1. بسم الله الرحمن الرحيم
285 PHL
Lab# 1
Introduction
Blood

2. blood is a tissue that circulates in the closed system of blood
vessels.
Functions:
Respiration
Nutrition
Excretion
Defense mechanisms
Regulation of acid base balance
Regulation of body temperature
Regulation of body water.

3. Composition: Cellular elements- RBCs, WBCs, Platelets
Plasma- Water + Solids
Solids: 1) Diffusible constituents:
a) Anabolic e.g. glucose, amino acids, and vitamins
b) Catabolic e.g. urea, uric acid, and creatinine.
2) Non diffusible constituents:
e.g. albumin, globulin, and fibrinogen.

4. Purpose of blood analysis:
Serum:
serum = Plasma- Blood clotting factors.
Purpose of blood analysis:
Diagnostic:
any change in the normal constituent, or presence
of abnormal constituent indicate disease state.
2) Prognostic.

5. Preparation of serum sample:
Obtain venous blood using empty centrifuge tube.
Allow the blood to clot.
Rime the clot.
Centrifuge.
Transfer the clean specimen tube using Pasteur pipette, don’t
disturb the clot.

6. Preparation of plasma sample:
Put some anticoagulant in a clean dry centrifuge tube.
Add the venous blood and mix.
Centrifuge.
Transfer the top clear plasma to a clean specimen tube.

7. are chemicals which prevent clotting of blood.
Anticoagulant:
are chemicals which prevent clotting of blood.
1.Heparin:
MOA. antithrombin “ prevent conversion of prothrombin to thrombin”
Disad. High cost.
2. Oxalates: Na, K, Li, or NH4 salts are used.
MOA. Forms insoluble salts with Ca ions.
Disad. - Inhibit lactate dehydrogenase
- Na, K salt should not be used in determination of electrolytes
as this lead to significant error.

8. 3. E.D.T.A: (ethylene di amine tetra acetic acid) MOA. Bind to Ca++.
Adv. Prevents clumping of platelets.
4. Na Fluoride: (enzyme poison)
MOA. Inhibit glycolysis, therefore its used in sample for determination
of blood sugar. ”can’t be used for determination of enzymes”
5. Tri Na Citrate:
used to determine ESR.
6. Acid Citrate Dextrose solution:
used mainly to study platelets and to preserve blood.

9. Deproteinization: Precipitate the proteins, Why?
→ because the proteins have certain U-V absorption and could
give false reading.
Also, proteins are colloids which make the solution
turbid and difficult to read on the spectrophotometer.

10. Deproteinization agents:
Trichloroacetic acid
Tungestic acid
Zinc hydroxide
Organic substances.

11. Diabetes mellitus: Type: IDDM NIDDM Diagnosis of Diabetes mellitus:
1) if the patient has symptoms:
measurement of fasting plasma glucose level
measurement of two hours postprandial plasma glucose level
2) if the patient has no symptoms:
Oral glucose tolerance test (OGTT)

12. Glucose conc. (mg/dl) Two hours after 75g glucose Fasting 65-140 mg/dl
Normal
mg/dl
mg/dl
Impaired glucose tolerance
>200 mg/dl
> 126mg/dl
Diabetes mellitus

13. Investigations for diagnosis of diabetes mellitus:
Glucose in the urine (glucose urea)
> 180 mg/dl.
2) Glycated proteins:
a) glycated hemoglobin
b) glycated albumin ( fructosamine)
3) Microalbuminuria:

14. Determination of blood glucose
Serum glucose is determined by tow methods:
1) Oxidation method “Enzymatic method”.
2) Alkaline copper reduction method “ Asatoor and king method”

15. Oxidation method “Enzymatic method”.
Principle:
Glucose is oxidized according to the following reaction:
Glucose glucose oxidase Gluconic acid + H2O2
H2O2 +phenol + amino-4-antipyrine peroxidase Quinoneimine + H2O

16. Procedure: Mix and incubate in the water bath at 37°C for 10 min, then
Read at wave length 505nm.
Standard
Sample -
0.1 ml
serum
standard
1 ml
Working reagent

17. Calculation: Serum glucose (mg/ dl) = A sample X standard conc.
A standard
Normal serum level:
mg/ dl.

18. Clinical Significance of determination of blood glucose:
Hyperglycemia: blood glucose level > 110 mg/dl.
Causes:
1) Diabetes mellitus
2) Hyperactivity of thyroid, adrenal, pituitary gland
3) Acromegaly
4) Asphyxia
5) Exercise or stress
6) Pancreatic carcinoma
7) Acute pancreatitis.

19. Clinical Significance of determination of blood glucose:
Hypoglycemia: blood glucose level <70 mg/dl.
Causes:
1) Overdose of insulin.
2) Hypoactivity of thyroid, adrenal, pituitary gland
3) Glycogen storage disease.

20. Renal glucose threshold:
The ability of renal tubules to reabsorb glucose
is 180 mg/dl “renal glucose threshold”
If blood sugar level rises above this value the glucose appears
in the urine “Glucosurea”