Lecture #2
The first step in all chemical analysis is the collection of a specimen. It may be a blood specimen, urine, stool, CSF, etc. Most of the analysis in clinical chemistry is done either on a whole blood, plasma or serum. Blood is by far the most frequent body fluid used for analytical purposes.
Amount of samples needed:
1. Macromethod – 1 ml and above
2. Micromethod – 0.1 – 0.9 ml
3. Ultramicromethod – 0.01 – 0.09 ml
4. Nanoliter method – 0.001 – 0.009 ml
General precautions in collecting blood samples:
1. Avoid prolonged application of tourniquet as this will result in stasis which may alter chemical values.
2. Do not extract blood from patients while they are receiving intravenous medication because this solution may influence the chemical analysis.
3. Syringes and needles used to extract blood must be chemically clean and dry.
4. Blood specimens obtained must be placed in appropriate containers for each specific test. The blood specimen collected with anticoagulants must be well mixed to prevent coagulation.
Specific precautions in collecting blood samples:
1. Fasting
a. The normal fasting hour is 8 to 14 hours.
b. Elevated blood glucose, potassium and lipids like cholesterol and neutral fats are seen in patients not under NPO (nothing per orem) and the reverse is true with inorganic phosphorous.
c. Prolonged fasting has been associated with elevation in serum bilirubin, plasma triglycerides, glycerol, free fatty acids and a decrease in plasma glucose.
2. Diet
a. After a meal, there is an increase in plasma potassium and triglycerides.
b. After a meal of high fat content, there is an increase alkaline phosphatase.
c. After a high meat protein diet, there is an increase serum urea, ammonia and urate but not creatinine.
d. High ratio of saturated to unsaturated fatty acids decreases serum cholesterol.
e. Diet rich in purines increases serum urate.
f. Presence of serotonin in bananas, pineapples, tomatoes and avocadoes elevates excretion of 5–hydroxyindoleacetic acid.
g. Caffeine increases plasma free fatty acids and catecholamines.
3. Exercise
a. Transient effects: increase in free fatty acids, alanine and lactate.
b. Long–term effects: increased creatinine kinase, aldolase, aspartate aminotransferase and lactate dehydrogenase.
c. Effects of long term physical training: increase in plasma testosterone, androstenedione and luetenizing hormone.
4. Alcohol ingestion
a. Increased plasma lactate, acetaldehyde, acetate and uric acid.
b. Higher plasma HDL cholesterol has been observed in chronic alcoholics as well as increase gamma glutamyl transferase (GGT) and mean corpuscular volume (MCV).
5. Tobacco smoking
a. Acute effects: increased plasma catecholamines and serum cortisol resulting to increased neutrophils, monocytes, free fatty acids and decreased eosinophils.
b. Chronic effects: increased blood hemoglobin concentration, MCV and WBC count.
6. Posture
a. A change of position from supine to upright causes an increase in albumin, total protein, enzymes, calcium, bilirubin, cholesterol, triglycerides and drugs bound to protein
7. Stress
a. Affects adrenal hormone secretion leading to anxiety than hyperventilation which causes:
(1) Disturbances in acid–base balance
(2) Increase in serum lactate
(3) Increase in plasma free fatty acid
8. Tourniquet application
a. Blood specimen for LDH should be drawn without tourniquet as this will cause an increase value.
b. Prolonged tourniquet application before venipuncture causes significant increases in enzymes, proteins and protein bound substances like cholesterol, triglycerides, calcium, bilirubin and iron.
Methods of collecting blood samples:
1. Skin Puncture
a. This is the method of choice in pediatric patients and is useful in adults with extreme obesity, severe burns and thrombotic tendencies. It is also preferred in geriatric patients.
b. Use for micromethod, ultramicromethod and nanoliter method.
c. Puncture site:
(1) Free margin of ear lobe
(2) Palmar surface of 3rd and 4th finger
(3) Lateral or medial plantar heel surface
d. Sites to avoid
(1) Edematous area
(2) Cyanotic area
(3) Scarred area
(4) Heavily calloused area
(5) Traumatized area
e. Cases wherein capillary puncture should be use:
(1) When venipuncture is impractical, e.g. in infants
and in cases of extensive burns.
(2) When small quantities of blood are required in
the examination
(3) When the patient is bed–ridden and the
operator feels that the skin or capillary puncture is easier to manipulate.
(4) When the blood examination requires free
flowing capillary blood.
f. Disadvantages of skin or capillary puncture:
(1) Small amount of blood is obtained and the
examination can’t be repeated.
(2) Capillary or peripheral blood frequently
hemolyzes.
