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من طرف Specimen collection N. M. ELIAS 1
SPECIMEN COLLECTION
Every biochemistry analysis should provide the answer to a question which the clinician has posed about the patient. In order to carry out biochemical analyses it's necessary that the laboratory be provided with both the correct specimen for the requested test and also information which will ensure that the right test is carried out and the result returned to the requesting clinician with the minimum of delay. Before collecting a specimen of any type, a laboratory specialist should label the container with patient's name, identification number, ward & bed number, and date and time of collection. All specimens should be treated as if they are potentially infectious.
Types of samples
A variety of specimens are used in biochemical analysis;
i- Body fluids such as blood, urine, spinal fluid, amniotic fluid, sputum, saliva, synovial (joint fluid) and peritoneal fluid,
ii- Feces,
iii- Tissues and cells,
iv- Calculi (stones) and other materials.
Collection of blood
Blood for analysis may be obtained from veins, arteries, or capillaries. Venous blood is usually the specimen of choice (can be collected via venipuncture). In young children, skin puncture is frequently used to obtain what is predominantly capillary blood; arterial puncture is used mainly for blood gas analyses.
Special precautions
1- Time of specimen collection is important for analysis of blood constituents that undergo marked diurnal variation (e.g. corticosieroids and iron) and for tests to monitor drug therapy (e.g., digoxin or prothrombin time).
Specimen collection N. M. ELIAS 2
2- Custody must be established in the collection and handling of specimens which are required for tests of blood alcohol levels and for tests with medico legal importance.
3- Before collecting any blood specimen, a phlebotomist should observe universal precautions (rubber gloves, a facemask, glasses, and gown).
Venipuncture
Site: the median cubital vein in the antecubital fossa (crook of the elbow), is the preferred site for collecting venous blood in adults, because the vein is both large and close to the surface of the skin.
Avoidance: an arm containing a cannula or arteriovenous fistula should not be used; if fluid is being infused intravenously into a limb, the fluid should be shut off for 3 – 5 min before a specimen is obtained and a suitable note made in the patient's chart and on the result report form. Specimens obtained from the opposite arm or in the same arm below the infusion site may be satisfactory for most tests except those in which analytes are contained in infused solutions (e.g., glucose or electrolytes).
Method: selection of a vein for puncture is facilitated by palpation.
1. Before performing a venipuncture, the phlebotomist should estimate the volume of blood to be drawn and select the appropriate tubes for the plasma or serum tests requested
2. The area around the intended puncture site should be cleaned with a prepackaged alcohol swab or a gauze pad saturated with 70 % isopropanol.
3. Once the skin has been cleansed, it should not be touched until after the venipuncture has been completed.
4. After the skin is cleansed, a tourniquet is applied 10 – 15 cm above the intended puncture site to obstruct the return of venous blood to the heart and to distend the veins.
Specimen collection N. M. ELIAS 3
5. An appropriate needle must also be selected. The most commonly used sizes are gauges 19 to 22 (1.06-0.71 mm outside diameter).
6. When blood collection is complete, the tourniquet should be released and the needle should be withdrawn.
7. When the needle has been removed, the patient should hold a dry gauze pad over the puncture site, with the arm raised to lessen the likelihood of leakage of blood.
8. The pad can subsequently be held in place by a bandage, which can be removed after 15 min.
9. The phlebotomist must separate the needle from the syringe or collection tube and discard it in a container designed specifically for this purpose.
Adverse effects of venous occlusion
1) When the flow of blood in the large veins is obstructed (venous stasis) by a tourniquet, the filtration pressure across the capillary walls is increased causing fluid and low-molecular-weight compounds to pass through the capillary wall. Within the short time of blood collection (1 – 3 minutes) the composition of blood slightly changes but marked changes have been observed after 3 min.
2) The concentration of protein-bound constituents is also influenced by stasis and, indeed, 3-min stasis may be enough to increase the concentration of protein or protein-bound constituents by 15%.
Pre -venipuncture avoidance
1. Pumping of the fist before venipuncture should be avoided because it causes an increase in the plasma K+, phosphate, and lactate concentrations (lowering PH of blood causes the plasma ionized Ca++ concentration to increase. The ionized Ca++ concentration reverts to normal 10 min after the tourniquet is released).
2. Stress associated with blood collection can have effects in patients at any age. Plasma concentrations of cortisol and GH may increase particularly
Specimen collection N. M. ELIAS 4
in young children who are frightened, struggling, and physically restrained. Adrenal stimulation may occur and lead to an increase in plasma glucose concentration or create increases in the serum activities of skeletal muscle enzymes.
Skin puncture
If only a small volume of blood is required for a blood test (e.g., a blood glucose test), a skin puncture may be used.
Avoidance: a skin puncture is time consuming, and a greater risk of infection is present than with venipuncture because the fingertip is more difficult to sterilize than the antecubital fossa.
Procedure: in an adult or grown child, blood may be obtained by puncturing the tip of a finger or by piercing an earlobe.
1. Before the skin is punctured, the skin is cleansed as for a venipuncture.
2. To improve circulation of the blood, the finger may be warmed by application of a warm, wet washcloth for 3 min prior to pricking.
3. The first drop of blood is wiped off and subsequent drops are transferred to the appropriate collection tube by gentle contact.
4. Filling should be done rapidly to prevent clotting, and introduction of air bubbles should be avoided. This is best done by holding the collection tube at only a slight angle to the skin surface.
