Hematology is the branch
of medical science that deals with the study of the clinical, morphologic and
laboratory disorders of the blood and the blood forming organs.
This study includes the
analyses of the concentration, structure and function of cells in the blood;
their precursors in the bone marrow; chemical constituents of plasma or serum
intimately linked with blood cell structure and function; and function of
platelets and proteins involved in hemostasis and blood coagulation.
**** THE BLOOD ****
Blood is vital, life–sustaining
fluid circulating constantly in a closed system of blood vessels; it is pumped
from the heart into arteries; from the arteries into the capillaries, and from
the capillaries into the veins to return back to the heart.
Blood is highly complex
liquid connective tissue in which the cellular elements are suspended in a
liquid supporting or ground substance.
FUNCTIONS OF THE
BLOOD
1. It transports oxygen from the
lungs to the tissues and carbon dioxide from the tissues to the lungs to be
eliminated.
2. It serves as a vehicle for
transport of food materials to the different tissues of the body.
3. It picks up metabolic end
products and delivers to other organ for use or elimination.
4. It distributes the heat produced
in active muscles and thus aids in the regulation of body temperature.
5. It transports hormones from the
glands in which they are produced to the target organs.
6. Through the presence of buffer
system, it helps maintain optimal pH of the acid – base equilibrium.
7. It regulates the water balance
through the effects of blood in the exchange of water between circulating fluid
and tissue fluid.
8. It serves in the mobilization of
defense mechanism by protecting the body against bacterial invasion and disease
through the activities of certain leukocytes and immune bodies in the blood
stream.
9. It maintains a degree of
irritability of the tissue cells so that functional activity can be carried on
satisfactorily.
**** COMPOSITION OF THE BLOOD ****
A. Fluid portion – approximately 55%
Blood
plasma is the fluid portion of the blood. It is a straw colored fluid
containing a variety of substances and with complex chemical composition. It is
the fluid portion that remains when coagulation is prevented. If blood is
allowed to clot, the liquid part that separates from the clot is called serum.
Constituents
of the liquid part:
a. Water
b. Inorganic constituents – sodium, calcium, potassium, magnesium, chloride,
bicarbonate, phosphate, sulfate
c. Organic constituents
1. Plasma proteins
(a)
Albumin –
its primary function is to cause osmotic pressure at the capillary membrane.
This pressure called colloid osmotic prevents the fluid of the plasma from
leaking out of the capillaries into the interstitial spaces.
(b)
Globulin –
alpha, beta and gamma globulins
Alpha
and beta globulins – transport substances by combining with them, acting as
substrates that react with other substances, transporting protein itself from
one part of the body to another.
Beta
and gamma globulins – protects the body against infection.
Gamma
globulins – antibodies that resist infection and toxicity thus providing the
body with immunity.
(c)
Fibrinogen –
plays a role in blood clotting
d. Internal secretions (hormones), antibodies and various enzymes as
amylases, proteases, lipases, etc.
e. Gases – oxygen, carbon dioxide, nitrogen
B. Solid portion – approximately 45%
The
solid portion consists of the cellular elements:
a. Red blood cells or erythrocytes
b. White blood cells or leukocytes
c. Hemoconia or Mueller’s blood dust; very small powdery
or dust like particles which are increased after eating fats
**** GENERAL CHARACTERISTICS OF THE BLOOD ****
A.
COLOR
Arterial
blood is bright red due to high oxygen content; venous blood is very dark or
purplish red due to low oxygen content. The colors of the blood from the
pulmonary veins and arteries are reversed since they carry blood from and to
the lungs capillaries for the elimination of carbon dioxide and pick up oxygen.
B.
VOLUME
It
varies with the size of the person, the total volume approximately 6 – 8% of
the total body weight. At birth, the total blood volume is around 250 – 350 ml.
After infancy, the volume increases gradually until adult life. The total blood
volume for adult men and women ranges from 60 – 80 ml/kg body weight.
As
a rule, the blood volume remains remarkably constant in an individual and rapid
adjustments take place within few hours after blood transfusion or intravenous
infusion. There is therefore no danger involved in taking one half liter of
blood for transfusion, and no risk in taking two ml tubes or more for analysis.
C.
