LABORATORY STANDARD OPERATING PROCEDURES ON THE FOLLOWING

1. SOURCES OF SPECIMEN
Several kinds of specimens are analyzed routinely in the clinical laboratory. The specimen most
often tested in blood. There are two general sources of blood for clinical laboratory tests
a. Peripheral or capillary blood
b. Venous blood
For small quantities of blood, for hematologic or micro-chemical determinations, capillary blood
is suitable. This is obtained from the capillary bed by puncture of the skin. The tip of the finger is
the site most commonly punctured.
For larger quantities of blood, a puncture is made directly into a vein (phlebotomy) using a sterile
syringe and needle collection system or vacuum tube and needle collection system, a vein in the
upper forearm or ante cubital fossa area is most often chosen for venipuncture as these veins are
easily palpable and fairly well fixed.
The specimen most often tested is urine and feces. Some cavity fluids are also being tested.
2. COLLECTION, RECEIVING, HANDLING AND STABILITY OF SPECIMEN
Collection Procedure:
A. Peripheral or Capillary Blood
For small quantities of blood required for most hematologic procedures and for micro-chemical
techniques requiring serum or plasma, an adequate blood sample may be obtained from the
capillary bed by puncture of the skin. From certain patients, such as babies, burned patients or
amputees, it may be necessary to draw only a very few amounts of blood. This blood is collected
into suitable capillary tubes or pipettes or used directly to prepare blood films.
Capillary blood is often used for bedside testing for glucose using one of several available meter
devices and accompanying reagent strips. The same procedure is done by countless diabetics on
their own blood to ascertain the level of their blood glucose.
● In adult and older children, the tip of the finger is punctured.
● In infants, the plantar surface of the heel or large toe is punctured.
In general, the ear lobe should be avoided for puncture because there is a slow flow of blood and
the concentration of cells and hemoglobin will be greater. Blood obtained by puncturing the ear
lobe has been found to contain a higher concentration of hemoglobin than the finger or venous
blood and also is not reliable for white blood contents. The earlobe is sometimes the desirable
source of blood for the preparation of blood films used to study leukocyte abnormalities since
larger cells are frequently trapped in the capillary bed because of the slow circulation. Blood
obtained by skin puncture of these types is generally called capillary blood but it is closer to
arteriolar blood in its composition. The results of tests for venous and capillary blood compare
well if the capillary blood is free flowing. To ensure the free flow of the capillary blood, the
finger must be warm.
Various types of disposable lancets or blades are used for skin puncture. Use of non- disposable
blades is not recommended because of the risk of infectious pathogens.
Precautions to note when obtaining capillary blood:
1. If the patient’s fingers are cold, slight rubbing may help to warm them.
2. The finger or heel must not be squeezed excessively, because tissue fluids may dilute the
blood sample or will cause the blood to clot faster than it normally would.
3. The first drop of blood is usually removed because it contains tissue fluid. Alcohol or
perspiration which will dilute the blood.
4. The tip of the blade should not touch anything until it punctures the skin of the patients.
Contaminated blades are discarded properly and new ones used. After the puncture is made,
the blade is discarded immediately.
5. Clean hands are essential when working with patients.
Skin Puncture Devices:
● A variety of automatic, spring-activated puncturing devices are commercially available.
Clean rapid incisions can be made of a consistent depth with the use of one of these
devices.
● A device such as a lancet held in place by a cooking lever. When released, the blade
penetrates the skin to a depth of 2-3 mm depending on the choice of platform used. The
platform regulates the depth of the puncture. Disposable blades and platforms or guards
are used with these devices. The device itself can be cleaned with a bleach or disinfectant
solution
● It is important that all blades and platforms be discarded in a container for sharps.
Transmission of blood borne infections must be prevented.
Procedure for Heel Puncture:
1. Perform the puncture on the most medial lateral portion of the plantar surface
2. Puncture no deeper than 2.4 mm
3. Do not perform punctures on the posterior curvature of the heel
4. Do not puncture through previous sites which may be infected
Notes:
● For infants under 3 months of age, the heel is the most commonly used site for obtaining
a blood sample. Any wound infliction device can result in serious injury when excessive
pressure and skin indentation accidentally allow the wound depth to reach the heel bones.
The foot must be held securely in place for the wound to puncture.
● The central portion and posterior curvature of the heel are not used because the bone lies
too close to the surface.
Procedure for Finger Puncture:
1. Assemble the necessary equipment; lancet device, alcohol pad, dry gauze, slides and capillary
tubes or other supplies necessary to receive the blood.
2. Be sure that the patient is seated comfortably.
3. Put on gloves
4. Choose an area for the puncture that is free from calluses, edema or cyanosis. Warm the
puncture site if it is coldly immersing it in warm water or by rubbing it.
5. Clean the skin of the puncture site on the third or fourth finger vigorously with a pad soaked
in alcohol solution; this will remove dirt and epithelial debris, increase the circulation and
leave the area sterile. Allow the area to air dry.
 6. Grasp the finger firmly and make a quick firm puncture about 2 to 3 mm deep with a sterile
disposable lancet or automated lancet device. This puncture should be made at right angles to
the fingerprint striations on the patient’s finger midway between the edge and the midpoint of
the fingertip. The puncture should not be made too far down on the finger and should not be
too close to the fingernail. A deep puncture hurts no more than a superficial one and it gives a
much more satisfactory flow of blood.
7. Discard the lancets in the “Sharp Disposal Container” (SDC) used lancets should never be
left lying in the work area. They should be discarded immediately after use and should not be
touched again.
8. Wipe away the first drop of blood, using a clean piece of dry gauze or tissue. This drop is
contaminated with tissue fluid and will interfere with some laboratory results if used. The
succeeding drops are used for tests.
9. If a good puncture has been made, the blood will flow freely. If it does not use gentle
pressure to make the blood form a round drop. Excessive squeezing will cause dilution of the
blood with tissue fluid.