5. In an infant younger than 1 year the lateral or medial plantar surface of the foot should be used for skin puncture; suitable areas are illustrated in the figure.
NO
YES
6. In older children, the plantar surface of the big toe may also be used, although blood collection from anywhere on the foot should be avoided in ambulatory patients.
Specimen collection N. M. ELIAS 5
Arterial Puncture
Site: the preferred sites of arterial puncture are, in order, the radial artery at the wrist, the brachial artery in the elbow, and the femoral artery in the groin.
Anticoagulants and preservatives for blood
Whole blood, plasma, and serum are the specimens of choice in clinical biochemistry laboratory. If whole blood or plasma is desired for testing, an anticoagulant must be added to the specimen and centrifugation can be done immediately after collection. Serum is obtained from coagulated blood.
Heparin:
Heparin causes the least interference with tests. It is a mucoitin polysulfuric acid and is available as sodium, potassium, lithium, and ammonium salts. This anticoagulant accelerates the action of antithrombin III, which neutralizes thrombin and thus prevents the formation of fibrin from fibrinogen.
Ethylenediaminetetraacctic Acid (EDTA):
The chelating agent EDTA is particularly useful for hematological examinations because it preserves the cellular components of blood. It is used as the disodium or dipotassium salt. EDTA prevents coagulation by binding calcium, which is essential to the clotting mechanism.
Sodium Fluoride:
Sodium fluoride is usually considered a preservative for blood glucose; however, it also acts as a weak anticoagulant. It exerts its preservative action by inhibiting the enzyme systems involved in glycolysis.
Citrate:
Sodium citrate solution is widely used for coagulation studies, because its effect is easily reversible by addition of Ca+2. It appears to preserve labile procoagulants, but it has little application in clinical chemistry.
Oxalates:
Specimen collection N. M. ELIAS 6
Sodium, potassium, ammonium, and lithium oxalates inhibit blood coagulation by forming rather insoluble complexes with calcium ions.
Iodoacetate:
Sodium iodoacetate is an effective antiglycolytic agent and a substitute for sodium fluoride. Because it has no effect on urease, it can be used when glucose and urea assays are performed on a single specimen. It inhibits creatine kinase but appears to have no other significant effects.
Advantages & disadvantages of plasma and serum usage:
o Use of plasma expedites analysis in medical emergencies because of harvest of serum requires a wait of 15 to 30 min for completion of coagulation before centrifugation.
o Furthermore, plasma yield from a given volume of whole blood is always greater than the yield of serum.
o When plasma is stored the fibrin clots or fragments formed and the subsequent risk of clogging sample probes of automated analytical instruments is a disadvantage.
o Plasma is also not a suitable specimen for electrophoretic analysis, because the presence of fibrinogen can confound interpretation of electrophoretic patterns.
Hemolysis
Serum shows visual evidence of hemolysis when the hemoglobin concentration exceeds 20 mg/dl.
Severe hemolysis causes a slight dilutional effect on those constituents that are present at a lower or higher concentration in RBCs than in plasma. Thus, plasma concentrations or activities of aldolase, total acid phosphatase, LDH, AST, ICD, K+, Mg, and phosphate are particularly increased by hemolysis.
An additional band due to hemoglobin may be observed on serum protein electrophoresis.
Specimen collection N. M. ELIAS 7
Storage of blood specimens
1) Plasma or serum should be separated from cells as soon as possible and certainly within 2 hrs to minimize the metabolism of cells.
2) If it's impossible to centrifuge a blood specimen within 2 hrs the specimen should be kept at room temperature rather than at 4 °C to minimize hemolysis.
3) If the specimen can't be analyzed at once, the separated serum should be generally stored in capped tubes at 4 °C until analysis both to:
i- Maintain stability of the specimen; and
ii- Reduce evaporation.
4) If a specimen for a particular test is sufficiently unstable at 4 °C, the serum specimen should be held at – 20 °C in a freezer capable of maintaining this temperature.
5) Specimens for bilirubin or carotene must be protected from both daylight and fluorescent light to avoid photodegradation.
6) Specimen tubes should be centrifuged with stoppers in place. Closure lowers evaporation and prevents aerosolization of infectious particles.
7) Removal of the stopper before centrifugation allow loss of CO2 and increase in blood PH.
Changes in blood metabolites on keeping
1. Glucose is converted to lactate, as a result of glycolysis occurring in blood cells specially RBCs. It's inhibited by fluoride but fluoride interferes with glucose oxidase and urease methods.
2. Glycolysis decreases serum glucose by approximately 5 – 7 % in 1 hr. (5 – 10 mg /dl) in uncentrifuged coagulated blood at room temperature.
3. The rate of glycolysis is higher in the presence of leukocytosis or bacterial contamination.
4. Several substances pass through RBCs membrane e.g. K+, LDH.
5. Loss of CO2, since the PCO2 of blood is much higher than air leading to
Specimen collection N. M. ELIAS 8
fall in plasma total CO2.