(3) Precision is poorer in capillary than in venous
blood because of variation in flow and dilution with interstitial fluid.
g. Procedure
(1) The site is first rubbed with gauze pad moistened
with 70% alcohol to remove dirt and epithelial debris.
(2) After the skin has dried and the circulation has
returned, a puncture 2 to 3mm deep is made with a disposable lancet (quick stab manner).
(3) The first drop of blood is discarded or wiped off
as it contains tissue juices.
(4) The second drop is use for examination using
disposable glass micropipettes.
(5) After the needed blood has been obtained, a pad
of sterile gauze is applied to the puncture and the patient is instructed to apply slight pressure until bleeding ceased.
2. Arterial puncture
a. Generally used in the determination of blood gases.
b. Puncture sites:
(1) radial artery – most preferred
(2) brachial artery
(3) femoral artery
(4) scalp arteries in infants
(5) catheterization of umbilical cords
c. Procedures:
(1) Identify the artery to be punctured by its pulsation.
(2) Aseptically clean the puncture site.
(3) An anesthetic is not usually required but a
theoretical error maybe possible owing to the
hyperventilation caused by the pain of the puncture.
(4) Using heparinized syringe, pierce the skin at an angle of 45 to 60 degrees and slowly approach the artery. Blood rushing into the syringe usually forces the plunger back.
(5) After obtaining the blood specimen, expel any aspirated air, remove the needle and place an air tight cap over the tip of the syringe, but the common practice is to force the point of the needle into a cork or rubber stopper.
(6) Apply compression on the puncture site for 2–5 minutes.
(7) Deliver the sample to the laboratory in a biohazard plastic bag with ice.
3. Venipuncture
a. Puncture site
(1) Antecubital fossa (vein in the arm at the bend of the elbow)
Median cephalic vein
Median basilic vein
(1) Veins of the dorsal surface of the hand
(2) Veins of the ankle
(3) External and internal jugular veins
(4) Femoral vein
(5) Superior longitudinal sinus
(6) Long saphenous vein
b. Methods of doing venipuncture
(1) Syringe method
(2) Vacutainer method
(3) Butterfly infusion method
c. Procedure:
(1) Preparing the materials needed.
(2) Applying the tourniquet
(3) Selecting the vein
(4) Applying the antiseptic
(5) Inserting the needle
(6) Withdrawing the blood
(7) Releasing the tourniquet
(8) Withdrawing the needle
(9) Preventing bleeding
d. Methods used to increase the amount of blood in the arm
(1) Using a tourniquet
(2) Having the patient close and open his fist
(3) Massaging the arm
(4) Slapping or thumbing the arm at the site of the
site of the puncture.
(5) Immersing the arm in warm water or covering
with a hot, wet towel for 5 or more minutes prior
to venipuncture.
e. Pointers in doing venipuncture
(1) To prevent hemoconcentration, the tourniquet pressure should not be maintained longer than necessary. The outer end of the tourniquet should be tucked under so that a slight pull will release it.
(2) To distend the veins, the patient is asked to open and close the fist several times.
(3) Even if not seen, veins can usually be felt beneath the skin. In fat persons, veins that show as blue streaks are usually too superficial and too small.
(4) Loosen the tourniquet if blood flows freely; otherwise, leave it in place until the desired amount is obtained. At this time, have the patient open his fist, release the tourniquet, withdraw the syringe and needle and apply gentle pressure to the puncture site with dry gauze or cotton.
(5) The operator must see that the patient’s condition is satisfactory before he is dismissed. If there is any sign of continued discomfort, anxiety, bleeding or shock, the patient should be kept lying down and seen by a physician.
(6) Hemolysis interferes with many examinations and therefore should be immunized.
f. Advantages of venipuncture
(1) Easier and more convenient to obtain an adequate volume of blood suitable for a variety of tests.
(2) It offers the fastest method of collecting samples from a large number of patients.
(3) It reduces the amount of and variety of apparatus to be carried to the hospital wards.
(4) It allows various tests to be repeated.
(5) It also allows the performance of additional tests that maybe repeated.
(6) It reduces the possibility of error resulting from dilution with tissue juices or construction of skin vessels by cold or emotion that may occur in taking blood by finger puncture.
g. Disadvantages of venipuncture
(1) Requires more time and skill.
(2) Require more equipment.
(3) More complications may arise.
(4) Hard to do on infants, children and obese
patients.
h. Advantages of vacutainer
(1) The system makes possible direct sampling from a vein, economically and efficiently.
(2) Disposable needles eliminate the hazard of serum hepatitis transmission, provided the phlebotomist uses proper drawing and needle disposable technique.
