Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
148
Medical Laboratory Technology: Volume 1
Figure 5.2 Collection of blood by venepuncture: (α–d) Tie the tourniquet, Prepare the site for
blood collection — (e–g) selection of commonly used veins, (h) Application of antiseptic; (i,j) Penetrate the needle, (k) Draw blood after releasing the tourniquet, (I, m)
Withdraw the needle and apply pressure with sterile cotton in order to stop bleeding
from the punctured spot, (n) Remove the needle from syringe, (o, p) Collect blood in
an appropriate container
Specimen Handling and Laboratory Records
149
Use of blood collection bottles
At the time of blood collection, collect the requisite amount of blood in the marked-bole and
mix the blood gently and thoroughly by swirling. Blood must be drawn to the appropriate
mark (2.5 or 5.0 mL). If the blood is less in volume, the anticoagulant concentration will be
more, resulting in distortion of cells. On the other hand, if the blood is more in quantity, the
anticoagulant may not be fully eective and cloing might occur which will aect the results.
The nal concentration of anticoagulant should be 1.5 ± 0.3 mg/mL.
Double oxalate
Fluoride combined with oxalate is recommended for blood glucose determination, which is
described later. Many laboratories use oxalated blood for coagulation studies but citrated
blood is recommended.
The anticoagulant solution contains 1.2% ammonium oxalate and 0.8% potassium oxalate.
Deliver 0.25 mL of the double oxalate solution into penicillin boles with a 2.5-mL mark and
0.50 mL in those with a 5.0 mL-mark.
Evaporate the anticoagulant solution in the blood collection boles in an oven (60°C) or in
an incubator (37°C). Put the rubber cap after drying the uid and then put the colour code on
the bole (grey is the colour of choice).
Sodium citrate
Prepare a 0.106 M solution of trisodium citrate in distilled water: This is equivalent to
2.73 g/100 mL solution of the trisodium salt. Sterilize it. This anticoagulant is used in solu-
tion form and is not dried inside the container. The penicillin boles are specially marked
for 2 and 10-mL marks. Give the colour code (blue is recommended). Add 0.4 and 1 mL of
the sterilized anticoagulant solution in boles marked 2 and 10-mL, respectively. After the
mixing of blood specimen with the anticoagulant, the ratio of anticoagulant solution and
whole blood comes to 1:4 and 1:9, respectively. The former is used for ESR (erythrocyte
sedimentation rate) and the laer for coagulation studies. The Westergren method now
modies the ESR method where EDTA-anticoagulated blood is used. By this modication,
a special blood collection procedure for haematological studies is avoided. The results are
not signicantly dierent so as to aect the clinical diagnosis. This will be further discussed
in the haematology section.
Sodium uoride–potassium oxalate
This anticoagulant is used for preparing blood specimens for the determination of glucose
and urea in plasma by non-enzymatic methods. Fluoride inhibits glycolic enzymes and there-
by prevents loss of glucose during transportation or delay in specimen handling. As uoride
is not a strong anticoagulant, it is mixed with oxalate. With the advent of newer enzymatic
methods, the use of this anticoagulant is now limited.
The anticoagulant is made of 1.2% sodium uoride (poison!) and 6.0% of potassium
oxalate. Grind 1.2 and 6 g of the respective salts into a ne powder. Quantitatively, transfer
the powder into a 100-mL volumetric ask, dissolve in 80 mL of distilled water and then
make it to a 100-mL volume. Add 0.25 mL of the above solution into the blood collection vials
with 5-mL mark and evaporate to dryness in an oven or incubator.
Heparin
This is rarely used in tropical countries and is limited to the determination of blood gases
and electrolytes. This chemical is expensive and is not very stable in hot climates. Use
commercially available heparinized blood collection tubes, which show the level of blood to
be collected. In addition, heparinized blood is unsuitable for microscopic studies since cells
are distorted more rapidly than in EDTA-anticoagulated blood.
150
Special note
1. All blood collecting tubes or vials must have airspace to facilitate mixing of reagents. As
a rule of thumb this should be a volume corresponding to about one-fth of the tube.
