Friday, 28 October 2016

SOPs OF BLOOD SMEAR AND STOOL






THICK BLOOD SMEAR PREPARATION FROM FINGER PRICK


Purpose
This procedure provides instructions for preparation of thick blood smears for hemoparasites from finger prick.
Materials
Reagents Supplies
•Absolute alcohol
•Cotton wool
•Gauze
•Protective gears
•Alcohol swab
•Jar
•Wooden applicator
•Drying rack
•Blood lancet
•Sharps container
Sample
Whole blood
Special Safety Precautions
All specimens must be regarded as potentially infectious.
Refer to Infection Prevention and Control Guidelines for additional safety considerations.
Quality Control
Refer to the Standard blood smear template.
Procedure
Follow the activities in the table below.
Step & Action
1.Label the slide with patient ID number using permanent marker
2.Clean the ring finger by using alcohol swab and allow to dry
3.Firmly press sterile disposable lancet to puncture the finger tip
 4.1 Wipe off the first drop of blood and slightly apply gentle pressure.
 4.2 Place a small drop of blood at the center of pre-cleaned scratch free,   labeled glass slide
5.By using a corner of another slide or an applicator stick spread the blood drop in a circular pattern making about 1.5cm2 Avoid making too thick smears
6.Allows the blood smear to air dry protected from direct sunlight, dust and insects
7.Transport the dried blood smear accompanied with the patient request form within an hour
References
       htpp.//www.dpd.cdc.gov
      Health Laboratory Safety and Waste Management manual
               

STOOL CULTURE COLLECTION PROCEDURE


Purpose
This procedure provides instructions for the collection of a urine specimen. 
Materials
Reagents, Supplies,  Equipment
Not applicable.
•Screw-capped stool containers
•Gloves
•Soap
•Permanent marking pens
•Transport box
Special Safety Precautions
All specimens must be regarded as potentially infectious
Refer to the Specimen Management and Waste Disposal Manual
Procedure
Follow the activities in the table below to collect a urine specimen.
Step &Action
1.Collection, Labeling and Registration
1.1.Properly identify the patient with at least the Patient’s name, PID, date of birth, and Doctor’s name.
1.2.Wash hands with soap and water
1.3.Wear gloves
1.4.Have all the needed specimen collection tools on hand Refer to specimen test catalog.
1.5.Explain the specimen collection procedure to the patient
1.5.1.Provide patient with a wide-mouth plastic, screw-capped stool container
1.5.2.Instruct patient to deposit approximately one spoonful (Spoon provided) into the screw-capped stool container.
1.5.3.Instruct patient to seal the specimen container tightly.
1.5.4.Ensure  the stool in the container is sent to the laboratory.
1.6.Label each specimen immediately with identifying information – patient identification (PID), date and time of collection.
1.7.Fill out lab request/report form for each specimen with date, time and initials of collector.
1.8.Check that PID on the specimen container matches with that on the lab request/report(Check specific name of form)  form.

NOTE: This labeling and matching is the primary responsibility of the collector and must be done carefully and precisely.
1.9.Place collected specimens in a transport container at an appropriate temperature, awaiting transport to the laboratory.
1.10.Dispose of gloves and wash hands after each specimen collection/handling.
1.11  Enter specimen information into specimen register book

References
               
      Practical Laboratory Manual for Health Centers in East Africa, AMREF, Jane Carter Orgenes  Lema  1994, pg. 170-171.

      District Laboratory Practice in Tropical Countries, Monica Cheesebrough Part I, 1998, 

Thursday, 20 October 2016

CARBOHYDRATE METABOLISM




Carbohydrate metabolism



Definitions (15 minutes)


ACTIVITY:

Define the following terms (Glycolysis, Glycogenesis, Glycogenolysis, Gluconeogenesis, Glucose Tolerance)
What is Carbohydrates Metabolism?



Definitions:
·         Glycolysis is the process whereby glucose molecule is hydrolyzed in the body in order to obtain ATP and Pyruvate.
·         Glycogenesis; the process of glycogen formation which  takes place in the liver and stored there.
·         Glycogenolysis is the process of splitting glycogen. Glycogen molecules do not remain in the cell permanently but are eventually broken apart (hydrolysed). Glycogenolysis alone can maintain homeostasis of blood glucose concentrate for only a few hours, since the body can store only small amount of glycogen.
Gluconeogenesis
·         Gluconeogenesis means the formation of the new glucose in the sense that it is made from protein or less frequently from the glycerol or fats which chiefly occurs in the liver.
·         The new glucose produced from protein or fats by gluconeogenesis diffuses out of the liver cells into the blood.
·         Glucose Tolerance is ability of the body to tolerate or cope with the standard dose of glucose.