(3) Rubber stoppers are color–coded to distinguish whether the tube contains a specific anticoagulant, is a plain tube or is a special tube made chemically clean.
(4) The self sealing stopper prevents problem due to evaporation and contamination.
(5) A prepackaged sterile unit that requires no prior preparation.
(6) A wide range of tube size and contained anticoagulants (2, 3, 5, 7, 10 and 15 ml).
i. The vacutainer blood collection system
(1) A detachable and disposable needle
(2) A reusable tube holder
(3) A disposable, color–coded, collecting tube (with
or without anticoagulant).
Complications in venipuncture and suggestion for their prevention:
I. Immediate local complication
a. Hemoconcentration – increased amount of cellular elements in the blood due to prolonged application of tourniquet (i.e., over 60 seconds).
Suggestion: Release the tourniquet immediately after sufficient blood has entered the syringe.
b. Failure of blood to enter the syringe
1. Excessive pull of plunger collapses the vein
Suggestion:
Slight back and forth movement reduces the force of aspiration
2.Piercing the outercoat of the vein without entering the lumen
Suggestion:
Withdraw the needle slightly and re-enter the vein.
3.Transfixation of veins – piercing through the walls of the veins.
Suggestion:
Withdraw the needle slightly and gently aspirate to see if blood enter. If this fails, the puncture may have to be reported.
c. Hemoconcentration or ecchymoses – subcutaneous effusion of blood resulting to discoloration, pain, swelling and tumor–like mass.
Suggestion: Repeat puncture on another site
d. Circulatory failure – failure of the blood to flow due to nervousness and other emotional factors.
Suggestion:
This is not beyond the control of the Medical Technologist. Call the physician.
e. Syncope – fainting due to sudden insufficiency of blood supply to the brain.
Suggestion: Let the patient lie flat on the bed and call a physician.
f. Continued bleeding – this may occur in patients with a hemorrhagic tendency.
Suggestion: Local pressure controls bleeding.
II. Late local complication
· These are under the care of a physician
III. Late general complications
a. Serum hepatitis
b. AIDS
Types of sample:
1. Whole blood – red fluid that circulates in the body.
2. Plasma – is the liquid portion of unclotted blood. It is the liquid part of the blood that separates after standing or centrifuging blood with an anticoagulant. It has fibrinogen.
3. Serum – is the liquid portion of clotted blood. It is the liquid portion of the blood that separates after standing or centrifuging blood without an anticoagulant.
Differences between plasma and serum:
Plasma:
a. Contains fibrinogen
b. Presence of lipemia clearing factor
c. Optically less clearer compared to serum
Serum:
a. No fibrinogen (because it is converted to fibrin)
b. Absence of lipemia clearing factor (because it is destroyed or co–precipitated in clotting process)
c. Optically clearer compared to plasma
Interferences in processing of blood sample:
I. Hemolysis – is the destruction of red blood cells.
A.This must be avoided because of the following reasons:
1. Most constituents, such as SGOT, LDH, Acid phosphatase and potassium are present in large amounts in erythrocytes so that hemolysis will significantly elevate the values of these substance in serum.
2. Invalidates determination due to color changes.
B.Other affected determination
1. Elevation of electrolytes like Na+, K+, Ca++, Mg++, Zn++ and I.P.
2. Elevation of glucose, cholesterol, phospholipids and others.
3. Increase in serum albumin by bromcresol green method.
4. Increase in serum bilirubin as determined spectrophotometrically.
5. Decrease in serum bilirubin as determined by diazotization reaction.
6. Release of adenylate kinase which causes an elevation of creatinine kinase.
C. Hemolysis can take place during:
1. Collection of blood:
a. Avoid excessive venous stasis through prolonged application of tourniquet.
b. Avoid moisture or contamination in needles, syringes or blood containers.
c. Avoid excessive traction on syringe plunger.
d. Avoid use of small lumen needles. Use a gauge 20 needle and the blood should be allowed to flow into the syringe with minimal traction.
e. Do not allow air to leak into the syringe during blood extraction.
2. Transfer of blood:
a. Do not expel blood from a syringe through a needle.
b. Do not allow the blood to fall into the container through the air.
c. Do not shake blood in container to mix with anticoagulant. It must be mixed by gentle inversion for at least 8 times.
d. Do not agitate blood that does not contain an anticoagulant.
e. Clotted blood should not be disturbed for at least 15 to 20 minutes.
3. Separation of blood:
a. Do not allow the red blood cells to be in contact with plasma or serum for a long time.
b. Do not refrigerate freshly collected blood before clotting has occurred.
c. Do not freeze whole blood because red cells will hemolyze on thawing (melting).
d. Do not overcentrifuge.
e. Separation of clot should be done by gently rimming/ringing.
