To investigate bleeding disorders, determination of platelets is required.
Normal range: 2,50,000 – 5,00,000/cu mm (ml).
Many times prolonged bleeding and poor clot formation is due to Thrombocytopenia (decreased platelet count).
It is also found in aplastic anemia, megaloblastic anemia, hypersplenism and acute leukemia.
Thrombocytosis (increased platelet count) is found in polycythemia vera, following splenectomy and in chronic myelogenous leukemia.
Splenectomy – is the surgical removal of the spleen.
Hypersplenism – in this spleen is enlarged.
Meaning of other terms is given in routine blood tests.

When a blood vessel is damaged, bleeding stops by a process called hemostasis. First, the blood vessel contracts which reduces blood flow. Second, platelets form a plug, and third, clot formation occurs.

In blood coagulation, thrombin which is formed from prothrombin acts on soluble fibrinogen in the presence of Ca++ ions to form numerous insoluble fibrin threads. These threads criss–cross with one another. Erythrocytes (RBC) and Leucocytes (WBC) are entangled in their interstices and clots are formed.
The routine coagulation test is carried out for:

1. Patients who have to undergo a major surgery.
2. Patients who have a history of spontaneous bleeding after trauma or surgery.

The routine hemorrhagic disorder tests include:

1. Bleeding time.
2. Clotting time.
3. Clot retraction and lysis time.
4. Prothrombin time.
5. Plasma recalcification time.
6. Partial thromboplastin time.
7. Activated partial thromboplastin time.
8. Thrombin time.
9. Fibrinogen determination.
10. Protamine sulphate test.

Normal Range

Bleeding time	1 – 5 minutes
Blood Clotting time	4 – 5 minutes
Clot reaction & lysis time	Of 50% of clot retraction occurs at the end of 1 hr at 37 C, it is abnormal. Normal lysis time is 72 hrs.
Prothrombin Time	14 (I) 2 seconds
Plasma recalcification time (RT)	Platelet rich plasma: 100 – 150 seconds Platelet poor plasma: 135 – 240 seconds
Partial thromboplastin time (PTT)	60 to 80 seconds
Activated partial thromboplastin time (APTT)	35 to 40 seconds
Thrombin Time	15 to 20 seconds
Fibrinogen	200 to 400 mg/dl
Protamine Sulphate test	if – to ++ is normal

1. Sickle Cell Anemia Screening.
2. Determination of Fetal Hemoglobin.
3. Determination of Osmotic Fragility of Red Blood Cells.
4. Determination of Lupus Erythematosus (LE) Cell.
5. Determination of Heinz Bodies.
6. Determination of Blood Parasites.
7. Determination of Glucose: 6 – Phosphate Dehydrogenase (46–PD).
8. Determination of Iron and Total Iron Binding Capacity (TIBC) in Serum.
9. Determination of Electrophoretic Fractionation of Hemoglobin.
10. Microscopic Examination of Bone Marrow and Detection of Iron.
11. Different tests to be performed in Different Bleeding Disorders.

Sickle Cell Anemia Screening

1. Normal blood does not have sickle cells.
2. Clinical findings in S C Anemia.
3. Normocytic, normochromic red cells.
4. Presence of nucleated RBC, sickle cells and Howell–Jolly bodies.
5. Increased reticulocytes.
6. Increased platelets.
7. Decrease in osmotic fragility of RBC.
8. Hemoglobin electrophoresis shows particular hemoglobin.
9. Positive sickle cell test.

Determination of Fetal Hemoglobin
High level of fetal hemoglobin (HBF) is found in B–thalassemia major patient. Mild but significant rise is observed in (HBF) in B–thalassemia trait, sickle cell disease and in other congenital or acquired hematological conditions. Various percentage of HBF observed.

Condition	HBF %
Normal	<1% (Adults)
B – thalassemia major	10–98%
Thalassemia	Reduced
Sickle cell trait	Normal
Sickle cell anemia	1–20%

Determination of Osmotic Fragility of Red Blood Cells
This is useful in determining hereditary spherocytosis, thalassemia and sick cell anemia. Here the Median Corpuscular Fragility (MCF) is determined.