10. Collect the specimens by holding a capillary tube to the blood drop or by touching the drop to
a clean glass slide. Rapid collection is necessary to prevent coagulation, especially when
several tests are to be done using blood from the same puncture site.
11. When the blood samples have been collected, have the patient hold a sterile dry piece of
gauze or cotton over the puncture site until bleeding has been stopped.
12. Remove gloves and wash hands.
Capillary Blood Specimen Containers:
● Once the skin has been punctured and blood is flowing, the capillary sample can be
collected in a variety of containers.
● For general, purposes, glass tubes with or without heparin can be used and the blood
allowed to flow into the tube by capillary action e.g., a heparinized capillary tube is used
to collect blood for doing the micro- hematocrit test in the hematology laboratory.
● Another capillary sampling device is called Unopette. It consists of a disposable capillary
pipette which measures a fixed volume and a reservoir of diluents appropriate to the test
being performed. This pipette and diluents system can be used with either capillary or
venous blood and the general system has been adapted for several chemical and
hematologic determinations.
● Another micro- container consists of a small plastic tube with a blood drop collector as
part of the lid. One such commercially available system is the micro- container. This
allows the specimen to be collected by the capillary action and results in a relatively large
amount of specimen in a single container. When the collection is finished, the lid with the
capillary tube is removed and discarded. The remaining container can be capped and
processed by centrifugation if serum is to be obtained. The micro- container is available
with various additives including serum separator gels.
B. Venous Blood
● The veins that are generally used for venipuncture are those in the forearm, wrist or
ankle. The first choice for a venipuncture site is a vein in the forearm. They are larger and
fuller than those in the wrist, hand or ankle region. These veins are used only if the
forearm site is not available.
● Venipuncture must be performed with great care and technical skills. The veins of the
patient are the main source of blood for testing and the entry point of the medications,
 intravenous solutions and blood transfusions. A patient has only a limited number of
accessible veins and it is important that everything possible be done to preserve their
good condition and availability. Part of this responsibility lies with the person doing the
blood drawing.
● Blood may be obtained directly from the vein (Phlebotomy) by using a sterile syringe and
needle or a vacuum tube and needle system. The use of the evacuated tube system allows
the blood to pass directly from the vein into the collection tube. Veins in the forearm are
most commonly used.
● The phlebotomy may be made by either the syringe method or the vacuum tube method.
In the syringe method, a needle is attached to a syringe and inserted into the vein. The
other end of the two way needle is inserted into the vein. Once it is in the vein, the needle
and the rubber stopper is pushed through the stopper to make a direct connection to the
vacuum tube. The vacuum tube creates suction, which draws the blood into the tube e.g.,
one commercial vacuum system is called vacutainer.
● When doing a venipuncture, the phlebotomy should remain in a standing position, which
gives the greatest freedom of movement. The patient should assume a comfortable
position. Bed patients should remain lying down and the ambulatory patients should be
seated comfortably. The seated patient should put an arm on the table or other firm
support and extend it for the phlebotomist.
BLOOD COLLECTION VARIABLES:
● The majority of clinical laboratory determinations are done on whole blood plasma or serum.
Many of those are done in the hematology or chemistry laboratories, but many other areas of the
laboratory require venous blood for testing.
● Most venous blood specimens are drawn from fasting patients. Most fasting blood is drawn in the
morning before breakfast. This means that the food from the previous meals has been completely
digested and absorbed and any excess has been stored.
● Food intake medication, activity and any time of the day can all influence the laboratory results
for blood specimens. Fasting state is one fact that is carefully noted especially for glucose and
phosphorus determinations; however an average meal has no significant effect on the
concentrations of most blood constituents, with certain exceptions such as tests for glucose,
phosphorus and triglycerides. Eating significantly affects blood glucose and triglycerides giving a
falsely high result and phosphorous giving a falsely low result.
● Because it is the most efficient time of the day to draw specimens for the laboratory, most blood
collection is done early in the morning and for this reason, most of the patients are in the fasting
state, fasting specimens however are not necessary for many laboratories' determination.
● High blood should not be collected while intravenous solutions are being administered if
possible.
● Other controllable biological variations in the blood include posture, whether the patient is lying
in bed or standing up, immobilization, resulting from prolonged best rest, exercise, circadian
variations, cyclical variations throughout the day, recent food indigestion e.g., caffeine effect,
smoking, nicotine effect, alcohol ingestion and administration of drugs. The concentration of
certain plasma constituents is affected by some of these factors and for laboratory tests, it is
important to take into consideration.
● Standardization of collection policies can minimize the effect of these variables on test values.
APPLICATION OF THE TOURNIQUET:
 ● The use of the tourniquet is desirable to enlarge the veins, so that they can be more prominent.
● A strip of flat tubing about 1 inch wide can serves as a tourniquet.
● It is applied around the arm just about the bend in the elbow and should be just tight enough to
stop the blood flow.
● The patient should also be instructed to clench the fist, to aid in building up the blood pressure in
the area of the puncture.
The proper way to apply a tourniquet is as follows:
1. Place the tourniquet under the patient’s arms just above the bend in the elbow.
2. Grasping the end of the tourniquet, pull up so the tension is applied to the tourniquet. This tension
should be maintained throughout the procedure.
3. With the proper tension, tuck a loop in the tourniquet from the top down. Do not tie a bow or a
knot. The loop must be made in such a way that it can easily be released when the tourniquet is to
be removed.
4. Do not leave the tourniquet on for a long period of time because this will cause a stoppage of the
circulation (stasis) prolonged stasis results in gross alterations in the blood constituents. Stasis
should be allowed for a minimum time only.