II. Lipemia or Lactescence – milky or lipemic plasma which occurs when blood samples are obtained 1 to 2 hours after eating a fatly meal. This is caused by a transient rise in chylomicrons following a meal containing fat. It causes interference with a large number of chemical analyses because of turbidity.
A.Affected determinations:
1. Bromsulpthalein test for proteins
2. Bromcresol green method for albumin
3. O–cresolpthalein method for calcium
4. Acid ammonium molybdate method for inorganic phosphorous.
5. Inhibits the activity of amylase, lipase, uricase and urease; decrease CK, bilirubin, total protein, SGPT & SGOT.
III. Icteric serum – yellowish serum due to bilirubin.
A.Affected determinations:
1. HABA method for albumin
2. Cholesterol assays using ferric chloride
3. O–toluidine method for glucose
4. Biuret method for total protein
5. Icterus index as Liver Function Test
IV. Concentration changes:
This may occur through dilution or evaporation. The sources of error are:
A. Use of syringe and needles rinsed in saline solutions (0.85% NaCl)
B. Use of liquid form of anticoagulant
C. Allowing the blood specimen in open container
D. Centrifuging blood specimen in open container
V. Composition changes:
This are brought about by:
A. Bacterial changes – such as formation of ammonia from urea.
1. This can be prevented by:
a. Sterile handling of blood
b. Prompt separation of cells from plasma or serum
c. Storing the specimen at 4 to 6oC or by freezing at –20oC.
B.Enzymatic changes – metabolic degradation of glucose and autodegradation (loss of enzyme activity). Glycolysis (breakdown of glucose into lactate and pyruvic acid) minimized by the use of anti–glycolytic agent such as sodium fluoride in conjunction with other anticoagulant like heparin.
C. Blood gas changes – loss of CO2
· Avoid rapid displacement or movement and mixing of separated fractions, e.g. plasma to avoid loss of gas through air specimen surface contacts.
D. Extravascular interchange
This is due to movements of substance between the cells and plasma or serum. This is minimized by prompt separation of the liquid component from the cells.
E.Changes in lipid concentration due to lipolysis
Changes that occur in blood upon long standing:
1. Bacterial changes
2. Enzymatic changes
3. Blood gas changes
4. Changes in phosphate due to hydrolysis of organic phosphate esters
5. Extravascular interchange
6. Changes in lipid concentration due to lipolysis.
Specimen variables
In general, blood for chemical analysis should be drawn while the patient is in the post absorptive state. An overnight fast is the usual procedure although a 6 hours fast is ample. During this time, there is no need to restrict water intake.
Common procedures that are significantly affected by eating:
1. Glucose – elevated
2. Inorganic phosphorous – decreased
3. Thymol turbidity test – increased
4. Triglycerides – increased
ANTICOAGULANTS
Anticoagulants are chemical agents or solutions that prevent blood from clotting. It is of great importance in the laboratory to determine the type of anticoagulant to be used and the proportion of anticoagulant to blood. Dry anticoagulants are more commonly used to avoid dilution which lowers the concentration of the formed elements in the blood. Anti–clotting mechanism can be done either chemically or mechanically.
Characteristics of an ideal anticoagulant:
1. It should not alter the size of the red blood cells.
2. It must not bring about hemolysis.
3. It must minimize deformation of leukocytes
4. It must be readily soluble in blood.
HEPARIN (Green top)
Heparin is present in most of the tissue of the body but in concentration less than that required to prevent the blood from clotting. It was first found in the liver and named hepar, the Greek word for that organ. In the body, it exists in highest concentration in the liver and the lungs. It is a mucoitin polysulfuric acid and is available as the sodium, potassium and ammonium salt.
Action of heparin as anticoagulant:
1. The heparin–protein complex inactivates thromboplastin, the first phase substance needed to start the series of clotting steps.
2. It has a direct antithrombin action, that is, it inactivates any molecule of thrombin in the presence of a cofactor located in the albumin fraction of the plasma.
Amount needed:
1. 1 mg of dry heparin or 0.1 ml of liquid heparin per 10 ml blood.
2. 0.1 ml to 0.2 mg of dry heparin per ml of blood.
Preparation of saturated heparin solution:
Powdered heparin – 1.0 g
Distilled water – 100 cc
Evaporate to dryness at 37 – 56oC
Advantages:
1. Excellent and natural anticoagulant usually prepared from lung of animals.
2. Does not alter the corpuscular size and therefore heparinized blood has been used as a standard for comparison of the effects of various inorganic anticoagulants.