VISCOSITY
The
relative viscosity in vitro at 18oC:
Whole
blood – 3.8 – 5.7
Plasma
– 1.67 – 2.35x
Serum
– 1.58 – 2.18x
D.
SPECIFIC GRAVITY
Whole
blood, men – 1.055 – 1.064
Whole
blood, women – 1.052 – 1.060
Plasma
– 1.025 – 1.029
Serum
– 1.024 – 1.028
E.
REACTION or pH
Alkaline
with pH range from 7.35 to 7.45
F.
OSMOLALITY
Serum
– 281 to 297 mOsm/kg H2O
**** COLLECTION OF BLOOD *****
The proper collection of
blood is essential for satisfactory examination. It is important therefore to
consider in detail various collection techniques.
Blood should be collected
in chemically clean or sterile containers, for blood is an excellent culture
medium for certain types of microorganisms. Containers should be thoroughly
cleaned, rinsed and dried after washing.
All specimens should be
labeled carefully at the time the blood is taken, giving the name of the
patient, date and other pertinent information.
The two sources of blood
for hematological tests are capillary or peripheral blood and venous blood.
Venous blood is preferred for most hematological examinations but peripheral
samples can be almost as satisfactory for some purposes if a free flow of blood
is obtained.
Capillary or Peripheral
Blood
Capillary blood is liable
to give discrepant results, and should only be used:
1. When venipuncture is impractical, e.g., infants and in cases of
extensive burns.
2. When small quantities of blood are required in the examination.
3. When the patient is bedridden and the operator feels that the skin or
capillary puncture is easier to manipulate.
4. When the blood examination requires free flowing capillary blood as in
clotting time and bleeding time determinations.
Sites
of skin puncture
1. Palmar surface of the tip of a finger, 3rd or 4th
finger of the left hand or the one which is less used.
2. Plantar surface of the great toe or heel of an infant especially in the
newborn.
3. Free margin of the ear lobe, not the side – is recommended for small
children, patients with poor veins, for some special tests and for patients who
are extremely edematous, especially in the extremities.
Equipment:
1.
Puncturing instruments
a. Disposable needle or lancets which are commercially available in sealed
containers
b. Hagedorn needle
c. Bard–Parker blade #11
d. Small scalper or blade
2.
Sterilizing agents
a. 70% alcohol
b. Ether
c. Alcohol–ether mixture
d. 1% alcohol solution of iodine
Advantages
of finger puncture:
1. The finger is accessible and could easily be managed.
2. Skin at the site is elastic and tends to close the wound easily.
3. It is preferably employed for preparing blood films or smears but the
blood must flow freely to avoid altered distribution of leukocytes
Advantages
of earlobe puncture:
1.
Skin is thin and puncture is less painful.
2.
Post–puncture tenderness is avoided.
3.
Less tissue juice.
Disadvantages
of skin or capillary puncture in general:
1. Small amount of blood is obtained and the examination can’t be
repeated.
2. Capillary or peripheral blood frequently hemolyzes.
3. Skin puncture yields more nearly arteriolar than capillary blood and
hematocrit, red cell count and hemoglobin content of true capillary blood are
significantly less than venous blood.
4. Platelet count is lower in capillary blood due to adhesion of platelets
to the site of skin puncture.
5. Fragility of red cells obtained from capillary blood is more than that
of venous blood.
6.
White blood cell count is higher in capillary blood than venous blood.
7.
Precision is poorer in capillary than in venous blood because of
variation in flow and dilution with interstitial fluid.
Technique
The
site is first rubbed vigorously with a gauze pad or cotton ball moistened with
70% alcohol or other sterilizing agent to remove dirt and epithelial debris and
to increase blood circulation. After the skin has dried, a puncture 2–3 mm deep
is made with the blade or lancet. A rapid, firm puncture should be made, but
with the control of the depth and site. With a sharp blade, the blade puncture
gives little pain. A deep puncture is no more painful than a superficial one
which makes repeated punctures necessary.
The
skin at the site of puncture should be dry. The first drop of blood, which
contains tissue juices, is wiped away. The second drop is used for examination.
The blood must not be pressed out, since this dilutes it with fluid from the
tissue juices. Moderate pressure some distance above puncture is allowable.
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 has ceased.
Pointers
in doing a skin puncture
1. An edematous or congested part should not be used.
2. Cold and cyanotic skin is not advisable to use – it gives false high
cell count.