6. Phosphate increases due to hydrolysis of organic ester phosphate compounds of erythrocytes.
7. Enzymes activities are lost on long keeping
8. Formation of ammonia from nitrogenous substances.
Urine collection
Normal urine is pale yellow, clean & transparent. It contains chloride, sulfate, calcium, ammonia, uric acid & creatinine, but there are many of the pathological constituents which are routinely tested in urine are protein, glucose, blood, ketone bodies and bile salts. In biochemistry laboratory, urine samples are assayed for either qualitatively or quantitatively analysis. Collecting the urine specimen is dependent on the type and purpose of the urine test itself. Since characteristics of the urine are affected by collection techniques and time of day, collection methods. Three familiar urine collection methods include
1- First (voided) morning specimen
The specimen is collected when the individual first awakens; it is preferred for routine urinalysis because it is more concentrated and therefore more likely to reveal abnormalities as well as the presence of formed substances. It is also relatively free of urine composition changes over the course of the day, i.e. dietary influences and of changes caused by physical activity, because the specimen is collected after a period of fasting and rest.
2- Random urine specimen
It's the most commonly requested specimen, which can be collected at any time.
There are no special preparation procedures or dietary restrictions for the first morning and random urine specimen collection methods.
Procedure for morning & random urine specimens
1. The patient is instructed to void directly into a clean, dry container.
Specimen collection N. M. ELIAS 9
2. All specimens should be covered tightly, labeled properly, and sent immediately to the laboratory. The label should be on the cup, not on the lid.
3. If a urine specimen is likely to be contaminated with drainage, vaginal discharge, or menstrual blood, a clean specimen must be obtained using the same procedure as for bacteriologic examination.
4. If the specimen cannot be delivered to the laboratory or tested within 1 hour, it should be refrigerated or have an appropriate preservative added.
Interfering Factors
1. Feces, discharges, vaginal secretions, and menstrual blood will contaminate the urine specimen. A clean voided specimen must be obtained .
2. If the specimen is not refrigerated within 1 hour of collection, the following changes in composition may occur :
a) Increased pH from the breakdown of urea to ammonia by urease-producing bacteria.
b) Decreased glucose from glycolysis and bacterial utilization.
c) Decreased ketones because of volatilization.
d) Decreased bilirubin from exposure to light.
e) Decreased urobilinogen as a result of its oxidation to urobilin.
f) Increased nitrite from bacterial reduction of nitrate.
g) Increased bacteria from bacterial reproduction.
h) Increased turbidity caused by bacterial growth and possible precipitation of amorphous material.
i) Disintegration of red blood cells (RBCs) and casts, particularly in dilute alkaline urine.
j) Changes in color caused by oxidation or reduction of metabolites
3- Other urine specimen collection methods
a) Timed specimens (2-hour & 24-hour) are collected when it is necessary to analyze urine that is excreted over a certain number of hours. The twenty-
Specimen collection N. M. ELIAS 10
four-hour timed collection method is fairly common. Substances excreted by the kidney are not excreted at the same rate during different periods of the day and night; therefore, a random urine specimen might not give an accurate picture of the processes taking place over a 24-hour period. For measurement of total urine protein, creatinine, electrolytes, and so forth, more accurate information is obtained from a long-term specimen.
Procedure
1. At the beginning of a 24-hour timed urine specimen collection (or any other timed specimen collection), the patient is asked to void. This first specimen is discarded, and the time is noted.
2. The time the test begins and the time the collection should end are marked on the container. As a reminder, it may be helpful to post a sign above the toilet (eg, 24-Hour Collection in Progress), with the beginning and ending times noted.
3. Do not predate and pretime requisitions for serial collections. It is difficult for some patients to void at specific times. Instead, mark the actual times of collection on containers.
4. Documentation of the exact times at which the specimens are obtained is crucial to many urine tests.
5. All urine voided over the next 24 hours is collected into a large container (usually glass or polyethylene) that is labeled with the patient’s name, the time-frame for collection, the test ordered, and other pertinent information.
6. Instruct the patient to urinate as near to the end of the collection period as possible.
7. To conclude the collection, the patient voids 24 hours after the first voiding.
8. All urine voided in a 24-hour period is collected into a suitable receptacle, wide-mouth container; a preservative is added or the collection is kept refrigerated, or both.
9. Test results are calculated on the basis of a 24-hour output.
Specimen collection N. M. ELIAS 11
Storage
1- Nonrefrigerated samples may be kept in a specified area or in the patient’s bathroom.
2- If refrigeration is necessary, the collection bottle must either be refrigerated immediately after the patient has voided or be placed into an iced container.
Interfering Factors
1. Failure of patient to follow the procedure. The patient should be given both verbal and written instructions.
2. Instruct the patient to use toilet paper after transferring the urine to container. Toilet paper placed in the specimen decreases the actual amount of urine available and contaminates the specimen.
3. The presence of feces contaminates the specimen. Patients should void first and transfer the urine to the collection receptacle before defecating.
4. If heavy menstrual flow or other discharges or secretions are present, the test may have to be postponed or an indwelling catheter may need to be inserted to keep the specimen free of contamination. In some cases, thorough cleansing of the perineal or urethral area before voiding may be sufficient.
b) Catheterized specimens are collected when voiding is difficult as with obstruction and severe urinary tract infection.
Procedure
1- The straight catheter method is accomplished by inserting a sterile, lubricated catheter into the urinary bladder and collecting the urine.
2- For patients who have an indwelling catheter, commonly called a Foley catheter, the urine specimen must not be collected from the urinary drainage bag.
3- The indwelling catheter is equipped with a specimen collection port that allows urine to be collected with a syringe before it reaches the drainage
Specimen collection N. M. ELIAS 12
bag. It may be necessary to clamp off the catheter for about 15 to 30 minutes before obtaining the sample.
