiaand Pneumonia