Noted by:

DR. SHELDON STEVEN C. AQUINO

Pathologist/ Head of Laboratory

GUIDELINES IN THE OPERATION AND MAINTENANCE OF THE LABORATORY

Resources and Facilities:

● Laboratory space, equipment, facility and supplies must be sufficient to ensure safe, accurate and
acceptable standards of performance.
● Temperature-dependent equipment such as refrigerators, and freezers ( standard, ultraflow or
liquid nitrogen) must be maintained at temperatures optimal for the storage or handling of each
type of regent or sample. They must be monitored and documented at appropriate intervals as
 determined at the direction of the laboratory director. Similarly, equipment requiring modified
atmospheres must be monitored and documented.
● The laboratory must be in compliance with all relevant safety codes to ensure the safe handling of
chemicals, radiation, recombinant, blood samples or other human tissues/ fluids and to ensure
their proper disposal as stipulated by the Department of Health & Department of Environment &
Natural Resources.
● All laboratories are required to participate in mandated laboratory inspection as required by the
Department of Health, Bureau of Health Facilities Services.
● All laboratory equipment should be maintained at appropriate intervals. The records of such
maintenance and repair must be kept as long as the equipment is in use.
● Adequate facilities for record storage must be available to the laboratory.
● A laboratory may engage the services of another laboratory to provide service for test systems not
available to the primary laboratory or to divert excess volume. In this event, the subcontracting
laboratory also must meet all applicable guidelines and standards stated in this document. The
identity of the subcontracting laboratory and that portion of the study for which it is responsible
must be noted clearly on the report.

Specimens and Intake Information:

● Specimen containers arriving in the laboratory must include two identifiers, which may be the
patient’s name, date of birth, hospital number, laboratory number or other unique identifiers. The
date of specimen collection and when appropriate, the time of collection should be included.
● When appropriate, specimen collection methods must not compromise aseptic technique.
● Specimen transport and handling must be in accordance with the Department of Health guidelines
with the express understanding that any human tissues and fluids may harbor infectious agents.
● Intake information to accompany the specimen must include sufficient clinical information to
ensure appropriate and accurate testing and interpretation of results (send-out).
● When appropriate, intake records should include the dates the specimen is obtained and received
in the laboratory and the quantity and qualitative condition of the specimen. All specimens for
cell culture should be received within 1 day of being obtained if at all possible (send- out.)
● Informed consent should be obtained as required by law and professional standards. The
laboratory should be available to assist in determining the appropriate level of informed consent,
which can be obtained from established guidelines, especially drug testing specimens.
● Specimen handling and processing methods should preclude contamination, tampering and
substitution.
● The laboratory should retain the original patient sample until all testing is completed and the
report has been signed out. The retention of a patient’s DNA should be in compliance with the
retention standards set by law.
● Re- use of patient DNA specimens. Subsequent use and retention is as allowed by the patient.