4- Clean the specimen port with antiseptic before aspirating the urine sample with a needle and syringe.
c) Special-purpose specimens include fasting specimens and postprandial specimens. For fasting specimens the patient voids and discards the first urine specimen at least four hours after food ingestion. The next voided urine is the fasting specimen. The postprandial specimen is urine voided two to three hours after a meal.
The midstream technique
1. It's necessary when a urine specimen must be free from contamination.
2. After thorough handwashing, the patient is instructed to cleanse the genital area with an antiseptic wipe, to void a small amount of urine into the toilet, to stop voiding midstream, and to collect a urine sample by voiding into the collection container. The specimen container must be free of feces, mucus, or vaginal discharge.
Preservatives
They are usually added to
a) Reduce bacterial action or chemical decomposition.
b) Solubilize constituents that might otherwise precipitate out of solution.
c) Decrease atmospheric oxidation of unstable compounds.
1. The most satisfactory preservation of urine specimens is refrigeration immediately following collection.
2. Urinary preservative tablets that contain a mixture of chemicals, such as potassium acid phosphate, sodium benzoate, benzoic acid, methenamine, sodium bicarbonate, and red mercuric oxide have been used for chemical and microscopic examination. Because these tablets contain Na+ and K+ salts, among others, they cannot be used for analysis of these analytes.
Specimen collection N. M. ELIAS 13
3. The preservative tablets act mainly by lowering the PH of the urine and by releasing formaldehyde.
4. Acidification PH 3 (sulfamic acid, boric acid are used to reduce PH) is widely used to preserve 24-hrs specimens and is particularly useful for specimens for Ca++, steroids, and vanillylmandelic acid determinations.
5. However, precipitation of urates occurs, thereby rendering a specimen unsuitable for measurement of uric acid.
6. Toluene is the only organic solvent that is still used as a preservative (large enough amount acts as a barrier between the air and the surface of the specimen).
7. A mild base, such as NaHCO3 or a small amount of NaOH, is used to preserve porphyrins, urobilinogen, and uric acid. A sufficient quantity should be added to adjust the PH 8 - 9.
COLLECTION OF OTHER SAMPLES
Feces
It is frequently analyzed in biochemistry laboratory to detect the presence of "hidden" or so-called occult blood, which is recognized as one of the most effective clues to the presence of a bleeding ulcer or a malignant disease in the GIT. Feces is composed of:
1- Products of digestion which are not absorbed.
2- Undigested food such as starch, granules, cellulose fibers, muscle fibers.
3- Changed bile pigments.
4- Cells of intestinal wall.
5- Products of foodstuff, e.g. indole, skatole, fatty acids
6- Bacteria
7- Water
The feces samples are collected in bedpan. The samples should be examined soon and watery samples can be preserved in refrigerator while solid samples Specimen collection N. M. ELIAS 14
can be dissolved in H2O and this can be preserved with formalin 2 ml / 10 ml DW. Feces samples can be collected by giving:
1) Diet containing 190 g carbohydrates, 111 g fats, 102 g proteins;
2) Charcoal, carmine, gentian violet dye (0.5 – 1 g) in the form of capsule; or
3) Chrominum trioxide capsule (500 mg capsule 3 times).
Normal biochemical values of feces
1) Weight for a day is ~ 60 – 200 g depending up on diet. Diet rich in meat & low in vegetables reduces feces outcome.
2) 70–80 % water contents, 25–45 g total solid, PH is 5–9; usually alkaline.
3) Odor or smell due to indole, skatole & H2S.
Spinal fluid
It is normally obtained from the lumbar region. Spinal fluid is examined when there is a question as to the presence of a cerebrovascular accident, meningitis, demyelinating disease, or meningeal involvement in malignant disease.
Synovial fluid aspiration (arthrocentesis)
Synovial fluid is withdrawn from joints to characterize the type of arthritis and to differentiate non-inflammatory effusions from inflammatory fluids.
Amniotic fluid aspiration (amniocentesis)
Requested for prenatal diagnosis of congenital disorders, assess fetal maturity, or to look for Rh isoimmunization or intrauterine infection.
Pleural, pericardial, and ascitic fluid collection
Pleural (thoracentesis), pericardial (pericardiocentesis), and peritoneal (paracentesis) cavities normally contain a small amount of serous fluid that lubricates the opposing parietal and visceral membrane surfaces. Inflammation or infections affecting the cavities cause fluid to accumulate. The fluid may be removed to determine if it is an effusion or an exudate, a distinction possible by protein or enzyme analysis.
Collection of saliva
Specimen collection N. M. ELIAS 15
Measurement of a drug in saliva has been suggested to estimate the free, pharmacologically active concentration of the drug in serum.
Specimen transport
1. It should be assumed that transport from a referring laboratory to a referral laboratory may take as long as 72 hours.
2. The tube used for holding a specimen (primary container) should be so constructed that the contents do not escape and should protect the specimens from adverse conditions.
3. The shipping or secondary container used to hold one or more specimen tubes or bottles must be constructed to prevent the tubes from knocking against each other. Corrugated cardboard, fiberboard, designed to fit around a single specimen tube may be used.
4. For transportation of frozen or refrigerated specimens, a special container must be used. The container walls should be 2.5 cm thick to provide effective insulation; should be vented to prevent buildup and possible explosion of carbon dioxide under pressure. Solid carbon dioxide (dry ice) is the most convenient refrigerant material for keeping specimens frozen, and temperatures as low as – 70 °C must be used.