Condition	MCF
Normal	0.45% or little lower.
Hereditary spherocytosis.	0.5%.
Sickle Cell Anemia and Thalassemia.	0.35% to 0.4%.

Determination of Lupus Erythematosus (LE) Cell
Lupus Erythematosus is a disorder of collagen. Collagen is a fibrous, insoluble protein in connective tissue. If the test shows the presence of LE cells, it indicates the disorder.

Determination of Heinz Bodies
It is useful in detecting metabolic defects caused due to the deficiency of G–6 – PD or glutathione. The test is positive even when a patient is undergoing therapy with drugs such as anti–malarials and suphonamides.

Detection of Blood Parasites
Given in Routine Hematological (Blood) Tests.

Determination of Glucose: 6 – Phosphate Dehydrogenase (G6 – PD)
Glucose – 6 – phosphate dehydrogenase deficiency is hereditary disorder. Presence of this enzyme is required in red blood cells to protect hemoglobin from oxidation. Absence of this enzyme is harmless unless red blood cells are exposed to antipyretics, anti–malarial drugs and sulphonamides.

Observations	Decolorization time
Normal	30 – 60 minutes.
4 – 6 PD deficient	140 minutes to 24 hours.
4 – 6 carriers	90 minutes to several hours.

Determination of Iron and Total Iron Binding Capacity (TIBC) in Serum
Normal Values:
Serum Iron: 60 – 150 mg/dl
Serum TIBC: 270 – 380 mg/dl

• Decrease in the serum iron level is due to chronic blood loss or nephrosis (any disorder of the kidney that is caused by any degenerative process other than infections).
• Increase in the serum iron level is due to hemolytic anemia, lead poisoning, pyridoxine deficiency, pernicious anemia and necrotic hepatitis (Necrosis means death of a small area of tissue within an organ).
• Decrease in serum TIBC is found in cirrhosis (a type of permanent and progressive liver damage), hemochromatosis and nephrosis.
• Increase in serum TIBC is found in different states of chronic iron deficiency and nephrosis.

Determination of Electrophoretic Fractionation of Hemoglobin
Hemoglobin is a conjugated protein when it is subjected to an electrical field, the different proteins depending upon their speeds towards the anode (at pH 8.9). Normal and abnormal hemoglobin have different velocities. This helps in identifying congenital disorders of hemoglobin.

Blood Group	Father	Mother	Possible Group Of Child
	A	A	A
	A	O	A or O
	A B	B	AB or A or B
	A	B	A or B or O or AB

This test is performed to detect presence of Rh antibodies or other antibodies in patient’s serum, in the following cases:

1. To check whether an Rh negative woman (married to Rh positive husband) has developed anti Rh antibodies.
2. Anti D may be produced in the blood of any Rh negative person by exposure to D antigen by
   • Transfusion of Rh positive blood.
   • Pregnancy, if the infant is Rh positive (if father is Rh positive).
   • Abortion of Rh positive fetus.

This test is performed to detect anti D antibody or other antibodies attached to red blood cells in the blood stream. It is done in the following conditions.

• When there is Rh +ve baby in the womb of a Rh –ve woman, to defect hemolytic anemia (described in hemolytic disease of newborn).
• Transfusion reactions.
• Drug induced red cell sensitization.
• Autoimmune hemolytic anemia.

This is done before transfusion of blood to a patient of the cross–matching as non–compatible blood should never be transfused. Minor cross match results are also important but in an emergency one can use blood with minor incompatibility.

The antiglobulin cross–match, if compatible, is a high degree of assurance that the transfused blood will be successfully accepted by the patient.

Semi–quantitative determination of Anti–D Antibody Titer
This is useful to defect hemolytic disease of the newborn.

Tests to be Performed and Criteria for Blood Transfusion
Most important tests performed are:

1. Blood grouping and Rh factor.
2. Antibody detection by indirect antiglobulin (Coomb’s) test for a typical, incomplete antibodies.