2. All tubes and vials must have rubber caps (stoppers) to facilitate transportation.
Medical Laboratory Technology: Volume 1
Blood collection by venepuncture
Supplies
• Needle: Sterilized sharp needles of bore size 18–20 gauge (medium, 1.2–0.9 mm) for
adults and 23 gauge (0.5 mm) for children are needed. The bevel length should be
medium (20 mm) for adults and short (15 mm) for children. The use of disposable
needles is recommended. In case of reusable ones, the needlepoint should be kept sharp
and assurance of their sterility ensured. Keep a stock of sterile needles in small glass
tubes and the point should rest on a pad of non–absorbent (sterile) coon wool and the
tube should be plugged with the same material. A sterilized needle should be aached
to the syringe under aseptic condition (Figure 5.2).
• Syringe: Syringes of dierent capacities, 2-, 5-, 10- and 20-mL, should be available. The
size of the syringe to be used depends on the amount of blood needed. There are different types of connections between the needle base and the syringe tip; always read
the manufacturer’s instructions before using them. Check the end of each syringe for
its right t for the needle. At any stage of assembly of the needle and syringe, do not
touch the needle tip and keep the assembled syringe and needle inside a sterilized tube
or covered with sterile gauze. With reusable glass syringes, the t of the plunger and
barrel and the integrity of the syringe tip should be checked. Disposable plastic syringes
come in a sterilized wrap and they have sharper needles for single use. Now-a-days,
sterilized disposable plastic syringe with needle is recommended due to life threatening
infections such as HIV.
• Following the use of a reusable syringe, it should be rinsed in cold water to remove the
blood. All glass syringes should be properly sterilized and perfectly dry before their
use. A wet syringe, the presence of detergent (not properly washed), the use of too ne
a needle and emptying the syringe without removing the needle are some of the causes
that lead to haemolysis. Haemolysed blood is unacceptable of a number of analyses
done in haematology, chemistry and serology.
• Tourniquet: This is a soft rubber tubing of 2–5 mm bore and 30–40 cm length. A at elas-
tic rubber strip can also be used. The tourniquet is applied to the patient’s arm to slow
the blood ow and make the veins more prominent. This helps to select the puncture
site for blood drawing.
Procedure
The procedure of venepuncture comprises of three steps—preparation, drawing blood and
clean up.
A. Preparation
1. Introduce yourself to the patient and be pleasant. Read the patient’s request form
carefully. Identify the patient by name, date of birth and accession number. If the patient
should be fasting before giving blood, inquire if the patient has done so.
2. Decide how much of blood is needed and arrange the correct tube(s) to be used for
each test. For example, if haemogram is requested, 2 mL of blood in EDTA-tube will
be sucient, whereas, if the blood is needed in the biochemistry laboratory, 10 mL of
blood in a plain container without any anticoagulant would be needed. This will yield
the required amount of seram.
Specimen Handling and Laboratory Records
151
3. Plan the sequence in which blood samples must be taken. For example, the rst 1 mL of
blood must be discarded when blood is taken for a coagulation assay. So, collect this in
the second tube, which will be used for other tests.
4. Before taking the blood, wash your hands with soap and water. If necessary, wear gloves
if the patient is suspected of harbouring an infection or contagion. The phlebotomist, to
minimize patient’s apprehension, should explain the procedure of blood collection.
5. Fix the needle on to the syringe, touching only the top of the needle. Test the needle and
syringe to make sure that the needle is not blocked and the syringe is airtight. Place the
end of the needle in the sterile tube until ready for use.
6. Ask the patient to sit alongside the table used for taking blood. The patient should sit on
a chair so that he/she can comfortably stretch horizontally the left or right arm. It also
applies to a patient in the horizontal position (in bed). The elbow may be supported by
an armrest.
7. The working arm (often the right arm) should be selected for blood collection, since on
this side veins are easily accessible. Lay the patient’s arm on the table, palm upwards
and support it by placing a small cushion under the elbow. If the patient is in bed, lay
his/her arm in an outstretched position. The correct site to take the blood is from the
vein at the bend of the elbow, at the point where the vein is thickest and most easily
visible. If possible, choose one of the branches forming a Y (median cubital or elbow
vein) just above their junction. This is the most common site for blood collection. Alternatively, you can choose basilic or cephalic veins (Figure 5.4).
Caution Never draw blood from a standing patient or patient siing on a high stool.
The phlebotomist should be prepared for the occasional patient who may faint and
should be trained to administer rst-aid techniques should this occur.
