excessive heat. Allow to dry in air for at least 8–12 hours
or for 2 hours in an incubator at 37°C. Keep free from dust
or contamination with excreta of flies or roaches. Stain as
soon as practicable, freshly stained material gives distinctly
Fix thin filmed end of slide in methanol for 2–3 minutes. The
thick films must not be fixed. Immerse slides for 30 minutes
in a mixture of 1 drop of concentrated Giemsa’s stain to
each mL of phosphate-buffered distilled water (pH 7.2).
Wash off with buffered water and dry in air. Examine with
oil immersion objective. Hematoxylin or methylene blue
sheaths (if present) stain more distinctly.
Concentration of Microfilariae
Hemolyze 10 mL or more of citrated blood with 50 mL of a
2% solution of a saponin in physiologic saline. Centrifuge
and examine the sediment. Chylous urine and scrotal
aspirates should always be centrifuged to attempt recovery
For a proper diagnosis of kala-azar, bone marrow smear
obtained by sternal puncture is essential. The Leishman’s
stain or Giemsa’s stain are used for this purpose.
Microscopic examination under oil immersion shows
Serum samples obtained from the patients are used for
following tests in certain parasitic infections:
1. Precipitation: It is done for trichinelliasis, hydatid
2. Flocculation test: Slide flocculation tests are useful for
trichinelliasis and schistosomiasis.
3. Complement fixation test: This test is used in several
parasitic infections for detecting complement fixing
antibodies, but this is of special value in cases of
schistosomiasis, trichinelliasis, hydatid disease,
nonpulmonary paragonimiasis, and toxoplasmosis.
4. Fluorescent antibody test (FAT): This test is applied to
demonstrate the presence of parasites or their specific
antibodies in tissue or in the blood of the patient, e.g.
Toxoplasma gondii and Entamoeba histolytica.
5. ELISA tests now are available for most parasitic
6. Immunochromatography technique based kits.
These are done to demonstrate allergic state in certain
parasitic diseases. The antigen of the suspected parasite
is injected intradermally and in positive cases, wheal
or induration results. These tests are useful for hydatid
disease (Casoni’s test), trichinelliasis, filariasis, chronic
schistosomiasis and toxoplasmosis.
It was first developed by Casoni in 1911. Intradermal
injection of 0.2 mL on flexor aspect of right arm of a fresh
sterile hydatid fluid (sterilized by Seitz filter) produces
within half an hour in all positive cases a large wheal (5
cm in diameter) with multiple pseudopodia, it fades in an
site of injection. A negative reaction does not exclude
echinococcal infection. The test usually becomes positive
8–12 weeks after infection and remains positive after
surgical removal of cyst from the patient.
Hydatid fluid from human cases (removed operatively)
or from animals (obtained from a slaughter house) is used
as an antigen. While 0.2 mL of antigen is injected to one
arm, sterile normal saline 0.2 mL is injected in the other arm
Muscle Biopsy for Trichinella spiralis
Small pieces of deltoid, biceps, or gastrocnemius
muscle are removed from the vicinity of their tendinous
attachment under local anesthesia.
1. Microscopically examine small pieces of muscle
compressed between 2 glass slides for encysting or
2. Digest muscle in artificial gastric juice (pepsin and
hydrochloric acid) and examine sediment for motile
The blood consists of a fluid of complicated and variable
cells—WBCs) and platelets. By using an anticoagulant, the
formed elements can be separated from plasma. When
blood coagulates, the fluid that remains after separation
of the clot is called serum. Serum = Plasma-fibrinogen.
The techniques of hematology are concerned mainly with
the cellular formed elements of blood, their number or
concentration, the relative distribution of various types of
cells and the structural or biochemical abnormalities that
For hematologic exercises, venous blood obtained from
a vein is better. However, for total and differential blood
counts and for hemoglobin estimation, blood can be taken
(i) The lobe of the ear, (ii) the palmar surfaces of the tip
of the finger, (iii) in infants, from the plantar surface of the
(a) heel or (b) the great toe. The puncture should be about
3 mm deep. An edematous or a congested part should not
be used. If the area to be punctured is cold and cyanotic,
warm it by massaging or else erroneous results may be
obtained. Clean the site with spirit or alcohol, let dry and
puncture. Wipe off the first drop of blood, never press out
blood. Having obtained the requisite amount of blood, let
the patient apply slight pressure over the area with sterile
Reassure the patient about what is to be done. Inspect the
veins, use a tourniquet if needed (Fig. 9.2). Use a syringe
of a size according to the amount of blood required.
Needles of gauge less than 22 should be used and be 1
to 1½ inches long. Instead of a tourniquet, one can use a
sphygmomanometer cuff, apply pressure that is midway
between systolic and diastolic pressure. Ask the patients
FIG. 9.1: Sites for obtaining-blood skin puncture
206 Concise Book of Medical Laboratory Technology: Methods and Interpretations
to open and close his fist several times. Take aseptic
precautions, and puncture the vein. Sometimes, it may
be difficult to obtain blood at the first instance, in such
a case, first the skin around the vein is punctured and
then the vein is punctured, the moment needle enters
the vein, blood flows back into the syringe. If still blood
has not been obtained, withdraw the needle and in many
instances blood would be obtained. Having withdrawn
blood, loosen the tourniquet and ask the patient to open
his fist. Let the patient apply a sterile gauze piece with
gentle pressure over the area. The patient should apply
pressure gently with three fingers. Why three fingers?
Simply because the site of skin puncture is not the same as
the site of venipuncture. Make sure that the bleeding has
stopped before the patient leaves. In infants, blood may be
secured from femoral or external jugular vein. Wash the
syringe after dispensing the blood in the right container.
Transfer blood from syringe into the container gently (not
through the needle). Ideally use disposable syringes and
Hematoma and syncope. Hematomas can be avoided
by applying adequate gentle pressure at the site and for
syncope a physician should be brought to take care. In any
case, let the patient lie down if he or she is already not and
raise the foot end. Some patients with a bleeding tendency
Thrombosis of the vein. Rarely thrombophlebitis may
occur. Apply Thrombophob if it happens. Seek professional medical help.
Transmission of serum hepatitis, AIDS by using contaminated needle and syringe.
1. If oozing from the puncture site is difficult to stop,
elevate area and apply a pressure dressing. Stay with
the patient until bleeding stops.
2. Never draw blood for any laboratory test from the
same extremity that is being used for IV fluids, IV
medications or blood transfusion.
3. In patients with leukemia or agranulocytosis and in
others with lowered resistance, the finger prick and
ear lobe puncture are more likely to cause infection
than venipuncture. If capillary sample is necessary
in these patients, the cleansing agent should remain
in contact with the skin for 7 to 10 minutes. Alcohol is
not bactericidal, povidone-iodine (Betadine) is the
cleansing agent of choice on leukemic patients.
On letting blood stand, it clots after some time.
Anticoagulants are agents that prevent clotting when mixed
with blood in an appropriate proportion. The common
pathway of the clotting mechanism is represented as
(insoluble) Thrombin + Ca++ (soluble)
Fibrin with blood cells with entrapped constituents
The thromboplastin released by damaged tissue, or
soluble fibrinogen into insoluble fibrin clot in the
presence of calcium ions. Anticoagulants commonly used
for hematological investigations are as follows:
Ethylenediamine tetra-acetic acid (EDTA), disodium
or potassium salt. EDTA acts by chelating calcium and
preserves cellular element better than oxalates. Eight (8)
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