3. Disposable lancets or needles are highly recommended.
4. Whichever site is used, make sure it is warm to guarantee dilated skin
vessels and thus ensure a free flow of blood.
5. For earlobe puncture, the ear must be rubbed with lint until warm and
pink. When the heel is used, it must be made warm by immersion in warm water or
by use of hot water compress. Otherwise, values significantly higher than in
venous blood may be obtained, especially in newborn.
6. After sterilizing the site, puncture through a dry area to be able to
obtain a whole rounded drop of blood.
7. First drop of blood is discarded because it contains tissue juices and
other particulate matters lying over the skin.
8. The ideal depth of skin is 2 to 3 mm.
9. Vigorous squeezing after the puncture should be avoided because it
causes the admixture of tissue juices. Moderate pressure some distance above
the puncture is allowable.
10. Free flow of blood is essential
to obtain reproducible results comparable to those with venous blood.
Venous blood
The venipuncture is in
most instances a relatively simple procedure. The patient’s life may depend on
vein potency and care must be taken to preserve these vessels. Hematomas or ecchymosis
are usually evidence of the operator’s poor technique or judgement.
Venous blood is preferred
for most hematological examinations because venipuncture is:
1. Easier and more convenient to obtain and 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 and variety of apparatus to be carried to the
hospital wards.
4. It allows various tests to be repeated.
5. It allows the performance of additional tests that may be requested.
6. It reduces the possibility of error resulting from dilution with tissue
juices or constriction of skin vessels by cold or emotion that may occur in
taking blood by finger puncture.
Sites of venipuncture
1. Antecubital fossa (vein in the arm at the bend of the elbow) – median
cephalic / median basilic vein
2. Veins of the dorsal surface of the hand
3. Veins of the ankle
4. External and internal jugular veins
5. Femoral vein
6. Superior longitudinal sinus
7. Long saphenous vein
Venous
blood is best drawn from an antecubital vein
Equipment
Equipment consists of
gauze pads or cotton balls, sterilizing agent, tourniquet and syringe with a
needle or a vacutainer.
Tourniquet may be:
1. Soft rubber tubing at least 2 – 5 cm in diameter and 18 inches in
length.
2. Blood pressure cuff inflated to
40 to 60 mmHg
3. Band–quet tourniquet – 1 inch wide rubber band
Needles
The gauge and length of
the needle are chosen for the specific task. The gauge number expresses the
diameter of the needle: the smaller the number, the larger the needle. The length
of the needle used depends upon the depth of the vein. The tip should be
inspected carefully. A blunt or bent tip will damage the patient’s vein and
often leads to failure.
Length:
30 – 40
mm
Diameter
or gauge: 0.9 mm (20 gauge)
1.0 mm (19 gauge)
1.1 mm (19 gauge)
1.2 mm (18 gauge)
0.6
mm (23 gauge)
0.5
mm (25 gauge)
1.625
mm (14 gauge)
Usually, for adults 20 or
19 gauge is suitable. For children under the age of five, 23 or 25 gauge needles
can be used.
Syringes
The syringe should be of
the proper size for the amount of blood to be drawn. Disposable plastic syringes
are widely used. With reusable glass syringes, the fit of the plunger and
barrel and the integrity of the syringe should be checked. Instead of syringes,
evacuated blood collection tubes may be employed. Evacuated tubes, e.g.,
Vacutainer sealed with a stopper, are supplied with a measured amount of
anticoagulant (or none) and sufficient vacuum to draw a pre–determined volume
of blood.
Venipuncture with vacuum
containing system is ideal in terms of direct sampling, economy and efficiency.
These systems provide flexibility in terms of specimen volume (2, 3, 5, 7 or 10
ml per tube) and anticoagulant as well as chemically clean or sterile
glassware.
Method to increase the
amount of blood in the arm and to distend the veins:
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 puncture
5. Immersing the arm in warm water or covering with a hot, wet towel for 5
or more minutes prior to venipuncture.
Factors involved in a
good venipuncture:
1. The venipuncturist
The
operator must be aware not inflict damage and preserve the veins for
innumerable future uses. The hematomas displaying all the colors in the site of
puncture testify to the operator’s lack of skill or judgement. As a rule, the
damage is only temporary but it may be long lasting or even permanent.
