I.

GENERAL RULES FOR LABORATORY EXAMINATIONS

A. Fill – out request form completely:

1. Patient’s Personal Identification, includes:

 Full Name ( Last, First, Middle )

 Age, Sex, Status

 Address

 Birthday

2. Patient’s Hospital Number

3. Date and Time of Admission

4. Clinical Diagnosis

5. Requesting Physician (with Signature)

6. Name of Staff Nurse Attendant

7. Name of Medical Technology on Duty

8. Desired Laboratory Request for Examination

B. Proper Handling of Specimen Collection:

1. Safety Measures Includes:

 Wearing Laboratory Gown/ Isolation Gown

 Wearing surgical mask, N95 etc.
 Wearing Face shield
 Using Disposable gloves

2. Preparation of Laboratory Instruments, Reagents, Specific Specimen Collection Prior to

Examination.

 Check for Expiration Date

C. Availability of Laboratory Test to Be Performed.

 Inform Resident On Duty, Staff Nurse, and Laboratory Staff.

D. Sanitary Infection Waste Management

 Sharp Instrument: Syringe , Needle, Lancet, Pricker, Uses Slides, Broken glasses,
Applicator Stick, Specimen Tubes, Vacutainer Tubes in proper labeled disposable
Waste container.

 Infectious Specimen: Urine, Stool, Blood Component ( Serum, Plasma ),

Whole Blood, Blood Fluids, Sputum Should be disposed in a proper Disposable
Waste Immediately.

E. Proper Usage and Labelling of Vacutainer tubes and Collection prior to test according to

Laboratory test requests: Name, Date, Time should be noted.

F. Ensure to Inform Patient and Patient’s Relatives or Watcher about:

 Identification of Laboratory Staff on Duty

 Purpose of Blood Collection.
 Re-check laboratory request for incomplete information about patient.
G. Record Results in appropriate and designated logbooks completely with Patients identification

Record, Date examined, and laboratory test result done.

H. Reporting of Result must provide complete information needed to identify patient’s record,

Laboratory result with reference range according to sex and age of patient.

I. Results must be released immediately at appropriate time upon receiving and given to the staff

Nurse on duty to be attached in patient’s chart for in patient.

Laboratory results for Outpatient must be released to patient and authorized relatives only.

II. LABORATORY PROCEDURE FOR SPECIMEN COLLECTION

1. CAPILLARY BLOOD COLLECTION

 Obtained from tip of a finger in adults, and from the great toe or heel in infants avoiding
Callus area

a. Apply aseptic technique with 70 % alcohol on desired area.

b. Dry area with sterile gauze.

c. Puncture area with sterile disposable blood lancet designed to penetrate not deeper
than 2mm.

d. Use sterile gauze to wipe away first drop of blood and collect sufficient subsequent
drops in a heparinized capillary tube.

e. Avoid squeezing the punctured area to prevent alteration of composition in the blood
specimen.

f. Wipe punctured area and cover with sterile gauze.

2. VENOUS BLOOD COLLECTION

 Obtained preferably from the antecubital fossa, avoiding hematoma.

a. Application of tourniquet on the upper arm less than 1 minute.

b. Cleanse Puncture site with 70 % alcohol

c. Dry with sterile gauze

d. Puncture readily accessible or visible vein with proper gauge of syringe needle with
bevel pointing upward, 45 degree angle.

e. Aspirate the plunger gently from the barrel allowing blood to flow.

f. When desire volume of blood is obtained, loosen tourniquet.

g. Apply pressure to the punctured site with sterile gauze.

h. Remove needle from punctured site immediately “fish out” the needle with the cover.

i. Transfer blood specimen to properly labeled vacutainer tubes, using anticougulated

using anticougulated tubes preferably EDTA, mix gently by inversion several times

j. Dispose sharp needles to appropriate labeled containers.

III. HEMATOLOGIC TECHNIQUES

A. SAMPLE COLLECTION
MACRO METHOD

 Collect Venous Blood into an anticoagulated vacuum tubes using sterile disposable 3 ml
or 5 ml syringe or vacutainer.

 Volume of whole blood is indicated in vacuum tubes, mix well but gently.
Label properly noting complete name, age, sex, date and time.