8. Fix the needle on to the syringe, touching only the top (syringe end) of the needle. Test
the needle and syringe to make sure that the needle is not blocked and the syringe is
airtight. Place the end of the needle in the sterile tube until ready for use.
B. Drawing Blood
1. Apply the tourniquet as described below.
a. Place the tourniquet under the patient’s arm just above the bend in the elbow. With the
right hand, wrap the tourniquet rmly around the arm and hold the ends.
b. With the left hand, pull one of the tourniquet’s ends across.
c. Loop the end under the main part of the tourniquet half way through in a slipknot. The
tourniquet should be just tight enough to slow down the blood ow and distend the
veins; however, it must not be so tight that the blood ow in the arteries is reduced. The
slipknot can be easily released when the tourniquet is to be removed.
2. Ask the patient to open and close his or her hand several times to swell the veins. Using
the index nger of your left hand, feel for the vein where you will introduce the needle.
Palpate the lled vein about 5–7 cm distal from the tourniquet and identify the site for
vein puncture (Figure 5.3).
3. Disinfect the site on the skin with a sterile gauze or coon swab soaked with 70% alcohol.
4. Take the syringe in your right hand, holding your index nger against the top of the
needle.
5. Position the needle with the bevel uppermost and hold it at a low angle (about 150–250)
to the skin surface with the tip directed towards the tourniquet. Make the venepuncture entering the centre of the vein without hesitation in the direction of the anatomical
course of the vein to avoid injury to the interior wall of the vessel, which would cause
pain to the patient.
Important Never approach a vein from the side.
152
Medical Laboratory Technology: Volume 1
6. You will feel the needle going through—the layer of skin, which is resistant, than the
wall of the vein, which is less resistant (more exible). So, there will be a sudden loss of
resistance as you penetrate the needle (Figure 5.4). Push the needle along the line of the
vein to a depth of 1.0–1.5 cm.
7. With your left hand pull back the piston of the syringe slowly. Blood should appear in
the syringe. Maintain a continuous blood ow, but avoid development of a strong negative pressure in the syringe chamber, which may cause collapse of the vein, foaming
and haemolysis of red blood cells.
Important The person collecting the blood sample must ensure that cloing does not
occur during collection (e.g., due to a lengthy procedure and/or to multiple aempts
with the same needle) and that there is adequate blood ow without turbulence.
8. Continue to withdraw the piston to ll the syringe with the required amount of blood.
9. After the requisite amount of blood is withdrawn, place a sterile dry gauze pad under
the needle still sticking in the vein.
10. Open the tourniquet, by pulling the looped end and allow blood to ow freely in the
vein. Do not pull the syringe and needle out prior to opening the tourniquet.
11. Apply a dry swab over the hidden point of the needle. Withdraw the needle in one rapid
movement from under the swab (Figure 5.3).
12. Ask the patient to press rmly on the coon wool swab for 3 min, keeping the arm
outstretched. Bending the arm back over the swab is not recommended because of the
risk of a haematoma.
13. Remove the needle from the syringe.
14. Expel the blood slowly out of the syringe gently down the side of the container tube
or vial. Fill specimen tubes or vials with the blood up to the mark. Immediately invert
tubes or vials that contain the anticoagulant several times to ensure complete mixing of
anticoagulants. The mixing should be thorough but gentle in order to prevent cloing
without causing haemolysis.
Note If several tubes are to be lled from the amount of blood drawn, the following
sequence is recommended:
• Blood culture tube (Bacteriology)
• Plain tube for serum (Biochemical analysis)
• Tube with anticoagulant (whole blood—haematology and coagulation)
• Other additives
15. Label tubes or boles clearly with the patient ID, date of specimen collection and the
accession number.
16. Before the patient leaves, re-inspect the venepuncture site to ascertain that the bleeding
has stopped. If the bleeding has stopped, apply an adhesive tape over the coon wool
swab on the wound; otherwise, continue to apply pressure until the bleeding stops. Do
not leave the patient until the bleeding stops.
C. Cleanup
1. Rinse the needle and syringe at once with cold water, then rinse in a disinfectant (see
disinfection procedure) and give a nal rinse. Prompt rinsing prevents cloing of blood
in the syringe or lumen of the needle. Many laboratories prefer to leave the syringe and
the needle in water with a mild detergent. Then they are washed later thoroughly prior
to sterilization.