Records:
● All patient test records of the patient’s laboratory testing must be accessible to the Head of the
Laboratory/ Pathologist.
● Files should be retrievable by both patient name and a second unique identifier (e.g., laboratory
accession number), soft and hard copy.
● Records must be maintained in a manner that will ensure privacy, security, integrity and access,
as required by law and professional standards.
● Laboratory records should be released only with the appropriate authorization for release.’
● Laboratory records that are reviewed as part of inspection or regulation should be treated in such
a way as to maintain patient confidentiality.
● The various components of records of each case should be maintained for time periods as shown
in the specialty sections or as required by specified in the Department of Health Specimen
 Retention Guide. In general, critical records of genetic testing are kept for 1 generation (20
years).
● The Laboratory Computer System must be validated to ensure proper functioning in all aspects of
the laboratory, including a security system that safeguards patient confidentiality issues, have
sufficient back- up and verification to allow uninterrupted functioning of the laboratory and
prevention of loss of data. Both hardware and software must be properly maintained and updated
as needed to maintain the functioning of the laboratory. The laboratory Administrative Officer is
responsible for the selection or development of the appropriate computer system for the
laboratory and must annually review the performance. The laboratory must maintain
documentation of all upgrades.

POLICY ON SECURITY OF SUPPLIES, SPECIMENS AND CONFIDENTIALITY OF

RECORDS
 
Laboratories contain valuable equipment, samples, work in progress, notes and data. They also
contain potentially hazardous materials, such as chemicals, biological agents, and radioactive
substances. All of these assets must be protected from unauthorized access or removal, theft, or
mishandling.

The laboratory staff has primary responsibility for protecting these assets. However, all
Laboratory Personnel who access the laboratory, must take precautions to protect
against unauthorized access or removal, theft, or mishandling of items found in the
laboratory. Security measures should correspond to the potential risks in the laboratory.

In order to safeguard the laboratory and the data/materials contained therein, all Laboratory
Personnel are, at a minimum, to comply with the security requirements.

Security Requirements:
 1. Limit access to the laboratory.  Keep laboratory doors closed and locked unless a
laboratory personnel is actually present.
2. Question the presence of unfamiliar/suspicious individuals in laboratories and/or building
common areas. Report all such persons and/or suspicious activity immediately.
3. Normally, laboratory building exterior doors are secured after designated working hours.
To minimize the likelihood of unauthorized access, all after-hours building users must:

a. Avoid providing building access to unauthorized persons.

b. Secure doors behind themselves
c. Report any building security issues immediately.
d. Do not give laboratory keys to non-laboratory personnel.

Requirements for Security Documents, Data, Equipment and Materials:

1. Secure important research documents, equipment and experimental materials (e.g., lab
notebooks, samples, hazardous substances) in locked areas.
2. Secure devices capable of storing sensitive information or data (such as computer disks,
magnetic tape, flash drives, smartphones, or tablets) in locked areas.
3. The Laboratory personnel must maintain a detailed, inventory of all chemical, biological,
radioactive or other regulated substances kept in the laboratory and have it readily
available for review.
4. Report potentially missing materials, documents, samples, etc., immediately upon
discovery.  
 
HEMATOLOGY SECTION

This section offers examinations ranging from cell counts, cell indices and determination of bleeding
parameters for the diagnosis of hematologic disorders, utilizing both automated and manual methods.
● CBC
● Cell counts (WBC count, RBC count, platelet count, reticulocyte count, differential count),
clotting parameters (clotting time, bleeding time, prothrombin time, partial thromboplastin
time, fibrin degradation determination, clot retraction time.

Pre- analytical Procedure:

1. Request form from the attending physician must be presented by the patient with the requested
laboratory examination.
2. Confirm patients’ status if he/ she is ready or qualified to undergo blood extraction.
3. Prepare the materials to be used, slide, capillets, EDTA tubes tunes (for send- out)
4. Prepare the materials needed for extraction; make sure the syringe needle is securely fitted to the
nozzle. Ready the tubes needed within reach.
5. Place the patient in a comfortable position in which he/she can hold the selected arm comfortably,
apply the tourniquet to locate the vein.
6. Prepare the arm by swabbing the area with 70% alcohol and make sure the skin is dry before
inserting the needle.
 7. For skin puncture, swab the area with 70% alcohol and make sure the skin is dry before pricking
and wipe off the first drop of blood.
8. As soon as sufficient blood is extracted, remove the tourniquet gently, withdraw the needle and
apply sterile cotton to the puncture site, instruct the patient to put pressure on the puncture site for
at least 5 minutes.
9. For skin puncture, collect blood using 3-4 capillary tubes and make a peripheral smear, wipe off
the blood and apply sterile cotton on the puncture area.
10. Make sure the patient is in good condition before leaving the laboratory.
11. Prepare the necessary reagents and equipment for the test.
12. Label the specimen properly.
13. Write the results in its proper form with the patient’s complete information.
14. Results must be available after 1-2 hours. Must be given only to the patient or the requesting
physician.
15. Record all results with the patient’s complete information in its designated logbook
16. Make sure that the patient is properly informed that the test done is for screening only and a
confirmation test can be done if a request comes from the physician.