SPECIMEN COLLECTION
Every biochemistry analysis should provide the answer to a question which the clinician has posed about the patient. In order to carry out biochemical analyses it's necessary that the laboratory be provided with both the correct specimen for the requested test and also information which will ensure that the right test is carried out and the result returned to the requesting clinician with the minimum of delay. Before collecting a specimen of any type, a laboratory specialist should label the container with patient's name, identification number, ward & bed number, and date and time of collection. All specimens should be treated as if they are potentially infectious.
Types of samples
A variety of specimens are used in biochemical analysis;
i- Body fluids such as blood, urine, spinal fluid, amniotic fluid, sputum, saliva, synovial (joint fluid) and peritoneal fluid,
ii- Feces,
iii- Tissues and cells,
iv- Calculi (stones) and other materials.
Collection of blood
Blood for analysis may be obtained from veins, arteries, or capillaries. Venous blood is usually the specimen of choice (can be collected via venipuncture). In young children, skin puncture is frequently used to obtain what is predominantly capillary blood; arterial puncture is used mainly for blood gas analyses.
Special precautions
1- Time of specimen collection is important for analysis of blood constituents that undergo marked diurnal variation (e.g. corticosieroids and iron) and for tests to monitor drug therapy (e.g., digoxin or prothrombin time).
Specimen collection N. M. ELIAS 2
2- Custody must be established in the collection and handling of specimens which are required for tests of blood alcohol levels and for tests with medico legal importance.
3- Before collecting any blood specimen, a phlebotomist should observe universal precautions (rubber gloves, a facemask, glasses, and gown).
Venipuncture
Site: the median cubital vein in the antecubital fossa (crook of the elbow), is the preferred site for collecting venous blood in adults, because the vein is both large and close to the surface of the skin.
Avoidance: an arm containing a cannula or arteriovenous fistula should not be used; if fluid is being infused intravenously into a limb, the fluid should be shut off for 3 – 5 min before a specimen is obtained and a suitable note made in the patient's chart and on the result report form. Specimens obtained from the opposite arm or in the same arm below the infusion site may be satisfactory for most tests except those in which analytes are contained in infused solutions (e.g., glucose or electrolytes).
Method: selection of a vein for puncture is facilitated by palpation.
1. Before performing a venipuncture, the phlebotomist should estimate the volume of blood to be drawn and select the appropriate tubes for the plasma or serum tests requested
2. The area around the intended puncture site should be cleaned with a prepackaged alcohol swab or a gauze pad saturated with 70 % isopropanol.
3. Once the skin has been cleansed, it should not be touched until after the venipuncture has been completed.
4. After the skin is cleansed, a tourniquet is applied 10 – 15 cm above the intended puncture site to obstruct the return of venous blood to the heart and to distend the veins.
Specimen collection N. M. ELIAS 3
5. An appropriate needle must also be selected. The most commonly used sizes are gauges 19 to 22 (1.06-0.71 mm outside diameter).
6. When blood collection is complete, the tourniquet should be released and the needle should be withdrawn.
7. When the needle has been removed, the patient should hold a dry gauze pad over the puncture site, with the arm raised to lessen the likelihood of leakage of blood.
8. The pad can subsequently be held in place by a bandage, which can be removed after 15 min.
9. The phlebotomist must separate the needle from the syringe or collection tube and discard it in a container designed specifically for this purpose.
Adverse effects of venous occlusion
1) When the flow of blood in the large veins is obstructed (venous stasis) by a tourniquet, the filtration pressure across the capillary walls is increased causing fluid and low-molecular-weight compounds to pass through the capillary wall. Within the short time of blood collection (1 – 3 minutes) the composition of blood slightly changes but marked changes have been observed after 3 min.
2) The concentration of protein-bound constituents is also influenced by stasis and, indeed, 3-min stasis may be enough to increase the concentration of protein or protein-bound constituents by 15%.
Pre -venipuncture avoidance
1. Pumping of the fist before venipuncture should be avoided because it causes an increase in the plasma K+, phosphate, and lactate concentrations (lowering PH of blood causes the plasma ionized Ca++ concentration to increase. The ionized Ca++ concentration reverts to normal 10 min after the tourniquet is released).
2. Stress associated with blood collection can have effects in patients at any age. Plasma concentrations of cortisol and GH may increase particularly
Specimen collection N. M. ELIAS 4
in young children who are frightened, struggling, and physically restrained. Adrenal stimulation may occur and lead to an increase in plasma glucose concentration or create increases in the serum activities of skeletal muscle enzymes.
Skin puncture
If only a small volume of blood is required for a blood test (e.g., a blood glucose test), a skin puncture may be used.
Avoidance: a skin puncture is time consuming, and a greater risk of infection is present than with venipuncture because the fingertip is more difficult to sterilize than the antecubital fossa.
Procedure: in an adult or grown child, blood may be obtained by puncturing the tip of a finger or by piercing an earlobe.
1. Before the skin is punctured, the skin is cleansed as for a venipuncture.
2. To improve circulation of the blood, the finger may be warmed by application of a warm, wet washcloth for 3 min prior to pricking.
3. The first drop of blood is wiped off and subsequent drops are transferred to the appropriate collection tube by gentle contact.
4. Filling should be done rapidly to prevent clotting, and introduction of air bubbles should be avoided. This is best done by holding the collection tube at only a slight angle to the skin surface.
