Collection and Preparation of
Blood Specimen
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Blood is one of the most common specimens studied in biochemical laboratories in search of blood disorders, metabolic disorders and infection.
Blood clots within a few minutes after it has been removed from the body, unless an anticoagulant is used which stops the process of clotting. Anticoagulated blood is also known as whole blood. Plasma (Fluid portion of unclotted blood) is obtained from the anticoagulated blood. Serum (Fluid portion of clotted blood) is obtained from clotted blood.
Usually, blood is obtained by vein puncture with the help of a sterilized dry syringe. For clinical biochemistry, mostly serum/plasma and occasionally whole blood is required.
Preparation of Specimen Bottles Using Different Anticoagulants
Anticoagulants are used when either whole blood or plasma is required. Most of the anticoagulants remove calcium which is one of the factors required in coagulation process. Heparin, however, directly interferes in the coagulation process by destroying thrombin and thromboplastin. Use of an anticoagulant largely depends upon the nature of clinical investigation, e.g. heparin is necessary for determination of blood gases (and pH) while sodium fluoride in addition to oxalate, is necessary for glucose estimation. Specific amount of anticoagulant is also recommended.
Containers of blood collection must be thoroughly cleaned with no trace of detergent. Specific amount of anticoagulant is added (preferably in solution form) and dried oven inside the container. For oxalate anticoagulant (which is most common in biochemistry lab), a high temperature is avoided as it changes oxalate into carbonate. All previously prepared containers can be stored at room temperature. For easy identification, the containers are labelled differently with coloured markers.
EDTA (Ethylene Diamine Tetraacetate)
10% solution of dipotassium salt of EDTA, 0.l ml of this is used/5 ml of blood (Conc. 2 mg/ml of whole blood).
Double Oxalate
1.2% of ammonium oxalate + 0.8% of potassium oxalate. Use 0.5 ml for 5 ml blood (Conc. 2 mg/ml of whole blood).
Sodium Citrate
0.106 M trisodium citrate in distilled water, sterilize it. This is used in solution form and is not dried inside the container. 1 ml of this solution is used for 9 ml blood (The ratio of anticoagulant solution and whole blood comes to 1 : 9, which is ideal for coagulation studies). Not very common for biochemical studies/purposes.
Sodium Fluoride-Potassium Oxalate
Used for blood sugar estimation (except in enzymatic method of sugar estimation).
1.2% sodium fluoride + 5% potassium oxalate. 0.25 ml is used/5 ml of blood (=3 mg sodium fluoride + 15 mg potassium oxalate).
[Caution: Fluoride is a poison].
Heparin
Limited to the determination of blood gases and electrolytes, 0.1-0.2 mg/ml of the blood.
Collection of Blood by Vein Puncture
Before proceeding to collect the blood from the patient, assemble all the necessary equipment which includes the container for blood collection, a needle, syringe, tourniquet, disinfectant (methylated spirit or 70% alcohol), swab and a tray of cold water to rinse the needle and syringe after drawing blood.
Needle Sterilized sharp needle of bore size 18 to 20 gauge for adults and 23 gauge for children
is used. Use of disposable needles is highly recommended. In case of reusable ones, the needle point should be kept sharp. Keep a stock of sterile needles in small glass tubes and the point should be on a pad of non-adsorbent cotton wool and the tube should be plugged with the same material. A sterile needle should be attached to the syringe under asceptic conditions.
Syringes Of different capacities, 2 ml, 5 ml, 10 ml and 20 ml are available. The size depends
upon the amount of blood needed. Check that the end of each syringe fits into the needle. At any stage of assembly of the needle and the syringe, do not touch the tip of the needle and keep the asembled syringe and needle inside a sterilized tube or covered with sterile gauze. With reusable glass syringes, the fit of the plunger and the barrel and the integrity of the syringe tip should be checked. Disposable plastic syringes come in a sterilized wrap and they have sharpened needles for single time use.
Following use, syringe should be dipped in cold water to remove the blood. All glass syringes should be properly sterilized and perfectly dry. A wet syringe, an improperly washed syringe. Use of a too fine needle, emptying the syringe without removing the needle are some of the causes that result in haemolysis.
Tourniquet It is a soft rubber tubing of 2.5 mm bore and 30-40 cm in length. A flat elastic
rubber strip can also be used. This is applied to the arm to slow the blood flow and make the veins more prominent. This helps to select the puncture site.
Collection and Preparation of Blood Specimen 53
Procedure of Blood Collection
Venous blood is frequently collected while the blood may be taken from any prominent vein, a vein on the front of the elbow or forarm is almost universally employed. The arm should be warm. This improves the circulation. The arm is extended and a rubber torniquets firmly applied a few inches above the elbow. The skin over the vein is cleaned by rubbing over with spirit or ether. A well sharpened sterile hypodermic needle fixed onto a syringe can be held steady by a thump of the other hand of the operator. When the needle enters the vein, the plunger is withdrawn slightly. If blood appears, the tourniquet is released. When the desired amount of blood has been drawn into the syringe, a small pad of wodsoaked with spirit or ether is placed on the arm where the needle was inserted, and the needle is withdrawn. This pad is held on firmly for a few minutes until bleeding stops. The needle is removed from the syringe and the blood transferred to an appropriate container, using minimum amount of pressure. The needle and syringe are immediately washed out with cold water to remove any remaining blood. The needle to prevent any infection must be stressed.