COMPLETE BLOOD COUNT

Principle:
Complete blood count is a series of tests and calculations that furnish information relative to the diagnosis
of many diseases. Complete blood count is often referred to as CBC. Complete blood count is often
referred to as CBC. Complete blood count consists of five series of tests:
● While cell count
● Hemoglobin concentration
● Hematocrit determination
● Differential white cell count
● Stained red cell examination

Reagents and Supplies:

● WBC diluting fluid
● WBC pipette
● Rubber sucking tube
● Sterile blood lancets
● Glass slides
● 70% alcohol
● Wright’s stain
 ● Capillary tube

Equipment:
● Microscope
● Hemocytometer
● Counting chamber (Neubauer)
● Tally counter
● Differential counter

Procedure:
1. Clean site with 70% alcohol
2. Prick/ extract using sterile blood lancet/ syringe
3. Suck blood to 0.5 mark of pipette
4. Suck diluting fluid to 11-mark, mix
5. Discard the first few drops, change counting chamber
Normal Values

Hemoglobin Male: 135-175 g/ L ESR Male: 0-10 mm/hr

Female: 120-160 g/L Female: 0-20 mm/hr

Children: (varies with age) Clotting Time 2-5 minutes

112-165 g/L

Newborn: 165-195/L Bleeding Time 1-3 minutes

Hematocrit Male: 0.40-0.54 Differential Count: Band Stab: 0.03-0.05

Female: 0.37-0.47 Segmenters: 0.54-0.65

Children (varies with age) Lymphocytes: 0.25-0.35

0.35- 0.49

Newborn: 0.49-0.54 Monocytes: 0.03-0.07

WBC 4.3- 10.8 x 109/ L Eosinophils: 0.01-0.03

Platelet 150-400 x 109/ L Basophils: 0.0- 0.75

RBC Male: 4.6- 6.2 x 1012/ L

Female: 4.2- 5.9 x 1012/ L

6. Count WBC in 4 corner large square

 Computation WBC in thousand/ mm3= WBC counted x 50
7. Hemoglobin
Sahli- Hellige hemometer
a. Place O I N H C I up to mark 2 of a graduated Sahli- Hellige hemometer
b. Suck blood up to the mark
c. Transfer blood sample into hemometer tube containing HCl let stand for 10 minutes
d. Place hemometer tube into the comparator block and distilled water drop by drop mix
contents of tube with use of stirrer
e. Compare the color of the diluted sample with the color standard tube. When the color of the
diluted sample matches that of the standard tube.
f. Read the height of the diluted sample directly on the gram scale of the hemometer tube.
g. Normal value= Male: 13.5- 17.5 g/ 100 ml, Female: 11.0= 16.0 g/ 100 ml

ESR DETERMINATION: 
WINTROBE AND LANDSBERG METHOD 
Procedure:
a. with a long-stemmed pasteur pipette, fill the wintrobe tube with oxalated blood up to the o mark of the
left red side. Avoid introducing bubbles. 
b. Let the tube stand in a perfectly vertical position for 1 hour.
c. Read ESR directly against the calibration of the tube after 1 hour. ESR is read at the upper left red side
of the wintrobe tube, or the height or amount of plasma should be determined. Hematocrit is read at the
lower right white side of the tube. 

Normal Values:
Male adult: 0-10 mm/hr. 
Female adult:0-20 mm/hr. 
Children: 0-13 mm/hr. 

BLEEDING TIME:

DUKE'S METHOD: 
a. Materials 
● 70% alcohol sponge
● sterile gauze
● disposable sterile blood lancets
● filter paper 
● stop watch
 b. Procedure 
1. Disinfect the site of puncture with 70% ethyl alcohol and allow to dry with sterile gauze. 
2. Puncture to a depth of 3 mm with a sterile disposable blood lancet.
  3. Immediately start the timer as soon as the first drop of blood appears.
4. Blot the drop of blood with filter paper every 30 secs, making sure that the filter paper does not
touch the wound.
5. Stop timer as soon as the last drop of blood disappears or as soon as bleeding stops.
 
Normal Values: 2-4 mins. 

CLOTTING TIME: 

SLIDE METHOD: 
a. Materials 
● 70% alcohol sponge
● sterile gauze
● disposable sterile blood lancets
● stop watch
● clean glass slides 
● needle

 b. Procedure 
1. Disinfect site of puncture with 70% alcohol sponge and dry.
2. Puncture to a depth of 3 mm
3. Wipe off 1st drop of blood.
 4. Start the timer as soon as the 2nd drop of blood appears. 
5. Transfer the 2nd drop of blood onto the center of glass slide. 

AUTOMATED PROCEDURES FOR CBC (SRFI) 

1. Before Analysis 

Check the Instrument as follows, 

a. Check the tubing, make sure it is in good condition and no distortion
b. Check cables and socket
c. Check the connection of accessories 

2. Start-up the instrument 

a. Switch the external printer if connected 

b. Turn on the instrument

c. The instrument will check if reagents are available and clean the liquid tubing. 