3. Best anticoagulant for prevention of hemolysis.
4. Used for electrolyte determination, ESR and tests for blood pigments.
5. Less toxic than sodium citrate and therefore is preferred as an anticoagulant for open heart surgery and exchange transfusion in which large quantities of blood are given rapidly.
6. Especially used for erythroblastosis fetalis.
Disadvantages:
1. Expensive
2. Temporary action – the anticoagulant effect in vivo is of short duration but if used in a adequate concentration in vitro it will prevent coagulation of refrigerated blood for 3 or more days.
3. Not satisfactory for serological test.
ETHYLENE DIAMINE TETRA ACETIC ACID (Lavander/Violet Top)
This is also known as versene or sequestrene which exist as disodium salts or dipotassium salts, respectively.
Action of EDTA as anticoagulant:
1. It combines with calcium and builds it in a non–ionized form which is called chelation or sequestration.
Amount needed:
1. 1–2 mg/ml of blood
2. 0.1 ml of 10% solution in water per 5 ml of blood.
Preparation of 10% sequestrene solution:
Sequestrene (dry powder) – 10 g
Distilled water – 100 ml
Advantages:
1. Preserves cellular elements of the blood and doesn’t cause significant changes in the size and shape of RBC and WBC.
2. May also be used in blood transfusion as it has few toxic effects.
Disadvantages:
1. It decreases plasma calcium levels and interferes with either sodium or potassium determination depending upon the form of EDTA used.
OXALATES (Black Top)
Action of oxalates as anticoagulant:
1. Oxalates such as sodium, potassium, ammonium or lithium inhibit blood coagulation by forming rather insoluble complexes with calcium ions which are necessary for coagulation.
a. Dried ammonium–potassium oxalates
Other names: Double oxalate
Wintrobe’s fluid
Balanced oxalate
Heller–Paul’s fluid
Preparation of the anticoagulant:
3 parts ammonium oxalate – 1.2 g
2 parts potassium oxalate – 0.3 g
distilled water – 100 cc
2 mg of the mixed oxalate per ml of blood is used as an anticoagulant
Advantages:
1. The balance action of this anticoagulant – ammonium swells the cells and the potassium shrinks the cells – preserves the cells from hemolysis thus it does not produce significant shrinkage or enlargement of red cells.
2. Cheap, easy to prepare and requires no dilution.
Disadvantages:
1. Cannot be used for chemistry examinations especially in the determination of urea, nitrogen or potassium.
2. Cannot be used for blood transfusion.
3. It affects calcium determinations by removing it and inhibit some enzymes like LDH, ACP, AMS and LPS.
b. Potassium oxalates
This is used as anticoagulant in the proportion of 1 to 2 mg per ml of blood. Dried sodium or lithium oxalate can be substituted for potassium oxalate.
Note:
Temperature in excess of 80oC should be avoided
during the drying of oxalates since at elevated
temperature, the oxalates will be converted to
carbonates which has no anticoagulant activity.
CITRATES (Blue Top)
Action of citrate as anticoagulant:
1. Citrates form a double salt with calcium and bind it in a non–ionized form.
Amount needed:
1. 5 mg/ml of blood
Advantages:
1. Citrate is widely used in collecting blood for transfusion since it is relatively non–toxic salt which is rapidly utilized by the body or excreted by the kidneys.
2. Buffered citrate is now commonly used because it helps stabilize plasma pH.
Disadvantages:
1. It decreases plasma calcium levels and inhibits amylase.
FLUORIDES (Gray Top)
Action of Fluoride as anticoagulant:
Fluoride forms a weakly dissociated calcium component and thus prevents coagulation. Although considered as a preservative for blood glucose determination, it also acts as a weak anticoagulant. When used as a preservative along with an anticoagulant as potassium oxalate, it is effective in a concentration of about 2 mg/ml of blood. It exerts its action by inhibiting the enzyme system involved in glycolysis.
When sodium fluoride is used as an anticoagulant, the concentration must be much greater, 6 to 10 mg/ml of blood. Fluoride acts as a powerful enzyme poison – hence it is also used as a preservative especially for blood and CSF sugar estimation where there is likely to be more than ½ to 1 hour delay between the taking of the specimen and actual analysis.
Amount needed:
1. 10 mg/ml of blood with 1 mg thymol
Disadvantages:
1. It inhibits the action of urease and glucose oxidase in the enzymatic method for glucose and urea, respectively.
2. It diminishes the activity of acid phosphatase and increase amylase activity.
SODIUM POLYANETHOLSULFONATE
This has no effect on the erythrocyte volume. It has been reported to interfere with potassium determination. It is used in a proportion of 1–2.5 mg/ml of blood.