Metabolism refers to the complex, interactive set of chemical process that makes life possible.
·         It is made up of two major processes, catabolism and anabolism. Each of these in turn consists of a series of enzyme-catalyzed chemical reactions known as metabolic pathway.
·         Catabolism releases energy in two forms heat and chemical energy. The amount heat generated is relatively large; so large in fact it would hardboiled cells if it were released in one large burst. Fortunately, this does not happen.
·         Heat is practically useless as an energy source for cell in that they cannot use it to do their work. However this heat is important in maintaining the homeostasis of body temperature
·         Chemical energy released by catabolism is more obviously useful. It cannot, however, be used directly for biological reactions. First it must be transferred to the high-energy molecule of adenosine triphosphate (ATP)
·         ATP is one of the most important compounds in the world because it supplies energy directly to the energy-using reactions of all cells in all kind of living organisms. Adding water (H2O) to ATP yield a phosphate group, adenosine diphosphate (ADP) and energy which is used for anabolism and other cell work.
·         The Citric acid cycle is the central metabolic pathway that completes the oxidative degradation of metabolites.


 Carbohydrates Metabolism

·         Carbohydrates are found in most of the foods that we eat. Complex carbohydrates – polysaccharides (such as starches in vegetables, grains and other plant tissue) are broken down into simpler carbohydrates before they are absorbed as Monosaccharides. Carbohydates (CHO) are named because they have one molecule of water for every carbon.  The basic unit is a monosaccharide which contains 4,5 or 6 carbons (and H2O)
·         Cellulose – a major compound of most plant tissue is an important exception to this principle. Since human do not make enzymes that chemically digest this complex carbohydrate (CHO), it passes through our system without being broken down. Also called dietary fibres or roughage, cellulose and other indigestible polysaccharides keep chyme thick enough for the digestive system to push it easily
·         The monosaccharide in the form of glucose is a 6 carbon carbohydrate that is the most useful to the typical human cell. Other important monosaccharide – fructose and galactose are usually converted by liver cells into glucose for use by other cells of the body.


·         Carbohydrate metabolism begins with the movement of glucose through cell membranes.
·         Glucose in the body undergoes one of three metabolic activities:
o   It is catabolised to produce Adenosine Triphosphate (ATP). This occurs in the peripheral tissues, like in the brain, muscle and kidney.
o   It is stored as glycogen. This storage occurs in liver and muscle.
o   It is converted to fatty acids. These are stored in adipose tissue as triglycerides.
·         If glucose is needed immediately upon entering the cells to supply energy, it begins the metabolic process called glycolysis (catabolism).
·         If glucose is needed immediately upon entering the cells to supply energy, it begins the metabolic process called glycolysis (catabolism).

·         Immediately on reaching the interior of a cell, glucose reacts with ATP to form glucose 6-phosphate. This step, prepares glucose for further metabolic reactions.
·         Glucokinase is an enzyme that facilitates conversion of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver, pancreas, gut, and brain of humans
·         The major reason for this step is to prevent glucose diffusion out of the cell.
·         Glycolysis is the first process of carbohydrate catabolism. It is a metabolic pathway found in the cytoplasm of cells that converts one molecule of glucose into two molecules of pyruvate, (pyruvic acid) and makes energy in the form of two net molecules of ATP. Glycolysis results in the net production of 2 ATP and 2 NADH ions.

         Glycolysis consists of a series of chemical reactions. A specific enzyme catalyzes each of these reactions. Carbohydrate metabolism starts with glycolysis, which releases energy from glucose or glycogen to form two molecules of pyruvate.
·         Glycolysis is an anaerobic process, that is, it does not use oxygen. It is the only process that provide cells with energy when their oxygen supply is inadequate or even absent
         Glycolysis is an essential process because it prepares glucose for second step in catabolism, namely the citric acid cycle.
         Glucose itself  cannot enter the cycle but it must be converted to pyruvic acid then to a compound called acetyl-CoA (coenzyme A) Then acetyl Co-A enters the Krebs cycle (or citric acid cycle), an oxygen-requiring process, through which they are completely oxidized.