3. Background Test 
a. First perform a background test in order to control cleanliness of the 
instrument
 b. The acceptable range of results for the background is; 

Parameter Background Range Unit

White Blood Cells S0.3 10.9/L

Red Blood Cells S0.03 10.12/L

Hemoglobin S0.2 g/L

Hematocrit S0.5 %

Platelet S10 10.9/L

If the background test data exceeds this range, repeat the above test step until it can be accepted. Run the
quality control test before the blood samples. 

4. Blood Sample 

• Venous Blood 
• Peripheral Blood 
WARNINGS: Be very careful when manipulating samples, controls and calibrators wear suitable
protective gears. Use EDTA anti- coagulated tubes.  
5. Counting Analysis 
● Input patient information 
● Put the sample cup under the probe, start pad 
● The instrument begins to analyze samples automatically, wait for the analysis result 
● The result will display on the window with the histogram of WBC, RBC and PLATELET 
NORMAL VALUES

Red Blood Cells 3.50-5.50 10.6/ uL

Hemoglobin 110-160 g/L

Hematocrit 36.0-48.0 %

White Blood Cells 5.0-10.9/L

Platelet 150-400 10.9/L

6.Pass the tip of the needle through the drop of blood every 30 seconds and note for the information of
fibrin strands.
7.Stop the timer as soon as fibrin strands are seen clinging at the tip of the needle.
Normal Values: 2- 5 minutes

PLATELET COUNT 

Direct Method or Hemocytometer Method 

REES AND ECHER'S 

Procedure:
1. Rinse RBC pipette first with Rees and Echer's diluting fluid by sucking in and out the diluting fluid. 
2. Suck blood to mark 0.5 of the RBC pipette
3. Suck diluting fluid to mark 101. 
4. Shake the pipet for 3-5 mins. to mix. 
5. Discard the first few drops. 
6. Charge the counting chamber and allow it to stand for at least 3 mins.
7. Count platelets in the 4 corner large squares (as in WBC count) 
8. Computation: 
Platelet ct/cu.mm.= platelet ctd. X 10 x 200 
4

9. Normal Values = 140,000 - 340, 000/mm3

CYANMETHEMOGLOBIN METHOD (HiCN)

● Also known as HEMOGLOBIN CYANIDE and FERRIHEMOGLOBIN CYANIDE

● Best method of HEMOGLOBIN DETERMINATION 

DRABKIN'S REAGENT: 
Sodium Bicarbonate (NaHCO3) ---------------------------------------------------1.00 gm 
Potassium Cyanide (KCN)----------------------------------------------------------0.05 gm 
Potassium Ferricyanide K3Fe (CN)6 --------------------------------------------- 0.20gm
Distilled water to -------------------------------------------------------------------- 1.000mL 

PROCEDURE: 
1. Place 5 mL of Drabkin's Reagent into a clean test tube. 
2. Add 0.02 mL of capillary or venous blood, rinsing the Sahli hemoglobin pipette at least 3 times. 
3. Mix thoroughly and let stand for at least 10 minutes before reading.
 4. Read in a spectrophotometer, wavelength 540 mu. Read equivalent hemoglobin from the calibration
curve or table. 
PROCEDURE= BTS 
1.Place 500 microliter of drabkin's reagent into a clean test tube 
2. Add 2 microliter of capillary or venous blood. 
3. Mix and stand for 10 mins. 
4. Read absorbance at 540 wavelength. 
Platelet count= Direct Method or Hemocytometer Method 

REES & ECHER'S:

Procedure:
Rinse RBC pipet first w/ Rees & Echer's rgt by sucking in & out. 
2. Suck blood to mark 0.5 at the RBC pipette
3. Suck diluting fluid to mark 101. 
4. Shake for 3-5 mins. 
5. Discard the first few drops. 
6. Charge the counting chamber, stand for 3 minutes. Count platelets in the 4 corner large squares as in
WBC count. 
7. Computation: 
Platelet ct/cu.mm.=platelet counted x 10 x 200 
4
8. Normal Values= 140,000 - 340,000/mm3
HEMOGLOBIN (BIOSYSTEM) 
Distilled Water (3) 490 uL
Reagent 30 uL 
Sample  6 uL
Incubate for 3 mins at room temperature 

NOTE: Sample probe is automatically cleaned internally and externally at the end of each test. 