PRESERVATION OF SPECIMEN
1. Physical – using temperatures.
Commonly employed reduced temperatures:
a. 4oC – refrigeration
b. –5 to –20oC – freezer
c. –70oC – dry ice
d. –142oC – liquid nitrogen
2. Chemical – use of preservative
Classification of chemical preservative according to function:
a. Enzyme inhibitor – prevents chemical changes such as glycolysis.
b. Bacteriostatic agents – interfere with and prevent microbial growth.
Microbial growth can be solved by:
(1) Collection and storage under sterile conditions
(2) Freezing of the sample
(3) Extreme alteration of pH
(4) Addition of an antimicrobial agent
Common preservatives:
a. Concentrated HCl – for catecholamines and VMA
b. Thymol, Toluene, CHCl3, boric acid – for urine
c. Fluoride – prevents glycolysis and other enzyme actions, should not be used when such as actions are involved
d. Sodium carbonate – for porphyrins
e. Petrolatum ether – to retard oxidation by formation of a protective layer
f. Antibiotics – 1 mg streptomycin base per 10 ml of blood for urea determination.
Processing of blood samples after collection:
1. Whole blood – if not done within one hour after collection, refrigerate at 4–5oC.
2. PFF – prepare at once (30 minutes after collection) and refrigerate.
3. Plasma or serum – separate serum and plasma after standing and refrigerate at 4–6oC or freeze at –20oC if analysis is delayed for more than four hours.
4. Specimen for blood NH3, gases or other unstable substances should be processed as soon as possible.
HANDLING OF CLINICAL SPECIMEN
The individual handling of the specimen (serum or plasma) will depend on the analysis that is to be done and the time that will elapse before the analysis is started.
1. Glucose – if test cannot be performed within 2 hours, the blood maybe refrigerated for a few hours.
2. PFF (drop of toluol added) – may be stored overnight in refrigerator. The use of fluoride can deter glycolysis for a short time.
3. Chromogens (urobilinogen, phorphobilinogen, etc.) – only fresh or suitably preserved specimen should be used.
4. Bilirubin, BSB, Chloride, Icterus index, Na or K, Proteins, Thymol turbidity – serum may be stored overnight in a refrigerator.
Important:
Bilirubin should always be stored in the dark.
5. Cholesterol and cholesterol esters – serum maybe stored in a refrigerator for a few hours.
6. Calcium – serum must be separated from the clot within 30 minutes, otherwise calcium diffuses into the cells. Serum can be kept overnight in a refrigerator.
7. Phosphorous – this is very unstable. Serum must be separated from the clot within 30 minutes and tested immediately.
8. Cephalin–cholesterol flocculation – serum cannot be preserved. Test must be started within 4 hours of specimen collection.
9. Amylase – amylase activity rapidly decreases after blood withdrawal. If test cannot be performed immediately, the serum or plasma maybe refrigerated for up to 3 hours only.
10. Lipase – test must be run within 2 hours of blood collection.
11. Phosphatase – serum (small drop of toluol added) maybe kept for 2 hours in a refrigerator. Serum for alkaline phosphatase will keep for a few days in a freezer. Acid phosphatase, on the other hand, deteriorates rapidly even in the frozen state. Sodium citrate added to serum and frozen can keep acid phosphatase for a short period.
12. Transaminase (GPT & GOT) – serum maybe stored overnight in the freezer.
13. T2 – serum should be frozen until ready to test.
14. PBI – can be kept at room temperature.
15. Carbon dioxide combining power – plasma (under oil) may be stored in a refrigerator overnight.
Note: When obtaining blood for CO2 determination, the tourniquet should be released before the blood is aspirated.
DEPROTEINIZATION OR PREPARATION OF PROTEIN FREE FILTRATE
Analytical methods for determination of various components of blood, urine and CSF require preliminary treatment of the fluid to render it free of protein.
Purpose of deproteinization:
1. Prevent foaming
2. Prevent turbidity
3. Prevent precipitate formation
4. Prevent direct interference with certain color reactions.
Methods of deproteinization:
1. Physical – includes adsorption onto adsorbents such as kaolin, ultrafiltration, microdiffusion and heat denaturation.
2. Chemical
a. Precipitation by addition of miscible solvents
Proteins can be precipitated from aqueous solution by the addition of organic solvents miscible with water. Solvents such as methanol, ethanol, acetone and ethyl ether lower solubility of the proteins and precipitate them from highly polar solutions.
b. Precipitation by “Salting Out”
Addition of neutral salts decreases the attraction of protein molecule for each other increasing their solubility. This is the “salting in” phenomenon. As the salt concentration is increased, a point is reached at which, because of the successful competition with the protein molecules for water molecules, the protein becomes dehydrated with a resultant decreased solubility. This precipitation is called “salting out.”