Therefore, venipuncture must be approached with extreme care and deliberation.
2. The patient and his veins
The
patient should be reassured with kind words. Self–assurance and poise will do
much to establish the proper rapport. The patient should be made comfortable
and the approach to his arm should be convenient for the operator.
The
veins should be checked and evaluated. When veins are deep and not felt
distinctly, use a tourniquet. Use any technique to make the veins more
prominent before you do the actual puncture.
3. Equipment
Syringe
size is determined by the volume of blood required. Disposable plastic syringes
are most widely used. The gauge and length of the needle used depend on the
size and depth of the vein. The gauge number varies inversely with the diameter
of the needle.
Pointers in doing
venipuncture:
1. To prevent hemoconcetration, 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 of blood 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
minimized.
HEMOLYSIS
Hemolysis is the
destruction of the red blood cells. It is a process which should be prevented
to take place during the collection of blood. It affects the results of various
hematological examinations. It can be prevented by observing the following:
1. Use of dry syringe and needle.
2. Use of clean dry glassware and not too thin needle.
3. Draw blood slowly, not faster than the vein is filling.
4. Avoid rough handling of blood.
5. Don’t eject blood from the syringe through the needle. Remove the
needle first.
6. Avoid frothing by ejecting blood gently down the side of the tube.
7. Avoid admixture of air.
8. Mix the blood with anticoagulant by gentle inversion, not by shaking.
9. Don’t freeze blood because the red cells will hemolyze on thawing.
10. Make sure that all solutions with
which blood is to be mixed or diluted are correctly prepared.
11. Stopper and store blood sealed,
if examination is to be delayed beyond 1 to 3 hours, store in a refrigerator.
12. Use of isotonic solutions.
Complications of
venipuncture and suggestions for their prevention:
A. Immediate local complications
1. 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
2. Failure of the blood to enter the syringe – this maybe due to:
a. Excessive pull of plunger
collapses the vein
Suggestion:
Slight back and forth movement reduces the force of aspiration
b. Piercing the outercoat of the
vein without entering the lumen
Suggestion:
Withdraw the needle slightly and reenter the vein
c. Transfixation of veins – piercing
through the walls of the veins
Suggestion:
Withdraw the needle slightly and gently aspirate to see if blood enters. If
This fails, the puncture
may have to be reported.
3. Hematoma / ecchymosis – subcutaneous effusion of blood resulting to
discoloration, pain, swelling and tumor–like mass.
Suggestion:
Repeat puncture on another site
4. Circulatory failure – failure of the blood to flow due to nervousness and other emotional
factors
Suggestion:
This is not beyond the control
of a medical technologist.
Call a physician
5. 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
6. Continued bleeding – this may occur in patients with hemorrhagic tendency.
Suggestion: Local pressure controls
bleeding
B. Late local complications
1. Thrombosis – clotting of blood inside the vessel due to trauma and
repeated infections.
2. Thrombophlebitis – inflammation of the vein in which a thrombus (blood
clot) is present.
Suggestion:
These complications are under
the care of a physician
C. Late general complications
Serum
hepatitis may be caused by transmission of the virus by contaminated needle or
syringe. The use of disposable needles has virtually eliminated the source of
transmitted disease.
**** ANTICOAGULANTS ****
Anticoagulants are
chemical agents that prevent blood from clotting.
If venous blood is to be
used for examination, an anticoagulant is required. The choice of anticoagulant
and the ratio of blood to anticoagulant are of great importance. Dry
anticoagulants are used to avoid dilution which lowers the concentration of the
formed elements in the blood. The anticoagulant selected must not alter the size
of the red cells, nor bring about their hemolysis, it must minimize disruption
of the leucocytes and it must be readily soluble in blood.
Since a measured amount of
dry anticoagulant is generally already present in the vessel used to receive
blood to be obtained by venipuncture, the operator must be certain that the
volume of blood required for the amount of anticoagulant is actually delivered
into the vessel and promptly mixed with the anticoagulant. If too much blood is
added, small clots may form; sometimes these are not easily noted but are
nevertheless, capable of altering the values. If too little blood is added, the
red cells may be crenated, thereby falsely reducing the volume of packed red
cells and the corpuscular indices derived therefrom.