If there is a clog or bubbles during the process, the information section would display the flag of "Clog,
Failure, or Bubble" 

FLAGS 
“B” means bubble in the test 
“C” means clog in the test
“L” means the result is lower than the limit 
“H” means the result is higher than the limit means
****** means inefficacy data 

6. Turn Off 

Please carry out the shutdown procedure after the test blood sample. The instrument will carry on the
maintenance and wash the measuring tube. 
The steps to follow:

● At the Blood Cell analyzer window, click shut down

● Present the Diluclair H18 for aspirations through the sample probe and press the start pad. 
● After this cycle the screen shows "Turn off the instrument now", turn off the power on the back
panel 
● Turn the printer power, clean the workbench and dispose the waste 

NOTE: Do not turn off the power instrument directly, without carrying on the shutdown procedure.
 CLINICAL MICROSCOPY & PARASITOLOGY
The section of clinical microscopy is concerned with the physicochemical microscope analysis of various
body fluids. These examinations are performed manually utilizing various reagents:
● Urinalysis 
● Fecalysis 
● Sperm Analysis 
● Fluid analysis (pleural, synovial ascetic, cerebrospinal)
● Pregnancy Test (Urine or Serum)
● Occult Blood Test in stool 

PRE-ANALYTICAL PROCEDURES
● Laboratory request form coming from the attending physician must be presented with complete
information of the patient.
 Urine Examination:
1. Give the patient a clean and or sterilized bottle 
2. Instruct the patient to catch the mid-stream of the urine and bring the specimen immediately to the
laboratory or if taken at home bring the specimen no longer than 1 hour.
3. Promptly label the specimen bottle and prepare the necessary materials for testing.
Fecal Examination 
1. Give the patient a clean and dry specimen cup with a cover and collect pea-size feces making sure that
the collected specimen is not contaminated or mixed with urine or water. Be sure to inform the patients to
bring the specimen to the laboratory no longer than 1 hour.
2. For infants and toddler patients make sure to inform their guardians to directly collect feces from the
anus, and do not submit specimen collected from diapers
3. Promptly label the specimen bottle and prepare the necessary materials for testing 

ANALYTICAL PROCEDURE
Urine Examination:
1. Microscopic examination of urine is done by noting its color and appearance.
2. Four parameters reagent is used to determine the specific gravity, Ph, urine glucose and
urine protein (others used 10 parameters) 
 3. For positive urine glucose and urine protein confirmatory test using Benedict test for
sugar and Exton's Test for protein 
4. For Microscopic examination, centrifuge 5-10 ml of urine for 5 minutes to separate the
sediments present 
5. Decant urine and transfer the remaining 0.5 ml urine with re-suspended sediments on a
glass slide and cover with coverslip 
6. Examine first under low power objective to have an overview of the urines general
composition before shifting to high power objective for sedimentation identification 
7. Microscopic examination notes the presence of red and white blood cells bacteria
epithelial cells, cuptals, cast and other sediments 
8. For Pregnancy tests a test kit is used.
Fecal Analysis:
1. Perform Microscopic observation of the color and consistency of the feces 
2. Put a drop of normal saline solution on a slide and emulsify stool mix and place a cover slip
3. Examine first under low power objective before shifting to high power objective 
4. Microscopic examination notes the presence of white and red blood cells muscle fibers fecal fat
and parasites
ROUTINE ANALYSIS
a. Urine should be examined while fresh or adequately preserved 
b. Immerse the reagent strip 
c. Centrifuge 10 - 15 m 140 RCF for 5 minutes
d. one drop of suspended sediments transferred to a microscope slide and covers is added
e. Microscope results should be correlated with the physical and the chemical findings to ensure
accuracy of report 

PREGNANCY TEST
One Step Rapid Test 
A. For Strips
Immerse the strips to sample. Take the strip out after 5 seconds and lay the strip flat. Read results
within 5 minutes.
B. For Cassette
Transfer 4 drops of sample into sample well. Read the results in 5 minutes.
 
SEMEN ANALYSIS 
Normal Values 
Volume---------------------------------------------------------------------- 2-5 ml 
Viscosity-------------------------------------------------------------------- Pour in droplets 
Liquefaction Time----------------------------------------------------------Within 30 minutes
pH----------------------------------------------------------------------------- 7.3-8.3 
Count------------------------------------------------------------------------- 20-160 million/ml 
Motility-----------------------------------------------------------------------> 50-60% within 3 hours 
Quality------------------------------------------------------------------------>20 or fair 
Morphology----------------------------------------------------------------- <30% abnormal forms 

Methods: Dilute the specimen 120 and count the sperm either in the 5 RBC squares or in two large WBC
squares
a. Using a 1:20 dilution counted in 5 RBC squares
Computation: Calculate the sperm count per milliliter sperm counted X 1,000,000=sperm/ ml
b. Using a 1:20 dilution counted in 2 WBC squares sperm counted X 100,000 = sperm/ml

OCCULT BLOOD
Guaiac Test
1. Place stool taken from different areas of the stool to the guaiac impregnated filter paper 
2. Add hydrogen peroxide to the filter paper with the stool applied on it. 
Positive result - blue color appears on the filter paper (presence of hemoglobin
pseudoperoxidase)
Occult blood test strip- see insert or brochure for procedure & result

LUGOL'S IODINE TEST (or NSS, Slide Method) 

Procedure:
1. Place one drop of Lugol's iodine in a glass slide
2. Mix pin head of stool specimen
3. Cover with coverslip and read at a low power field in the microscope for any presence of ova or
parasite
Sulfate Floatation Method