Other tests performed on collected blood are

1. Hepatitis – B surface antigen.
2. AIDS antibodies.
3. Syphilis (VDRL) test.
4. Malarial parasites.

Criteria accepted for selection of donor

• The age should be above 18 – 60 years.
• Hemoglobin should not be less than 12.5 g/dl.
• Interval of 16 weeks should be there between two donations.
• Pulse rate should be regular and between to 78 – 100/min.
• Accepted Blood Pressure.
  • Systolic: between 90 – 150 mm Hg.
  • Diastolic: between 50 – 100 mm Hg.
• Body temperature of donor should be normal.
• Absence of any chronic disease.

The donor is rejected if

1. There is a history of viral hepatitis or AIDS.
2. There is a history of malaria for the last three years.
3. If the patient has received immunization against rabies, mumps, rubella, small pox within two months.
4. If the donor has undergone dental surgery within 72 hours.

Glucose tolerance means the ability of the body to utilize glucose in blood circulation. Glucose tolerance is reduced in diabetes mellitus and in certain endocrine gland (Endocrine Gland: a ductless body organ which produces hormones that affect and help control various other organs. There are several such glands – thyroid, parathyroid, ovaries, testes, adrenals, pineal, pituitary and pancreas islet cells) disorders like hyperthyroidism, hyperpituitarism and hyperadrenalism.

Blood sugar in the case of a normal person remains fairly constant throughout the day. There is temporary rise in blood sugar levels after food which depends upon the type of food consumed. This increase remains up to two to three hours and then returns to normal. Diminished glucose tolerance is observed when the ability of the body to utilize glucose decreases. The rise in blood sugar or glucose is greater than in a normal person. Also, a return of blood glucose levels to the normal fasting level is slow.
This is observed in 1.

1. Diabetes mellitus.
2. Hyperactivity of thyroid, pituitary and adrenals.
3. Injection of cortisone like hormone.
4. Increased secretion of the growth hormone.
5. Very severe liver disease.
6. Glycogen storage disease of the liver due to the limited capacity of a person to store excess glycogen.
7. Severe infection of staphylococcal bacilli or even common cold.

Time	Fasting	1/2 hour	1 hour	1 1/2 hours	2 hours	2 1/2 hours
		After taking glucose
3 Blood Glucose gm/dl	70	130	145	105	75	80
Urine Glucose – Absent throughout the test
Normal renal threshold for glucose = 150 – 170 mg/dl.

Raised renal threshold after ingestion of glucose up to 250 – 300 mg/dl occurs with increasing age and prolonged diabetes mellitus. Lowered renal threshold (130 – 150 gm/dl) is observed in abnormality of tubular re–absorption of glucose.
Extended glucose tolerance curve (instead of two and a half hours goes up to four to five hours) is observed in insulin secreting tumors of the pancreas. It is also observed in Simmond’s disease which causes hypoglycemia (means reduction of sugar or glucose levels. It drops to below 60 mg/dl and affects brain cells).

The red blood cells of a normal person and children above six months old contain three genetically determined hemoglobin species HbA – 90%, HbA2 – 2.5% and HbF – 0.5% of total hemoglobin content of RBC. Besides these hemoglobins, other variants of hemoglobin present are HbAla – 1.6%, HbAlb – 0.8% and HbAlc – 4%. They are products of non–enzymatic, post–synthetic transformation of HbA. Their rate of formation depends upon the lifespan of RBCs and its mean hexose concentration. These are collectively measured as HbA – abc or HbAl. HbAl indicates the average blood sugar concentration for an extended time period. It remains unaffected by the short term fluctuations in blood sugar levels. HbAl levels term fluctuations in blood sugar level. HbAl levels reflect carbohydrate imbalance better than fasting glucose concentration or the GTT (Glucose Tolerance Test). The determination of HbAl is a convenient and suitable test as it helps evaluate the adequacy of diabetic control in the prevention of various diabetic complications.
Normal Range: 4 – 7%.