Salts containing multivalent anions are the most efficient precipitating agents. Commonly used salts are sodium sulfate, ammonium sulfate, magnesium salts, phosphates and citrates. Salting out technique have limited application in clinical chemistry.
c. Precipitation by shaking with chloroform
Protein can be removed from solution by adding 0.25 volume CHCl3 + 0.1 volume of any foam–preventing substances, such as amyl alcohol to the protein solution, shaking for 15–60 minutes, followed by centrifugation. The mixture separates into 2 layers, the upper being the aqueous layer and the lower consisting of a CHCl3–protein gel.
d.Precipitation dependent on insoluble salt formation.
Precipitation may be accomplished by addition of:
(1)Anionic precipitants such as tungstic acid, trichloroacetic acid and perchloric acid.
(2)Cationic precipitants such as Zn++, Hg++, Fe++ and Pb++.
The Anionic Precipitants:
Among the acid precipitants that have been used in clinical chemistry are picric acid, molybdic, phosphotungstic, tungstomolybdic, sulfosalicylic, tungstic, metaphosphoric and perchloric.
1. Tungstic acid – introduced by Folin–Wu. This is especially used for determination of non–protein nitrogen compound.
Principle:
The total protein of blood are removed after lysis of the cells by precipitation with tungstic acid, followed by filtration or centrifugation.
a. Folin–Wu method
Whole blood plasma or serum CSF
Volume of specimen 1 ml 1 ml 1 ml
Distilled water 7 ml 8 ml 8.5 ml
Sodium tungstate, 10% 1 ml 0.5 ml 0.25 ml
2/3 N H2SO4 1 ml 0.5 ml 0.25 ml
Mix, shake and allow to stand until the color changes to chocolate brown (about 15 to 30 minutes) and is filtered or centrifuged. The dilution of the sample is 1:10.
Less amount of precipitating agent are needed for serum, plasma and CSF due to lesser amount of proteins in these samples.
Complete precipitation takes place when:
1. Change of color from red to brown signifying change of hemoglobin to methemoglobin.
2. Absence of foam formation.
3. Presence of sound against a stopper when the mixture is shaken. If a brown color does not appear, 10% sulfuric acid is added drop by drop, shaking vigorously after each drop, until the change is complete.
b. Haden’s modification
1. The difference is in the reversal of the order of the addition of sodium tungstate and sulfuric acid.
2. Another difference is, it uses N/12 H2SO4 instead of 2/3 N H2SO4 to decrease the number of solutions to be added and there is no necessity for delaying filtration.
Preparation:
1 volume of whole blood
8 volumes of N/12 sulfuric acid
1 volume of 10% Na2WO4
Mix, shake vigorously and filter
Advantages of the Haden’s modification over Folin–Wu method:
1. Reaction is practically immediate and there is no necessity for delaying filtration.
2. More rapid filtration
3. Larger volume of filtrate obtained.
c. Van Slyke method – sodium tungstate and sulfuric acid are premixed before blood is added.
Preparation:
1 volume of blood
9 volume of deproteinizing reagent (consists of water, sodium tungstate and sulfuric acid).
The Van Slyke method is recommended when large amount of filtrate preparation are desired.
Disadvantages of this method:
1. Less stable, lasting only 2 weeks
2. Longer waiting time for the precipitate to become chocolate brown.
The tungstic acid filtrate is slightly acidic. It contains all the NPN constituents and reducing substances and is used for the estimation of these substances.
2. Trichloroacetic Acid Method (TCA) – used for
inorganic phosphorous
Preparation:
1 ml of serum or plasma
9 ml of 2.5% TCA or 5% TCA
The 2.5% TCA occasionally produces incomplete precipitation of protein so that the strength of TCA was increased to 5%.
The mixture is set aside for about 20 minutes before filtering or centrifuging. Filtration is rapid. The pH of the filtrate is about 1. The volume of the filtrate obtained is much greater than the filtrate obtained with tungstic acid precipitant.
TCA filtrate is useful when an acid medium is necessary:
a. To hold a particular substance in solution, e.g. inorganic phosphate and calcium.
b. To preserve unstable substances, e.g. ascorbic acid is relatively stable in acid solution, whereas in neutral or alkaline solution, it is oxidized rapidly to dehydroascorbic acid.