Since the formed elements
of the blood tend to settle upon standing, it is essential that, prior to
sampling for blood counting and other examinations, the venous blood be gently
but thoroughly mixed.
The specimen must also be
checked for visible autoagglutination of red cells and aggregation of
platelets, and for the presence of clots. Insufficient attention paid to these
simple matters may vitiate the significance of blood counts and other
examinations performed by the most costly apparatus or by trained medical
technologist.
A.
Heparin
Heparin
is present in most tissues 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 from hepar, the Greek word for that organ. In the body, it exists, in
the highest concentration in the liver and the lungs. It is a mucoitin
polysulfuric acid and is available as the sodium, potassium and ammonium salts.
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.
Formula
1
mg of dry heparin or 0.1 ml of liquid heparin per 10 ml blood
0.1 – 0.2 mg of dry heparin per ml of
blood
Preparation of saturated heparin solution
Powdered
heparin – 1.0 gram
Distilled
water – 100 cc
Evaporate
to dryness at 37oC – 56oC
Advantages
a. Excellent and natural anticoagulant usually prepared from lungs of
animals.
b. 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.
c. Best anticoagulant for prevention of hemolysis and is therefore
recommended for osmotic fragility tests.
d. Less toxic than sodium citrate and therefore is preferred as an
anticoagulant for open heart surgery and exchange transfusions in which large
quantities of blood are given rapidly.
e. Used for electrolyte determination, erythrocyte sedimentation rate and
test for blood pigments.
f. Especially used for erythroblastosis fetalis
Disadvantages
a. Expensive
b. Not recommended for study of morphology of the cells; it gives a blue
background on a blood smear stained with a Romanowsky dye, e.g. Wright’s stain.
c. Temporary action – the anticoagulant effect in vivo is of short
duration but if used in adequate concentration in vitro will prevent
coagulation of refrigerated blood for 3 or more days.
d. Not satisfactory for serological tests.
e. Not to be used for white cell and platelet counts as it tends to cause
the white cells and platelets to clump.
B.
Sequestrene
This
is also known as ethylenediaminetetraacetic acid (EDTA) or versene
Action of EDTA as anticoagulant
It
acts as a chelating agent which combines with calcium and binds it in a
non–ionized form. The disodium or dipotassium salt of EDTA is an effective
anticoagulant in the proportion of 1 to 2 mg / ml of blood or 0.1 ml of 10%
solution in water per 5 ml of blood.
Preparation of 10% sequestrene solution
Sequestrene
(dry powder) – 10 grams
Distilled
water – 100 ml
Advantages
a. Preserves cellular elements of the blood and doesn’t cause significant
changes in the size and shape of RBC and WBC.
b. Satisfactory for erythrocyte sedimentation rate, hematocrit, cell
counting, electrophoresis, fetal hemoglobin and blood grouping procedures.
c. Prevents agglutination of platelets and is preferred for thrombocyte
studies. Platelet counting is still possible after several hours since it tends
to prevent surface adhesion and clumping of platelets.
d. Recommended for blood smears because it prevents formation of artifacts
even on prolonged standing.
e. May also be used in blood transfusion as it has a few toxic effects.
Disadvantages
It is not suitable for use in the investigation of
coagulation disorders especially in the
estimation Prothrombin time.
C.
Oxalates
Action of oxalates as anticoagulant
Oxalates
such as sodium, potassium, ammonium or lithium inhibit blood coagulation by
forming rather insoluble complexes with calcium ions which are necessary for
coagulation.
1. Dried ammonium–potassium oxalate
Other
names: Double oxalate Wintrobe’s fluid
Balanced oxalate Heller – Paul fluid
Preparation of the anticoagulant
3
parts of ammonium oxalate – 1.2 grams
2
parts of potassium oxalate – 0.3 grams
Distilled
water – 100 cc
Advantages
a. The balanced 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.
b. May be used for erythrocyte
sedimentation rate, cell counts, hemoglobin, hematocrit determination and mean
corpuscular values.
c. Cheap, easy to prepare and
requires no dilution.
Disadvantages
a. Cannot be used for chemistry examination especially in the
determination of urea, nitrogen or potassium
b. Cannot be used for blood transfusion
c. Causes distortion of cells and clumping of platelets
d. Not recommended for morphological studies because it causes clover
leafing of leucocyte nuclei, crenation of red cells, vacuolation in the
cytoplasmic of granulocytes, phagocytosis of oxalate crystals, artifact
formation in the nuclei of lymphocytes and monocytes and other malformations
develop rapidly.