Concentration of blood sugar (glucose) remains steady up to 100 mg/dl during 24 hours. After partaking of food, it increases up to 140 – 150 mg/dl.
The ways by which glucose is added to the blood:

1. Absorption from the intestine.
2. By the breakdown of liver glycogen (glycogen is a starch like carbohydrate) to glucose.
3. By gluconeogenesis (a form of glucose from non–carbohydrates).

The ways by which glucose is removed from the blood:

1. By synthesis of fats e.g. triglycerides.
2. Conversion to liver glycogen.
3. Conversion to muscle glycogen.
4. In synthesis of glycoproteins and lactose etc.

When these processes are performed in the right balance, the blood sugar level remains within the normal limits of about 70 – 110 mg/dl. throughout the day. (Average 100 mg/dl).

• The liver plays an important part by taking glucose from the blood and converting it into glycogen.
• Releasing glucose from glycogen and converting pyruvate to glucose.
• Muscle glycogen does not contribute directly to blood sugar. Glycogenolysis in the muscle produces locate, which is converted into glucose in the liver.
• Kidney plays its part by the re–absorption of glucose when the blood glucose level is below 150 – 170 mg/dl (threshold level).

In normal individuals, the blood glucose level does not rise above the threshold level.
The following hormones play a part:
A. Insulin: It is secreted by the pancreas. It controls blood sugar in the following ways:

• By increasing transport of glucose across the cell membrane.
• By promoting oxidation of glucose or glycogen to pyruvate and lactate.
• By increasing the formation of glucose from non–carbohydrates.
• By decreasing conversion of glycogen to glucose.
• By inhibiting conversion of protein to glucose and favoring a synthesis of protein from amino acids.
• By promoting the transfer of potassium phosphate and amino acids into the cells.

Glucose is also regulated by other hormones than insulin but to a lesser extent. These hormones antagonize the action of insulin. They are:
B. Thyroxine (secreted by the thyroid gland).
C. Glucagon (secreted by alpha cells of the pancreas).
D. Growth hormone (secreted by the pituitary gland).
E. Glucocorticoids (secreted by the adrenal cortex).
F. Epinephrine (secreted by the adrenal medulla).
These hormones increase blood sugar levels by:

1. By increasing absorption of glucose from the intestine.
2. Decreasing the oxidation of glucose or glycogen to pyruvate and lactate.
3. Preventing synthesis of glycogen.
4. Stimulating synthesis of glycogen from glucose.
5. Stimulating the formation of glucose from non–carbohydrates.

Glucose travels throughout body and when it comes in contact with beta cells of pancreas, they secrete insulin in response to increased levels of glucose in the blood. This insulin attaches itself to each cell surface to make glucose molecules enter the cells to be used for energy. The cells require very little energy and the rest is stored as adenosine triphosphate (ATP). This is then used by the body. Everything in excess is stored by the muscles, the liver etc. In case of a normal person, the fasting blood sugar level is 70 – 110 mg/dl. It does not fall below 70 mg/dl due to the antagonizing effect of other hormones mentioned above. When the blood sugar rises to a relatively high level, the kidney filters it through glomeruli (network of tiny blood vessels) but it returns to the blood by the re–absorption system of its tubules.

Diabetes Mellitus
Diabetes Mellitus occurs when blood glucose is too high due to very less insulin or none at all. Sometimes, the pancreas produces normal amounts of insulin but the body needs more than the normal. As a result of this, the body suffers from a lack of energy. People with diabetes often complain of weakness and tiredness.
Characteristics of diabetes mellitus

• Raised fasting blood sugar levels.
• The amount of urine passed is increased often up to 5 to 6 liters of pale urine.
• Thirst.
• Due to lack of insulin glucose is not utilized by cells. Energy is obtained through:
  • Body proteins.
  • Fats.

This causes muscular weakness and results in weight loss. Also, the end product of protein is urea which increases above the normal range and causes discomfort.

• In severe cases, more fat is used for energy. The excessive oxidation of fatty acids increases ketone bodies in the blood and there is also an increase in cholesterol synthesis.
• In severe cases, the plasma bicarbonate decreases which results in a fall of blood pH and leads to a loss of sodium ions.

The last two conditions may result in diabetic coma.