5. In an infant younger than 1 year the lateral or medial plantar surface of the foot should be used for skin puncture; suitable areas are illustrated in the figure.
NO
YES
6. In older children, the plantar surface of the big toe may also be used, although blood collection from anywhere on the foot should be avoided in ambulatory patients.
Specimen collection N. M. ELIAS 5
Arterial Puncture
Site: the preferred sites of arterial puncture are, in order, the radial artery at the wrist, the brachial artery in the elbow, and the femoral artery in the groin.
Anticoagulants and preservatives for blood
Whole blood, plasma, and serum are the specimens of choice in clinical biochemistry laboratory. If whole blood or plasma is desired for testing, an anticoagulant must be added to the specimen and centrifugation can be done immediately after collection. Serum is obtained from coagulated blood.
Heparin:
Heparin causes the least interference with tests. It is a mucoitin polysulfuric acid and is available as sodium, potassium, lithium, and ammonium salts. This anticoagulant accelerates the action of antithrombin III, which neutralizes thrombin and thus prevents the formation of fibrin from fibrinogen.
Ethylenediaminetetraacctic Acid (EDTA):
The chelating agent EDTA is particularly useful for hematological examinations because it preserves the cellular components of blood. It is used as the disodium or dipotassium salt. EDTA prevents coagulation by binding calcium, which is essential to the clotting mechanism.
Sodium Fluoride:
Sodium fluoride is usually considered a preservative for blood glucose; however, it also acts as a weak anticoagulant. It exerts its preservative action by inhibiting the enzyme systems involved in glycolysis.
Citrate:
Sodium citrate solution is widely used for coagulation studies, because its effect is easily reversible by addition of Ca+2. It appears to preserve labile procoagulants, but it has little application in clinical chemistry.
Oxalates:
Specimen collection N. M. ELIAS 6
Sodium, potassium, ammonium, and lithium oxalates inhibit blood coagulation by forming rather insoluble complexes with calcium ions.
Iodoacetate:
Sodium iodoacetate is an effective antiglycolytic agent and a substitute for sodium fluoride. Because it has no effect on urease, it can be used when glucose and urea assays are performed on a single specimen. It inhibits creatine kinase but appears to have no other significant effects.
Advantages & disadvantages of plasma and serum usage:
o Use of plasma expedites analysis in medical emergencies because of harvest of serum requires a wait of 15 to 30 min for completion of coagulation before centrifugation.
o Furthermore, plasma yield from a given volume of whole blood is always greater than the yield of serum.
o When plasma is stored the fibrin clots or fragments formed and the subsequent risk of clogging sample probes of automated analytical instruments is a disadvantage.
o Plasma is also not a suitable specimen for electrophoretic analysis, because the presence of fibrinogen can confound interpretation of electrophoretic patterns.
Hemolysis
Serum shows visual evidence of hemolysis when the hemoglobin concentration exceeds 20 mg/dl.
Severe hemolysis causes a slight dilutional effect on those constituents that are present at a lower or higher concentration in RBCs than in plasma. Thus, plasma concentrations or activities of aldolase, total acid phosphatase, LDH, AST, ICD, K+, Mg, and phosphate are particularly increased by hemolysis.
An additional band due to hemoglobin may be observed on serum protein electrophoresis.
Specimen collection N. M. ELIAS 7
Storage of blood specimens
1) Plasma or serum should be separated from cells as soon as possible and certainly within 2 hrs to minimize the metabolism of cells.
2) If it's impossible to centrifuge a blood specimen within 2 hrs the specimen should be kept at room temperature rather than at 4 °C to minimize hemolysis.
3) If the specimen can't be analyzed at once, the separated serum should be generally stored in capped tubes at 4 °C until analysis both to:
i- Maintain stability of the specimen; and
ii- Reduce evaporation.
4) If a specimen for a particular test is sufficiently unstable at 4 °C, the serum specimen should be held at – 20 °C in a freezer capable of maintaining this temperature.
5) Specimens for bilirubin or carotene must be protected from both daylight and fluorescent light to avoid photodegradation.
6) Specimen tubes should be centrifuged with stoppers in place. Closure lowers evaporation and prevents aerosolization of infectious particles.
7) Removal of the stopper before centrifugation allow loss of CO2 and increase in blood PH.
Changes in blood metabolites on keeping
1. Glucose is converted to lactate, as a result of glycolysis occurring in blood cells specially RBCs. It's inhibited by fluoride but fluoride interferes with glucose oxidase and urease methods.
2. Glycolysis decreases serum glucose by approximately 5 – 7 % in 1 hr. (5 – 10 mg /dl) in uncentrifuged coagulated blood at room temperature.
3. The rate of glycolysis is higher in the presence of leukocytosis or bacterial contamination.
4. Several substances pass through RBCs membrane e.g. K+, LDH.
5. Loss of CO2, since the PCO2 of blood is much higher than air leading to
Specimen collection N. M. ELIAS 8
fall in plasma total CO2.