ZINC SULFATE CENTRIFUGAL FLOTATION TECHNIQUE

1. Place one gram of fecal material in a 15 ml conical centrifuge tube.
2. Add 8 drops of Lugol's iodine and mix well (optional)
3. Add 7-8 ml of zinc sulfate fecal flotation solution until there is a slightly positive meniscus
 4. Cover the top of the tube with the coverslip
5. Centrifuge (using a swinging head centrifuge) at 1500-2000 rpm for 5 minutes
6. Remove the coverslip and place it on a clean slide for microscopic examination
7. Examine the entire area under the coverslip for the presence of egg, cyst, oocyst, or larvae at a
magnification of 100x

CONFIRMATORY TEST FOR URINE TOTAL PROTEIN

Principle:
Proteins in urine are coagulated by heat and the degree of coagulation is directly proportional to the
amount of proteins present. Coagulation can be further enhanced when drops of acetic acid are added.
Procedure:
1. Pour 2-3 ml of urine into a 13X100mm glass tube and hold it using a tube holder.
2. Check the urine pH 7 or <3.
3. Adjust to between 4-5 using 3% acetic acid.
4. Heat the upper half of the column of urine in a flame until it boils.
5. Look for the appearance of cloudiness in the heated portion and contrast it with the lower portion
of the tube.
6. Appearance of cloudiness in the upper portion indicates the presence of proteins.
7. Add 2-3 drops 3% acetic to the precipitate and observe.
8. If the precipitate disappears, it indicates the presence of phosphates and carbonate (later produces
effervescence when the precipitate disappears). Persistence of the precipitate shows the presence
of albumin.
9. On adding 2-3 drops of concentration HNO2, if the precipitate disappears, the presence of the
mucin nucleoprotein is suggested.
RESULT: This test may be used as semi- quantitative, as follows:
COLOR CHANGE RESULT

No cloudiness Negative

Faint cloudiness (may be observed only if the tube is held Trace

against a black background)

Define non- granular cloud without flocculation 1+

Heavy and granular cloud without flocculation 2+

Dense cloud with marked flocculation 3+

Thick curvy flocculation and coagulation 4+

Interpretation and quality control:
This test is sensitive enough to detect protein down to a concentration of 2-3 mg%. For quality control
dilute 22g% of human albumin solution to get a concentration of 5 mg/dL. Use this as a test and check the
reliability and sensitivity of this method. 
NOTE: If alkaline urine is boiled, the protein may be converted into the so-called alkaline metaprotein,
which is not coagulated by heat. Therefore, it is always better to acidify the urine before doing this test.
If too much acetic acid is added, the protein may be converted to the so-called “acid metaprotein,” which
is also not coagulated by heat. Therefore, the urine should be only mildly acidic.

BENEDICT'S TEST 
How to perform a Benedict's Test:
BENEDICT'S TEST FOR GLUCOSE
Procedure:
1. Using a clean 50ml beaker obtains 10mL of a 1% (w/v) glucose solution.
2. Clearly label four clean 13X100mm test tubes for your standards.
3. Mix the appropriate amount of glucose solution, distilled water and Benedict's solution in
properly labeled test tubes as indicated by the following table:

TEST TUBE ML OF GLUCOSE ML OF WATER ML OF BENEDICTS

SOLUTION

1 0 5.0 1.0

2 0.5 4.5 1.0

3 1.0 4.0 1.0

4 2.0 3.0 1.0

4. Mix the contents of each tube by gently shaking the test tube back and forth.
5. Place the tubes in the test tube rack and set the rack in the water bath. Caution, the water is very
hot. 
6. Incubate the tubes for 20 minutes 
7. Remove your test tubes and allow them to cool.
8. Transfer the contents of the 15ml. centrifuge tubes of the 15ml. Centrifuge tubes and spin for 2
minutes
 ● It is important that the solution is clear for absorbance measurements. If there is solid matter
suspended in the solution, the light sent through the sample will be scattered and will cause errors
in the measurements.

9. Decant the supernatant into clean labeled test tubes. Be careful that any sediment remains in the
pellet at the bottom of the centrifuge
10. Determine the OD of each sample using the spectrophotometer set to read at 735 nm. Use
distilled water to set the 100% T.
BENEDICTS REAGENT - (Also called Benedict's Solution) is a reagent used as a test for the presence
of reducing sugars (such as glucose, lactose and fructose, but not sucrose) in a solution.
Simplified Procedure:
Benedict's Reagent can be used to test for the presence of glucose in urine. If glucose is found to be
present in urine, then this is an indication of diabetes.
Procedure:
1. Place 5.0 ml of benedict's qualitative solution in a test tube.
2. Add 0.5ml of urine in the test tube.
3. Boil in water bath for 5 minutes
Interpretation of Results:
No precipitate: Negative (-)
Green: Trace (-/+)
Yellow: (+)
Orange: (++)
Red: (+++)