Types of Diabetes
There are two major types of diabetes. Type I and Type II
Type I Diabetes
It is insulin dependent diabetes mellitus (IDDM) or “Juvenile diabetes.” This occurs when beta cells of the pancreas do not function to produce insulin. Patients take supplementary insulin by injections because if taken orally it will get digested, as insulin is a protein in nature. Insulin is either obtained from the pancreas of cattle or pigs, or produced synthetically, using recombinant DNA (deoxyribonucleic acid). This synthetic insulin does not stimulate the formation of antibodies against insulin as the animal insulin may do.
Most often young people get this type of diabetes and the onset is fast. Sometimes, older people also get Type I diabetes which has a slow onset and acts like Type II for a long period.
Type II Diabetes
This is non–insulin dependent diabetes mellitus (NIDDM). The body produces some insulin but the requirement is more. It may be due to reduction in the number of insulin producing beta cells. Due to this, glucose is unable to enter the cells as interaction between insulin and its receptor is less effective. People with this type of diabetes are usually above their ideal weight. Overeating also contributes to it, so diet is primary treatment for this type of diabetes. Generally people above the age of 40 get this type of diabetes, but it may be found in younger people also. About 85% of all people who have diabetes, have Type II diabetes.

Other Types of Diabetes
Some people are not diabetic but may have an increased risk of developing diabetes.
Pre–diabetes
A person both of whose parents are diabetic, or who has an identical twin who develops diabetes, although he/she may not have diabetes initially is considered a prospect for the disease.
Latent diabetes
Some people have elevated blood glucose levels only in certain times of stress e.g. in pregnancy or during severe infection. They suffer from diabetes but become normal when pregnancy ends or after they recover from the infection.

Test	Normal Values
Creatine Phospokinase	Male	16 – 110 U/L
	Female	16 – 94 U/L
Creatine Kinase (MB)	Normally, CK–MB activity is less than 5 U/lt. And accounts for less than 3 per cent of the total CPK activity. Myocardial infarction is suggested by CK – MB > 5% of Total CK Activity.
Lactate Dehydrogenase	160 – 319 U/lt. (At 30° C)
Serum Amylase	Up to 95 U/lt
Serum Lipase	20 – 300 U/lt. (At 30° C)
Fasting Blood Sugar	80 – 120 mgs/dl
Post Prandial Blood Sugar	up to 160 mgs/dl
Glycosylated Hemoglobin	< 7%	Normal
	7 – 8%	Excellent Control
	8 – 9%	Good Control
	9 – 10%	Fair Control
	> 10%	Poor Control
Serum Cerruloplasmin	0.20 – 0.55 gms/lt
Serum Iron	40 – 130 micrograms/dl
Serum Iron Binding Capacity	210 – 385 micrograms/dl
Plasma Fibrinogen	200 – 400 mgs/dl
Serum Ferritin	Male	20 – 400 ng/ml
	Female	10 – 130 ng/ml
Serum Ammonia	Normal: 11–35 Umol/lt
Serum Uric Acid	2 – 7 mgs/dl
Serum Acid Phosphatase	Up to 12 U/lt
Leptospira IgM	< 10	Negative
	10 – 20	Low Positive
	> 20	Positive
Tuberculosis IgG	Less than 225 Units/ml
Tuberculosis IgA	Less than 350 Units/ml
Fructosamine	Normal Range	1.6 – 2.6 m mol/l
	Satisfactory Control	2.6 – 3.2 m mol/l
	Mediocre Control	3.6 – 3.7 m mol/l
	Poor Control	3.7 – 4.5 m mol/l
Carcinoembryonic Antigen (CEA)	Smokers	Not detectable to 5.2 ng/ml
Non–smokers	Not detectable to 3.4 ng/ml
Alpha Feto Protein	0 – 20 IU/ml
	In Pregnancy
	Maternal Serum	Gestational Age
	15 weeks	8.9 – 61.1
	16 weeks	10.2 – 56.2
	17 weeks	15.6 – 59.3
	18 weeks	16.5 – 79.3
	19 weeks	18.9 – 86.1
	20 weeks	18.5 – 103.9