1. Blood Physiology

2. FUNCTIONS OF BLOOD Transport
O2, CO2, nutrient, hormones, waste product
Homoeostasis
Regulation of body temperature, pH
Protecting against infections
White Blood Cells, Antibodies
Blood clotting prevent blood loss

3. BLOOD COMPOSITION Cellular components Red Blood Cells (Erythrocytes)
White Blood Cells (Leucocytes)
Platelets (Thrombocytes)
Plasma
98% water, ions, plasma proteins (Albumin, globulin, Fibrinogen)
Same ionic composition as interstitial fluid

4. Blood Composition

5. Blood Composition

6. Collection of SERUM
after collection of the blood, its put in a dry and clean test tube for 5 – 15 min under room temperature to allow coagulation, after this put the test tube in centrifuge for 10 min. at 3000 r.p.m. the supernatant represent the serum, this withdraw to another tube to performing the tests.

7. Collection of PLASMA
, after collection of blood its put in an-anticoagulant tube (to avoid coagulation) mix gently by inverting the tube not shaking, leave the sample for period of time, notice the yellow – colored liquid above, this is the plasma.
Also can use the centrifuge to obtain all the plasma, which withdraw to another tube to perform the tests.

8. Anticoagulants
Anticoagulant are chemical reagents that ceased the series of the reactions that lead to the blood clotting .

9. The commonly used anticoagulants are the following:-
EDTA (Ethylene Diamine Tetra Acetic acid)
Sodium citrate
Oxalate
Sodium fluoride
Heparin
Warfarin (oral anticoagulant)

10. CLOTTING TIME To determine the clotting time of a subject.
Requirements: 
Fine capillary glass tubes of about 10 mm length, cotton, lancet, stop watch.

11. 1. Capillary Tube Method:
Procedure:
i. Clean the tip of a finger with spirit.
ii. Puncture it upto 3 mm deep with a disposable needle.
iii. Start the stopwatch.
iv. Fill two capillary tube with free flowing blood form the puncture after wiping the first drip of blood.
v. Keep these tubes at body temperature.
vi. After 2 minutes, start breaking the capillary tube at 1 cm distance to see whether a thin fibrin stand is formed between the two broken ends.
vii. Stop the watch and calculate the time from average of the tow capillary tubes.

12. Disadvantages:
(i) Method is insensitive.
(ii) Method is unreliable.
Advantages:
It can be performed when venous blood cannot be obtained.
Normal clotting time is 1-5 minutes.

13. 2. Lee and White Method:
i. After cleaning the forearm, make a venepuncture an draw 3 ml of blood in a silicon-sided glass or plast syringe.
ii. Start the stopwatch.
iii. Transfer 1 ml of blood each into 3 glass tubes which at kept 37° C in a water bath (Fig. 31.1)
iv. After 3 minutes tilt the tubes one by one every 30 second.
v. The clotting time is taken when the tubes can be title without spilling of their contents.
vi. Calculate the clotting time by average of 3 tube.

14. Advantages:
(i) More accurate and standard method.
(ii) Test can be run with control.
Disadvantages:
(i) It is also a rough method.
(ii) There can be contamination of syringe or tube.
Normal clotting time is 5-10 minutes.

15. Procedure: Capillary tube method: (Wright’s method)
Under sterile precautions make a sufficiently deep prick in the finger tip. Note the time when bleeding starts (start the stop watch).
Touch the blood drop at the finger tip using one end of the capillary tube kept tilted downwards. The tube gets easily filled by capillary action. After about two minutes start snapping off small lengths of the tube, at intervals of 15 seconds, each time noting whether the fibrin thread is formed between the snapped ends. Note the time (stop the stop watch) when the fibrin thread is first seen.

16. Normal clotting time
Clotting time is the interval between the moment when bleeding starts and the moment when the fibrin thread is first seen.
Normal value is 3to 10 minutes.
Bleeding time and clotting time are not the same. Bleeding time depends on the integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time remains normal.
Clotting time is also prolonged in conditions like vitamin K deficiency, liver diseases, disseminated intravascular coagulation, overdosage of anticoagulants etc.

17. Complete blood count (CBC)
1- Hemoglobin (Hb) determination.
2- Hematocrite of Packed Cell Volume (P.C.V.)
3- Erythrocyte Sedimentation Rate (E. S. R.)
4- Red Blood Cells Count (R.B.C count)
5- White Blood Cells Count (W.B.C count)
6- Differential W.B.C count
7- Platelets count

20. Hemoglobin (Hb) Determination By Sahli s method (acid hematin method)
The method is based upon conversion of Hb to acid Hematin compound (brown color) by using acid. By HAEMOMETER

21. Reagents & equipments:-
Hb pipette.
Stirrer (glass rod)
Standard Hb comparative
Graduated tube (Hb tube) 140%

22. Procedure:-
The specimen to be used is capillary blood or EDTA anticoagulant blood.
1- fill Hb tube with (0.1 N) HCL till 20 marks
2- Withdraw the blood sample by Hb pipette to 0.02 ml. mark (20μl).
3- Add the blood sample to the Hb tube quickly and mix well by the stirrer.
4- Used the acid to dilute the mixture of Hb until a match is seen with brown glass Hb standard.
5- Read the lower level of the fluid on % report Hb in g\ 100 ml. or (g \ dl.) of blood unit.

23. Normal ranges:-
Male adult: 14.0 – g / dl.
Female adult: 11.5 – 16.5 g / dl.
New born: 13.5 – 19.5 g / dl.

24. Determination of Hematocrite (Packed Cell Volume; P.C.V.)
P.C.V. may be defined as the percentage of the packed red cell volume to the total amount of blood.
Microhematocrite Method :-
Anticoagulant whole blood is centrifuged, and total volume of the red cells mass is expressed as a percentage of the total blood volume or decimal fraction. Ex. (42%).

25. Reagents & Equipments:-
Heparinized capillary tubes coated with either heparin or EDTA.
2- Microhematocrite centrifuge speed r.p.m.
3- Hematocrite reader (%)
4- Artificial clay
Specimen:-
The specimen is heparinized capillary tube or venous blood which adds to EDTA tube (whole blood)

26. Microhematocrite centrifuge speed 10000 r.p.m.

27. Procedure
1- The capillary tube used should not coated with anticoagulant (non – heparinized capillary tube) why? Because the excess anticoagulant may cause cell shrinkage & produce false low values. Ensure that is not air bubbles. 1-Leave at least 15 mm of capillary tube is empty. 2- Sealed the tube by artificial clay. 3- Put the tubes in centrifuge with the sealed ends toward the outside of the holder for 5 min. at r.p.m. 4- Read with reader & report the result as percentage or decimal fraction.

28. Δ Now look at the tube the blood is separated into 3 layers :- 1- Column of R.B.Cs at the bottom. 2- Narrow middle zone of buffy coat consists of W.B.Cs & Platelets. 3- Fluid of plasma at the top. Normal range:- Male adult: 36 – 52 % Female adult: 33 – 47 %

29. Above normal range of PVC
The Polycythemia (increase R.B.Cs numbers) and (dehydration) occur in sever diarrhea, sever burn, vomiting or drinking too little of water and use diuretics, because the loose of the fluids lead to decease the volume of plasma compared with R.B.Cs.

30. Below normal range of PVC
occur in anemia and leukemia because the disorder in bone marrow function that leads to low numbers of R.B.Cs or in sever bleeding and in pregnancy.