2. Place the rinsed needles and syringes in small glass tubes plugged with non-absorbent
coon wool and sterilize in an autoclave (see Sterilization). If dry-heat sterilization is
done in an oven, do not use coon wool.
3. Never use a needle or syringe on another person before it has been re-sterilized. Dispos-
able needles must only be used once, as they cannot be re-sterilized.
Specimen Handling and Laboratory Records
153
Special Notes
Patient preparation
For the measurement of certain analytes, it should be ascertained that the patient has fasted
for 12 h. Inquire from the patient if he/she followed the physician’s instructions.
Figure 5.3 Basic steps in venepuncture
Figure 5.4 Cross-section of puncture site during blood collection by venepuncture method
154
Medical Laboratory Technology: Volume 1
Preventing haematoma during venepuncture
• Preferably use veins in the elbow area and only major veins.
• Be careful that the bevel of the needle is fully inside the vein.
• Be careful not to transverse the vein.
• Loosen the tourniquet and ensure haemostasis with a dry sterile coon ball before pulling out the needle.
Special Circumstances
• When patients are receiving a transfusion or infusion, blood should never be collected
near the infusion site or from the catheter but from the opposite arm. If neither arm
is free, an ankle vein is the site of choice for the venepuncture. The rationale for this
is to avoid collecting any substance that has been infused (e.g., glucose, potassium
gentamicin and heparin) and which could give rise to misleading interpretation of
results.
• In the weak or elderly patients, the venous pressure may be so low that the pressure of
the needle may collapse the vein. Take extra care to draw the blood slowly.
• If the patient’s clothing is too tight above the venepuncture site, it slows down
the ow of blood and it may also cause haematoma. Loosen the cloth before drawing
blood.
Capillary puncture
A capillary is a small blood vessel connecting the small arteries (arterioles) to the small
veins (venules). The capillary blood is obtained by skin puncture. It provides only small
quantities of blood specimens for making a blood smear or haematocrit determination.
Skin puncture specimen is preferred over venepuncture specimen for the microscopic
study of a blood smear avoiding the patient’s traumatic experience during venepuncture.
However the small amount of specimen collected by skin puncture is inadequate for most
routine tests.
Capillary blood samples are more dicult to handle than venous blood samples and
should only be used in special circumstances, e.g., neonates and infants, control check
for diabetes, severe anaemic patients, blood gas analysis and in certain patients in whom
venous blood sampling is dicult. Squeezing of the nger and subsequent dilution with
interstitial uid is the major reason for errors in capillary sampling, e.g., a decrease of
concentrations of components and particulate maer (e.g., blood cells and parasites) and
haemolysis. To reduce this, remove the rst drop of blood before collecting the sample
with a free ow of blood. Capillary blood is also used for preparing thin and thick blood
lms for the diagnosis of malaria and other blood parasites (Borrelia, trypanosomes,
microlariae).
Site for capillary blood collection (Figure 5.5)
• Ear-lobes in adults and children
• Fingertips in adults and children
Note Finger punctures should not be performed in infants, as there is a risk of injuring
the bone. Puncturing the plantar surfaces of the heel of small infants can also damage the
heel bone. Use big toe and medial and lateral positions of the planter surface of the foot of a
neonate.
Specimen Handling and Laboratory Records
Figure 5.5 Sites for skin puncture: (a) Toe puncture for infants, (b and c) Ear puncture, (d) Finger
puncture
155
Supplies
• Coon ball and sterile gauze pad.
• Capillary tubes (may be coated with heparin to avoid blood cloing prior to the labora-
tory investigation).
• Disinfectant (ethanol or isopropanol, 70%).
• Lancet or needle, sterile.
Procedure (Figure 5.6)
1. Check the things you need. Allow the patient to sit comfortably.
2. Find a spot on the middle nger or ring nger of the left hand. The spot is located on the
side of the nger, which is less sensitive than the tip. Toe puncture and ear lobe punc-
ture are done in case of infants. Avoid previously punctured sites.
3. Increase the blood ow by rubbing or carefully warming the skin in order to enhance
the accumulation of capillary blood similar in composition to that of arterial blood. Use
of warm water may be advisable for old patients. Cold skin might prevent a free ow of
blood which is essential.