6. Phosphate increases due to hydrolysis of organic ester phosphate compounds of erythrocytes.
7. Enzymes activities are lost on long keeping
8. Formation of ammonia from nitrogenous substances.
Urine collection
Normal urine is pale yellow, clean & transparent. It contains chloride, sulfate, calcium, ammonia, uric acid & creatinine, but there are many of the pathological constituents which are routinely tested in urine are protein, glucose, blood, ketone bodies and bile salts. In biochemistry laboratory, urine samples are assayed for either qualitatively or quantitatively analysis. Collecting the urine specimen is dependent on the type and purpose of the urine test itself. Since characteristics of the urine are affected by collection techniques and time of day, collection methods. Three familiar urine collection methods include
1- First (voided) morning specimen
The specimen is collected when the individual first awakens; it is preferred for routine urinalysis because it is more concentrated and therefore more likely to reveal abnormalities as well as the presence of formed substances. It is also relatively free of urine composition changes over the course of the day, i.e. dietary influences and of changes caused by physical activity, because the specimen is collected after a period of fasting and rest.
2- Random urine specimen
It's the most commonly requested specimen, which can be collected at any time.
There are no special preparation procedures or dietary restrictions for the first morning and random urine specimen collection methods.
Procedure for morning & random urine specimens
1. The patient is instructed to void directly into a clean, dry container.
Specimen collection N. M. ELIAS 9
2. All specimens should be covered tightly, labeled properly, and sent immediately to the laboratory. The label should be on the cup, not on the lid.
3. If a urine specimen is likely to be contaminated with drainage, vaginal discharge, or menstrual blood, a clean specimen must be obtained using the same procedure as for bacteriologic examination.
4. If the specimen cannot be delivered to the laboratory or tested within 1 hour, it should be refrigerated or have an appropriate preservative added.
Interfering Factors
1. Feces, discharges, vaginal secretions, and menstrual blood will contaminate the urine specimen. A clean voided specimen must be obtained .
2. If the specimen is not refrigerated within 1 hour of collection, the following changes in composition may occur :
a) Increased pH from the breakdown of urea to ammonia by urease-producing bacteria.
b) Decreased glucose from glycolysis and bacterial utilization.
c) Decreased ketones because of volatilization.
d) Decreased bilirubin from exposure to light.
e) Decreased urobilinogen as a result of its oxidation to urobilin.
f) Increased nitrite from bacterial reduction of nitrate.
g) Increased bacteria from bacterial reproduction.
h) Increased turbidity caused by bacterial growth and possible precipitation of amorphous material.
i) Disintegration of red blood cells (RBCs) and casts, particularly in dilute alkaline urine.
j) Changes in color caused by oxidation or reduction of metabolites
3- Other urine specimen collection methods
a) Timed specimens (2-hour & 24-hour) are collected when it is necessary to analyze urine that is excreted over a certain number of hours. The twenty-
Specimen collection N. M. ELIAS 10
four-hour timed collection method is fairly common. Substances excreted by the kidney are not excreted at the same rate during different periods of the day and night; therefore, a random urine specimen might not give an accurate picture of the processes taking place over a 24-hour period. For measurement of total urine protein, creatinine, electrolytes, and so forth, more accurate information is obtained from a long-term specimen.
Procedure
1. At the beginning of a 24-hour timed urine specimen collection (or any other timed specimen collection), the patient is asked to void. This first specimen is discarded, and the time is noted.
2. The time the test begins and the time the collection should end are marked on the container. As a reminder, it may be helpful to post a sign above the toilet (eg, 24-Hour Collection in Progress), with the beginning and ending times noted.
3. Do not predate and pretime requisitions for serial collections. It is difficult for some patients to void at specific times. Instead, mark the actual times of collection on containers.
4. Documentation of the exact times at which the specimens are obtained is crucial to many urine tests.
5. All urine voided over the next 24 hours is collected into a large container (usually glass or polyethylene) that is labeled with the patient’s name, the time-frame for collection, the test ordered, and other pertinent information.
6. Instruct the patient to urinate as near to the end of the collection period as possible.
7. To conclude the collection, the patient voids 24 hours after the first voiding.
8. All urine voided in a 24-hour period is collected into a suitable receptacle, wide-mouth container; a preservative is added or the collection is kept refrigerated, or both.
9. Test results are calculated on the basis of a 24-hour output.
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Storage
1- Nonrefrigerated samples may be kept in a specified area or in the patient’s bathroom.
2- If refrigeration is necessary, the collection bottle must either be refrigerated immediately after the patient has voided or be placed into an iced container.
Interfering Factors
1. Failure of patient to follow the procedure. The patient should be given both verbal and written instructions.
2. Instruct the patient to use toilet paper after transferring the urine to container. Toilet paper placed in the specimen decreases the actual amount of urine available and contaminates the specimen.
3. The presence of feces contaminates the specimen. Patients should void first and transfer the urine to the collection receptacle before defecating.
4. If heavy menstrual flow or other discharges or secretions are present, the test may have to be postponed or an indwelling catheter may need to be inserted to keep the specimen free of contamination. In some cases, thorough cleansing of the perineal or urethral area before voiding may be sufficient.
b) Catheterized specimens are collected when voiding is difficult as with obstruction and severe urinary tract infection.
Procedure
1- The straight catheter method is accomplished by inserting a sterile, lubricated catheter into the urinary bladder and collecting the urine.
2- For patients who have an indwelling catheter, commonly called a Foley catheter, the urine specimen must not be collected from the urinary drainage bag.
3- The indwelling catheter is equipped with a specimen collection port that allows urine to be collected with a syringe before it reaches the drainage
Specimen collection N. M. ELIAS 12
bag. It may be necessary to clamp off the catheter for about 15 to 30 minutes before obtaining the sample.