Capillary Puncture
A capillary is a small blood vessel connecting the small arteries (arterioles) to the small veins (venules). Capillary blood is obtained by skin puncture which is recommended for babies and where vein puncture is difficult (old, prolonged illness). In adults and older children, the tip of the finger is punctured and in infants, the heel is chosen.
Arterial Blood
It is rearely examined. It is taken for blood gas determinations. It is most commonly obtained by inserting a needle into radial, brachial or femoral artery usually under a local anesthesia.
Separation of Serum and Plasma
Serum
Non-anticoagulated blood yields serum, the fluid portion of clotted blood in order to obtain serum is collected in a plain tube without anticoagulant and allowed to clot for 30 min at room temp. The tube can preferable be kept slanted. When the clot separates out, it is centrifuged (2500 rpm for 10 min) and serum be separated. Separation must not be delayed much because prolonged contact of the cells with the serum will cause the release of the red cells constituents into the serum composition. If the centrifuge is not available, refrigerate the blood for several hours, the clot will separate from the serum which can be removed with the help of a pasteur pipette.
Plasma
Appropriate aanticoagulant should be mixed with the blood by gentle rotation. Excessive amount of anticoagulant should not be used. Separation of plasma should be done by centrifuging at low to moderate speed. If the plasma is not required immediately, the cells can be allowed to sediment out. The upper fluid is removed and finally cleared of any remaining cells by centrifugation.
Preparation of Protein-free Filterate
Use of Different Protein Precipitating Agents
The first step in majority of biochemical determination made on blood is to remove the proteins as these interfere in many ways in the actual investigation. For this purpose, many substances are used, chiefly acids, heavy metal, ion in presence of suitable alkali, organic solvents, etc. As acids, e.g. tungstic acid and trichloroacetic acid are mostly used, others include perchloric, tungstomolybdic, phosphotungstic and sulphosalicylic acid. Choice of method depends on nature of estimation.
Few Common Methods are:
a. Folin method Tungstic acid is prepared by adding 10% sodium tungstate and 2/3 NH2SO4. To prepare the protein free filterate (PFF), mix 1 volume of blood with volume of D/W,
add 1 volume of 10% sodium tungstate and 1 volume of 2/3 NH2SO4. Shaking well during
the addition of H2SO4. Allow to stand for 10 minutes and centrifuge at 2000 rpm for 5 min.
b. Modified folin method/Maden method Mix 1 volume of whole blood to 1 volume of 10% sodium tungstate and 8 volume of N/12 H2SO4. Mix well. Keep for 10 min and centrifuge. c. Somogyi’s method In its original form, the additions are as follows: 4.2 ml isotonic sodium sulphate + 0.2 ml blood + 0.3 ml 10% zinc sulphate + 0.3 ml (N/S) sodium hydroxide. Mix well. Keep for 10 min and centrifuge. In addition to proteins saccharides (non-sugar reducing substances) are also precipitated. This method or its modification is particularly used for true sugar estimation.
COLLECTION AND PRESERVATION OF URINE SPECIMENS Collection of Urine Specimens
For most qualitative tests, any fresh specimen is suitable.
Because composition of urine varies during the days, quantitative estimation is usually carried out on 24 hr collection. While the specimen is being collected and during the period before it is analysed, it is essential either to keep the specimen refrigerated or to have a preservative in the collection container, so that unstable substances may be preserved and the growth of bacteria prevented. The preservative used will depend on the substance which is to be determined.
Preservatives
Hydrochloric Acid
50 ml of 2 M HCl is adequate for a 24-hr specimen suitable in particular for the determination of Ca, P, N, ammonia, VMA estimation. It can also be used when sodium, potassium, urea estimations are required but it is not suitable for uric acid and protein estimation as these may be precipitated.
Collection and Preparation of Blood Specimen 55 Toluene
10 ml of toluene is commonly used for 24 hr collection for Na, K, uric acid, creatinine and protein analysis but is not suitable for determination of Ca, P, VMA, ammonia, etc.
Hibitane (Chlorhexidine diacetate)
It is a preservative when glucose is to be estimated in urine since it inhibits the action of bacteria but does not interfere with the analysis either by enzyme or reduction methods. 5 ml of a 5% aq solution is added to the container. Chloroform, light petroleum, thymol and formalin are used as urine preservatives. A few and one of these is put into a 2.5 l container and given for collection.
Urine Analysis
A routine biochemical examination of urine consists of the following tests:
Physical Tests
Colour Urine is normally clear and pale yellow in colour due to a pigment, urochrome, said to
be a compound of urobilin, urobilinogen and a peptide substance.
Odour Freshly passed urine has a characteristic of aromatic odour, said to be due to volatile
organic acids. When the urine is allowed to stand, decomposition of urea by bacteria occurs, acid ammonia is evolved.
Reaction Freshly passed normal urine is usually slightly acidic with a pH about 6.0 and range
of 4.8-6.8. The pH is determined by simply dipping the pH indicator strips into the urine and comparing the colour change of the strip against the colour guide supplied with the indicator papers.
Volume Volume of urine passed by a normal person an average ranges from 1200-1500 ml/
day.
Specific Gravity
Specific gravity of normal urine ranges from 1.008 to 1.030. It is determined by using urinometer as follows:
Mix urine well. Take urine in specific gravity cylinder. Then put the urinometer gently in the urine. Take care it does not touch the wall of the cylinder. Take reading of the lower meniscus of the urine touching the calibration viewed at eye-level.
If the volume of urine is not sufficient to the cylinder, report as q.n.s. (quantity not sufficient). The physical test of urine is valuable if fresh urine is used. Upon standing, almost all the physical properties are changed.