The cationic precipitants:
Somogyi methods: for true glucose and BUN determination
1. Barium Hydroxide – Zinc sulfate method
Preparation: 1 ml whole blood
5 ml distilled water
2 ml 0.3 N Ba(OH)2
2 ml 5% ZnSO4
2.
Preparation:
1 volume blood
7 volume distilled water
1 volume 10% Zinc Sulfate (ZnSO4 + H2O)
1 volume 0.5 N NaOH
For plasma or serum, Somogyi recommended:
1 volume sample
8 volume water
0.5 volume of zinc sulfate
0.5 volume of sodium hydroxide solution
Somogyi noted that Zn++ alone fails to give complete precipitation of proteins from serum or plasma and that replacing NaOH with Ba(OH)2 results in complete precipitation due to the absorptive capacity of the BaSO4 that is formed.
The zinc hydroxide, aside from precipitating proteins also removes many of the non–protein nitrogen substances like uric acid, ergothionine, glutathione, creatinine, etc.
3.Copper sulfate–sodium hydroxide method or Copper sulfate–sodium tungstate method
Preparation:
1 ml whole blood
7 ml distilled water
2 ml 7% CuSO4 + 2H2O
For serum or plasma, use CuSO4 + 5H20 and 6% Na2WO4 + 2H2O
Advantages of Copper method:
a. Gives more complete precipitation of serum or plasma.
b. Excess reagent is precipitated along with the protein.
In all the above preparation of PFF, the final volume is 10 ml and therefore every 1 ml of PFF is equal to 0.1 ml of blood.
Test for complete precipitation:
1. Metallic click
2. Absence of bubbles or foam
Causes of brownish unclear filtrate:
1. Insufficient acid
2. Impure tungstate
3. Too much oxalate
HYGIENIC PRACTICES
The following practices must always be employed:
1. Always practice universal precautions with all specimens at all times (i.e. treat all specimens as if they were infected).
2. Lab coats must always be worn in the work area.
3. Lab coats should not be removed from the laboratory area except for laundering.
4. Keep lab coats away from dining or food areas.
5. Cuts and scratches on hands and arms should be well covered and protected.
6. Always wash hands after removing gloves and before leaving the laboratory.
7. Eating, drinking and smoking in the work areas is a serious health risk and must not be practiced.
8. Long hair should be pinned up or covered.
9. Always wear eye protection when generating droplets or aerosols.
10. Avoid activities such as applying make–up and combing hair in the laboratory.
11. Protective shoes that completely cover feet should be worn (no open–toed shoes or sandals).
12. Always wear gloves when handling blood or body fluids.
13. If gloves are in short supply, gloved hands can be washed frequently and wiped with disinfectant throughout the day.
WORKSPACE
A clean, organized workspace is a safer work space:
1. Reagents should be well–labeled, in proper containers and stored properly (example: store acids and bases in a separate cabinet below bench – top level).
2. Keep bench top free from clutter (non–essentials).
3. Do not place materials near the edges of counters or shelves.
4. Clean countertops with disinfectant before and after each work days using one of the following:
Suggested disinfectants:
a. Sodium hypochlorite, 0.1%–0.5% available chlorine
b. Ethanol, 70%
c. Isopropyl alcohol, 70%
d. Formaldehyde, 4%
e. Glutaraldehyde, 2%
f. Hydrogen peroxide, 6%
g. Povidone iodine, 2.5%
5.Restrict entry to authorized personnel only; receive visitors outside of the laboratory area.
6.Immediately contain spills and wipe contaminated surface with one of the disinfectants listed above.
7.Avoid use of sharp instruments whenever possible (scalpels, needles, scissors).
8.Pest control prevents known and unknown vectors which carry disease.
9.Breakable materials (glass) should be used only when absolutely necessary and should be stored safely.
10. Periodically clean doors, handles, telephones with disinfectants.
11. Each lab should have a first aid kit available.
12. Report accidents immediately to the supervisor.
13. An eye wash station should be available for accidental exposure from splashes.
BIOHAZARDOUS MATERIAL
Proper handling prevents infection to yourself and others:
1. Biohazards warning signs must be plainly posted.
2. All laboratory waste must be properly decontaminated before disposal. Autoclave at 121oC at one atmosphere for 10 minutes or incinerate.
3. Do not bury infectious waste.
4. Use a separate puncture resistant container for glass waste and needles.
5. Never recap a needle with two hands. Place the cap on the counter and insert needle/syringe into puncture resistant container for disposal.
6. Spills must be immediately wiped up with one of the disinfectants listed under “work space.”
7. Liquid wastes should be decontaminated by autoclaving or by adding sufficient sodium hypochlorite before discarding.
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