·         During short bursts of strenuous activity, muscle cells use pyruvate and lactate from anaerobic respiration to supplement the ATP production from the slower aerobic respiration.
The citric acid cycle
  • The citric acid cycle is the final stage for the metabolism of Carbohydrates, Fats, and Amino acids.
  • This is also called the Krebs cycle or the Tricarboxylic acid cycle or Oxidative cycle.
  • In carbohydrate catabolism (the breakdown of sugars), the Citric Acid Cycle follows Glycolysis, which breaks down Glucose (a six –carbon molecule) into Pyruvate (a three-carbon molecule).
  • Glycolysis is the first stage of the whole human respiration cycle and the whole process is to break down Glucose or Glycogen into Pyruvic acid through enzymatic reaction within the cytoplasm of the cells.
  • The process results in the formation of two molecules of ATP (three if the starting product was Glycogen). Without the presence of oxygen, pyruvic acid is changed to Lactic acid and the energy production process ends.        
  • The citric acid cycle is an 8-step process The net energy gain from one cycle is 3 NADH, 1 FADH, and 1 ATP. Thus, the total amount of energy yield from one whole glucose molecule (2 pyruvate molecules) is 6 NADH, 2 FADH, and 2 ATP.  This is a vital part of our life.


 Glucose Tolerance

·         Glucose Tolerance is the ability of the body to tolerate or cope with the standard dose of glucose.

 Control of Glucose is by many mechanisms and hormones

·          The complex mechanism that normally maintains homeostasis of blood glucose concentration consists of hormonal and neural devices.
·         Five endocrine glands are involved at least 8 hormones are secreted by those glands, which functions as the key parts of the glucose homeostasis mechanisms.
The glands are:
·         Pancreatic islets
·         Adrenal medulla
·         Adrenal cortex
·         Anterior pituitary gland
·         Thyroid gland
The pancreatic islets
·         β-cells of the pancreatic islets secrete the most well known sugar-regulating hormone of all – insulin.  It is known as a glucose lowering hormone. 
·         Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor, which in turn starts many activities. In this way it decreases blood glucose level by moving the glucose into cells.
·         It also increases the activity of enzymes of glucose metabolism.
·         Insulin deficiency will result into increase glucose level in blood, slow glycogenesis and low glycogen storage decrease glucose catabolism.





All the other hormones describe are typically glucose raising:
Pancreatic alpha cells
·         α-cell of the pancreatic islets secretes the sugar-regulating hormone glucagon.
·         Glucagon tends to increase blood glucose level
·         Glucagon increases the activity of an enzyme that accelerates liver glycogenolysis and releases more of its product, glucose into the blood
Adrenal medulla.
·         Epinephrine (adrenaline) is a hormone secreted in large amount by the adrenal medulla in times of emotional or physical stress
·         Epinephrine accelerates both liver and muscle glycogenolysis. Whereas, glucagon accelerates  only  liver glycogenolysis
·         Both hormone increase the blood glucose level
Adrenal cortex
·         Adrenocorticotropic hormone (ACTH) and glucocorticoids (e.g. cortisone) are two hormone that increase blood glucose concentration. 
·         Glucocorticoids accelerate gluconeogenesis.

Anterior pituitary gland
·         Growth hormone (GH) made by the anterior pituitary also increase blood glucose level but by different mechanism
·         GH causes the shift from CHO to fat metabolism.
Thyroid Gland
·         TSH from the anterior pituitary gland and its target secretion, thyroid hormone (T3 and T4) has a complex effect on metabolism.
·         Some of these raise and some lower the glucose level.
·         One of the effects of the thyroid is to accelerate catabolism, and since glucose is the body’s preferred fuel the result may be decrease in the blood level

Glucose intolerance
·         Disorders that affect glucose tolerance often are from abnormalities in the endocrine glands or tissues listed above.
·         Hyperglycemia means to have frequently high blood glucose levels.  It is often due to lack of functional insulin from ineffective or destruction of pancreatic beta islet cells.   With lack of insulin, there is decrease in glucose uptake and metabolism causing excess glycogenolysis and the switching of metabolism to fatty acid metabolism for energy.
·         Hyperglycemia from less common causes can result from excessive secretion of epinephrine, corticosteroids, glucagon or thyroid hormones.
·         Hypoglycemia is less common but may result from excessive insulin release from a islet cell tumour or from a patient who receives too much insulin during therapy.