2. Potassium oxalate
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 carbonate which
has no anticoagulant property.
D. Citrates
Action as anticoagulant
Citrate
forms a double salt with calcium and binds it in a non–ionized form.
Formula
1
part of 3.8% disodium citrate and 4 parts of blood is the usual proportion for
routine use especially for erythrocyte sedimentation rate determination
(Westergren method).
1
part of 3.8% or 3.2% aqueous solution of di– or tri–sodium citrate and 9 parts
of blood is the proportion for investigation of the clotting mechanism.
Buffered
citrate (sodium citrate and citric acid) – stabilizes plasma pH
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. Trisodium citrate is used for
blood coagulation and platelet function studies.
3. Buffered citrate is now commonly
used because it helps stabilize plasma pH.
For
better long term preservation of red cell for enzyme studies and for the study
of hemolytic disorders and for transfusion purposes, citrate is used in
combination with dextrose in the form of Acid Citrate Dextrose (ACD), Citrate
Phosphate Dextrose (CPD) or Alsever’s solution. Dextrose prolongs the survival
of red cells.
Standard preparation of Acid Citrate Dextrose (ACD)
Trisodium citrate – 1.32 grams
Citric acid – 0.48 grams
Dextrose – 1.4 grams
Distilled water –
100 ml
1 ml ACD is sufficient to prevent
coagulation of 4 ml blood
Preparation of Citrate Phosphate Dextrose (CPD)
Trisodium citrate – 30
grams
Sodium dihydrogen phosphate,
monohydrate – 0.15 grams
Dextrose – 2
grams
Distilled water q.s.ad – 1
liter
Preparation of Alsever’s solution
Glucose
– 20.5
grams
Trisodium
citrate, dihydrate – 8
grams
NaCl
– 4.2
grams
Water,
distilled q.s.ad. – 1 liter
4
parts blood are mixed with 1 part of Alsever’s solution
E. Fluoride
Action 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 anticoagulant, the concentration must be much
greater, 6 – 10 mg/ml blood. As a general rule, fluoride should not be used
when collecting blood specimens for enzyme determination or when using an
enzyme as a reagent in a test, e.g. urease method for the determination of urea.
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.
10
mg sodium fluoride per ml blood plus 1 thymol
**** BLOOD PREPARATION COMMONLY EMPLOYED IN HEMATOLOGY
****
Before blood is examined,
it usually undergoes some form of preparation. The kind of blood preparation
depends on the examination to be done. However, there are some examinations
which require no such preparation but instead; blood is examined as it flows
freely from the puncture as in bleeding time determination.
A. Diluted Blood
For
cell counts, e.g. white cell count, red blood count, platelet count (direct
method) and eosinophil count, the dilution is made by sucking a measured
portion of blood and diluting fluid into a pipette. For hemoglobin
determination and other special examinations, the dilution is done in a
standard test tube.
Keeping
time: Diluted blood is not very
stable. For the platelet count, it will only keep for about 30 minutes. For
other tests, the diluted blood will keep for about 2 hours.
B. Blood smear
Blood
smear may be prepared with the use of two glass slides or two cover glasses or
with a slide and a cover glass.
Blood
smear is used in the following tests:
1. Differential leukocyte count
2. Morphologic study of normal and
abnormal white cells, red cells and platelets
3. Reticulocyte count
4. Platelet count (indirect method)
5. Lupus erythematosus cell (L.E.)
examination
6. Bone marrow examination – bone
marrow film
Keeping
time: The blood smear should be
stained within 2 hours after it is made. Once stained, it may be kept for about
2 years.
C. Whole blood or Non–clotted blood
If
whole blood is needed in the examination, blood should be prevented from
clotting by adding an anticoagulant. Depending on the anticoagulant used, blood
may be oxalated, sequestrenized, heparinized or citrated.
Non–clotted
blood is used in those tests which require blood plasma and therefore may also
be used in preparing dilute blood.
Keeping
time:
The
keeping time of non–clotted blood depends upon the examination and the
anticoagulant used; it may be kept for 1 hour to even 24 to 48 hours provided
precautionary measures like refrigeration and prevention of hemolysis are
observed.