4. Cleanse the puncture site with a sterile gauze pad soaked with 70% alcohol.
5. Dry the puncture site with a sterile gauze pad (or air dry), so that residual alcohol will
not mix with out-owing blood; otherwise haemolysis may occur. Wet puncture site
also does not allow the oozing out blood to form into a well-rounded drop, which is
necessary for good results especially when the drop is used for smearing on the slide.
6. Grab the nger rmly and quickly puncture the site with a sterile lancet. The puncture
should be 2–3 mm deep. A deep puncture hurts no more than a supercial one and it
gives considerably more satisfactory ow of blood. If a good puncture has been made,
the blood ows freely. If it does not, use gentle pressure to make the blood form a round
drop. Excessive squeezing causes dilution of the blood with tissue uid. Discard the
lancet in the appropriate disposable container. Used lancets should never be left lying
on the work area.
7. Wipe away the rst drop of blood with dry sterile coon wool or gauze. The rst drop
of blood is contaminated with tissue uid and will interfere with the laboratory results
if used. The succeeding drops are used for laboratory tests.
8. Collect the out owing blood in a capillary tube by holding the tube to the blood drop.
This will be used for the haematocrit determination or sucking into the Sahli pipee for
haemoglobin determination or for blood count. By touching the blood drop on a clean
slide, smear can be made on the site. About 20 μL, of blood will probably be needed in
completing laboratory requirements.
9. After blood collection, press the puncture site with a dry sterile gauze pad to stop further
bleeding.
156
Medical Laboratory Technology: Volume 1
Figure 5.6 Finger stick technique: (a) Site for nger stick, (b) Disinfect the site, (c) Puncture, (d) Wipe
o the rst drop of blood, (e) Gently squeeze the nger and produce a large rounded drop
of blood, (f) Drawing up the blood for cell count, (g) Place a drop on the microscope slide
for preparing smear, and nally, (h) Press a wad of cotton on the puncture site in order to
stop the bleeding. Fix the cotton with an adhesive tape if necessary.
Things to remember
• To avoid infection do not puncture the same site twice.
• Never touch the tip of a sterile blade or keep it on the table. If a disposable lancet is not
available, you can use broken glass chips or needle sterilized in alcohol.
• Do not touch the blood; use rubber gloves, if available. All specimens are potentially
hazardous. Hepatitis В viras and AIDS virus (acquired immune deciency syndrome)
are transmied through the blood and other body uids.
Specimen Handling and Laboratory Records
157
Processing of blood specimens
Blood specimens collected by venepuncture are occasionally processed in the collection area
or they are sent directly to the testing areas for further processing and testing. Early processing or separation of blood components is recommended for more reliable results. Plasma
is the liquid portion of anticoagulated blood, while serum is the liquid portion of cloed
blood. Plasma contains brinogen along with the soluble ingredients of the uid component
of blood. Serum, on the other hand, has all chemicals of plasma uid, except the brinogen
that is used up in the clot formation.
Preparation of plasma
1. Collect blood in a tube with dry anticoagulant. Make sure the tube is not wet. This will
cause haemolysis.
2. Mix instantly by gentle inversion. This yields whole blood.
3. Whole blood is now centrifuged at 2500 rpm for 10 min. This separates the cellular
component of the blood, which goes to the boom as red-coloured, from its liquid component of the blood, which stays at the top.
4. Take out the liquid portion (plasma) into a separate tube for further use. Plasma is commonly used in coagulation laboratory and in blood grouping.
Preparation of serum with centrifugation
1. Collect blood in a plain tube without any anticoagulant.
2. Allow the blood to clot for 30–60 min at room temperature (Figure 5.7).
3. Release the clot from the wall of the tube by means of an applicator stick.
4. Centrifuge at 2500 rpm for 10 min.
5. The liquid portion at the top is the serum. Transfer the serum into another test tube for
further testing.
6. If electric centrifuge is not available, one can obtain serum with the help of a hand
centrifuge.
Figure 5.7 Preparation of serum following centrifugation of clotted blood: (a and b) Allow the blood to
clot for 30–60 min at room temperature, (c) Release the clot from the wall of the tube by
means of an applicator stick, (d) Centrifuge, and (e and f) Transfer the serum to another
test tube