4- Clean the specimen port with antiseptic before aspirating the urine sample with a needle and syringe.
c) Special-purpose specimens include fasting specimens and postprandial specimens. For fasting specimens the patient voids and discards the first urine specimen at least four hours after food ingestion. The next voided urine is the fasting specimen. The postprandial specimen is urine voided two to three hours after a meal.
The midstream technique
1. It's necessary when a urine specimen must be free from contamination.
2. After thorough handwashing, the patient is instructed to cleanse the genital area with an antiseptic wipe, to void a small amount of urine into the toilet, to stop voiding midstream, and to collect a urine sample by voiding into the collection container. The specimen container must be free of feces, mucus, or vaginal discharge.
Preservatives
They are usually added to
a) Reduce bacterial action or chemical decomposition.
b) Solubilize constituents that might otherwise precipitate out of solution.
c) Decrease atmospheric oxidation of unstable compounds.
1. The most satisfactory preservation of urine specimens is refrigeration immediately following collection.
2. Urinary preservative tablets that contain a mixture of chemicals, such as potassium acid phosphate, sodium benzoate, benzoic acid, methenamine, sodium bicarbonate, and red mercuric oxide have been used for chemical and microscopic examination. Because these tablets contain Na+ and K+ salts, among others, they cannot be used for analysis of these analytes.
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3. The preservative tablets act mainly by lowering the PH of the urine and by releasing formaldehyde.
4. Acidification PH 3 (sulfamic acid, boric acid are used to reduce PH) is widely used to preserve 24-hrs specimens and is particularly useful for specimens for Ca++, steroids, and vanillylmandelic acid determinations.
5. However, precipitation of urates occurs, thereby rendering a specimen unsuitable for measurement of uric acid.
6. Toluene is the only organic solvent that is still used as a preservative (large enough amount acts as a barrier between the air and the surface of the specimen).
7. A mild base, such as NaHCO3 or a small amount of NaOH, is used to preserve porphyrins, urobilinogen, and uric acid. A sufficient quantity should be added to adjust the PH 8 - 9.
COLLECTION OF OTHER SAMPLES
Feces
It is frequently analyzed in biochemistry laboratory to detect the presence of "hidden" or so-called occult blood, which is recognized as one of the most effective clues to the presence of a bleeding ulcer or a malignant disease in the GIT. Feces is composed of:
1- Products of digestion which are not absorbed.
2- Undigested food such as starch, granules, cellulose fibers, muscle fibers.
3- Changed bile pigments.
4- Cells of intestinal wall.
5- Products of foodstuff, e.g. indole, skatole, fatty acids
6- Bacteria
7- Water
The feces samples are collected in bedpan. The samples should be examined soon and watery samples can be preserved in refrigerator while solid samples Specimen collection N. M. ELIAS 14
can be dissolved in H2O and this can be preserved with formalin 2 ml / 10 ml DW. Feces samples can be collected by giving:
1) Diet containing 190 g carbohydrates, 111 g fats, 102 g proteins;
2) Charcoal, carmine, gentian violet dye (0.5 – 1 g) in the form of capsule; or
3) Chrominum trioxide capsule (500 mg capsule 3 times).
Normal biochemical values of feces
1) Weight for a day is ~ 60 – 200 g depending up on diet. Diet rich in meat & low in vegetables reduces feces outcome.
2) 70–80 % water contents, 25–45 g total solid, PH is 5–9; usually alkaline.
3) Odor or smell due to indole, skatole & H2S.
Spinal fluid
It is normally obtained from the lumbar region. Spinal fluid is examined when there is a question as to the presence of a cerebrovascular accident, meningitis, demyelinating disease, or meningeal involvement in malignant disease.
Synovial fluid aspiration (arthrocentesis)
Synovial fluid is withdrawn from joints to characterize the type of arthritis and to differentiate non-inflammatory effusions from inflammatory fluids.
Amniotic fluid aspiration (amniocentesis)
Requested for prenatal diagnosis of congenital disorders, assess fetal maturity, or to look for Rh isoimmunization or intrauterine infection.
Pleural, pericardial, and ascitic fluid collection
Pleural (thoracentesis), pericardial (pericardiocentesis), and peritoneal (paracentesis) cavities normally contain a small amount of serous fluid that lubricates the opposing parietal and visceral membrane surfaces. Inflammation or infections affecting the cavities cause fluid to accumulate. The fluid may be removed to determine if it is an effusion or an exudate, a distinction possible by protein or enzyme analysis.
Collection of saliva
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Measurement of a drug in saliva has been suggested to estimate the free, pharmacologically active concentration of the drug in serum.
Specimen transport
1. It should be assumed that transport from a referring laboratory to a referral laboratory may take as long as 72 hours.
2. The tube used for holding a specimen (primary container) should be so constructed that the contents do not escape and should protect the specimens from adverse conditions.
3. The shipping or secondary container used to hold one or more specimen tubes or bottles must be constructed to prevent the tubes from knocking against each other. Corrugated cardboard, fiberboard, designed to fit around a single specimen tube may be used.
4. For transportation of frozen or refrigerated specimens, a special container must be used. The container walls should be 2.5 cm thick to provide effective insulation; should be vented to prevent buildup and possible explosion of carbon dioxide under pressure. Solid carbon dioxide (dry ice) is the most convenient refrigerant material for keeping specimens frozen, and temperatures as low as – 70 °C must be used.