D. Blood plasma
When
clotted blood is centrifuged, the liquid portion which separates is called
plasma. It is prepared with a special type of anticoagulant and is used in the
following tests:
1.
Prothrombin Time
2.
Partial Thromboplastin Time
3.
Fibrinogen Deficiency Test
4.
Thromboplastin Generation Time
5.
Other tests for coagulation disorders
Keeping
time: None, because it should be prepared directly after the blood is withdrawn
and should be used immediately.
E. Blood serum
When
blood is placed in a test tube and allowed to clot, after sometime, the clot
retract an expresses a fluid called serum. This serum is pipetted off and used
in the following tests:
1. Prothrombin Time
2. Partial Thromboplastin Time
3. Thromboplastin Generation Time
4. Other tests for coagulation
disorders
Keeping
time: It should be kept within 2
hours after prepping; otherwise, cells may lose their clumping or agglutinating
power.
F. Cell suspension
When
an isotonic solution like normal saline solution (NSS) is placed in a test tube
a few drops of blood are added, the preparation is called cell suspension. This
may also be prepared by allowing the cells to suspend in their own serum. This
kind of blood preparation however is seldom used in Hematology.
Keeping
time: It should be kept within 2
hours after preparing, otherwise, cells may lose their clumping or
agglutinating power.
G. Defibrinated blood
Defibrination
(removal of fibrin from the blood) is done by the:
1. Use of applicator sticks in which 3 to 4 paper clips are attached to
one end and is twirled in the blood sample until all fibrin thread are attached
to the clips.
2. Use of glass beads – about 25 ml blood in an Erlenmayer flask
containing 20 (3 – 4) glass beads. Flasks are rotated until the beads are
covered with fibrin and cease to make a rattling sound.
3. Use of 0.5 cm length of glass rod which is held in place by thrusting
into the bore of a stopper or cotton. Blood is placed into the flask and the
rod is fused inside; this is then rotated in a figure of 8 motions for 5 – 10
minutes until all fibrin threads adhere to the rod.
After
removing the fibrin strands by means of any of the methods previously
described, the resulting blood preparation is called defibrinated blood which
is used for morphologic study of white cells, red cells and for lupus
erythematosus cell (L.E. examination)
H. Blood collected in siliconized glassware
The
glassware is first coated with a suitable water dilution of a water soluble silicon
concentrate by immersion in the solution for 5 seconds or longer. Then the
glassware are rinsed well with water and then dried at room temperature for 24
hours or in hot air over at 100oC for 10 minutes.
This
blood collected in silicon coated glassware is used in various platelet
examinations. It should be immediately used.
I. Buffy coat preparation
A
number of methods are available for the concentration of leukocytes, platelets
and nucleated cells which collect above the column of packed red cells. This
buffy layer is sometimes called the “white cell concentrate.”
Venous
blood, anticoagulated or defibrinated is centrifuged for 15 minutes in a
Wintrobe hematocrit tube at 3000 rpm. The supernatant liquid is thrown off and
the buffy layer is pipetted and ejected on one or two clean slides. After
emulsifying the buffy coat in a drop of the patient’s serum or plasma, films
are spread, allowed to dry in air, fixed and stained in the usual way.
When
leucocytes are scanty or if many slides are to be made, it is worthwhile
centrifuging the blood twice; first, centrifuge 5 ml blood and then fill a
hematocrit tube with upper cell layers of the previously centrifuged sample and
recentrifuge.
As
an alternative to centrifugation, the blood may be allowed to settle, with the
help of sedimentation–enhancing agents such as fibrinogen, dextran, gum acacia,
Ficoll or methyl cellulose.
Uses of buffy coat
1. In the diagnosis of various cases of leukopenia.
2. In the diagnosis of various cases of leukemia.
3. In searching for blasts and other immature cells, megakaryocytes,
nucleated red cells, phagocyte and atypical cells.
4. In the examination of lupus erythematosus cells.
5. In searching for megaloblasts in pernicious anemia.
6. In searching for nucleated red cells and immature granulocytes in
metastatic malignancy.
7. In determining the presence of erythrophagocytosis in hemolytic anemia
and chronic granulomatous infection.
2 comments:
thank you for doing this
Very nice post
Post a Comment