 Avoid hitting several times to prevent hematoma.

 Avoid cyanotic area to prevent hemolysis specimen.

 Avoid prolonged tourniquet application to prevent hemoconcentration/venous statis.

MICRO METHOD

 Whole blood is collected into a heparinized capillary tube by finger puncture.

 The Capillary tube should be at least two-thirds (2/3) full.

 Avoid squeezing too hard.

ANTICOAGULATED VACUTAINER TUBES:

1. PURPLE/ VIOLET/ LAVANDER ( EDTA TUBES)

 For hematologic test, CBC,ABO/RH grouping

 For whole blood

2. GREEN ( LITHIUM/SODIUM HEPARINE TUBE )

 For troponin determination, ionized calcium, Electrolytes

 For Arterial Blood Gas, hormones, ammonia level

3. RED ( PLAIN TUBE AND COAGULANT TUBE )

 For serology, chemistry, drug monitoring, food poisoning, hormonal assay.

 For serum samples

4. LIGHT BLUE ( SODUIM CITRATE TUBE )

 For Coagulation studies ( PT, APTT,FIBRINOGEN LEVEL)

 For plasma and whole blood samples

5. GRAY ( POTASSIUM OXALATE, SODUIM FLOURIDE TUBE )

 For glucose levels ( GLUCOSE TOLERANCE TEST, OGTT, OGCT)

 For alcohol and lactic acid testing

 6. ROYAL BLUE ( PLAIN TUBE )

 Special tube free from trace elements for aluminum, arsenic, chromium,
Copper, nickel, and zinc

 For serum sample.

7. TAN / BROWN ( HEPARIN

 For determination of LEAD level, plasma sample.

B. PREPARATION OF BLOOD SMEAR

 A well prepared smear of blood, which is properly stained, is essential to determine all
details of the cell present for accurate identification.

1. The glass slides must be scrupulously clean. Slides should not be left uncovered on top of the
counter. Dirt and grease will ruin a smear. It is best to keep the slides covered and in a drawer
until ready for use. Using a micro hematocrit tube or small pipet, place a drop of well mixed EDTA
anticoagulated blood 3-4 mm in diameter near the frosted end of the slide or 1 ⁄4 inch from one
end of an unfrosted slide.

2. Rest the spreader slide at a 25° angle on the slide to be made. Draw in carefully back to the drop
of blood. The drop should flow to the edges of the spreader slide approximately 1-2 mm in depth.
Keep the spreader slide at a 25° angle with light but firm pressure against the horizontal slide.
Increasing the angle results in a thicker smear, whereas a smaller angle gives a thin smear.

3. Draw the spreader slide rapidly and smoothly over the entire length of the smear slide pulling a
thin even film behind it. The smear should cover 1 /2 to 3 ⁄4 of the slide and finish with a
“feathered” edge.

4. If the smear is too thick the cells are too packed and detail is lost; if the smear is too thin,
insufficient number of cells will be present to count and morphological characteristics will also be
lost to some degree. In either situation, red blood cells and white blood cells may appear distorted.

5. The smear must be stained with a good quality of stain to observe true morphological
characteristic, color of cytoplasm, nuclear structure and color of granules if present .

C. BLOOD CELL COUNT: HEMOCYTOMETER METHOD

1. WHITE BLOOD CELL COUNT

 Whole blood is mixed with a weak acid solution to dilute the blood and hemolyze red
blood cells.

 REAGENTS : WBC DILUTING FLUID

a) 2 % ACETIC ACID IN DISTILLED WATER

b) 1 % HYDROCHOLIC ACID IN DISTILLED WATER
c) TURK’S DILUTING FLUID
 GLACIAL ACETIC ACID – 3 ml
 1 % AQUEOUS GENTIAN VIOLET – 1ml
 DISTILLED WATER- 1 ml

2. RED BLOOD CELL COUNT

 Whole blood is diluted with an isotonic solution diluting fluid to facilitate counting and
prevent lysis of the red blood cells.
  REAGENTS : RBC DILUTING FLUID

1. 0.85 % NaCI or Formol Citrate

 Sodium Citrate – 3 gm
 Formaldehyde - 1 ml
 Distilled water - 100 ml

 DILUTION FOR WBC AND RBC COUNT :

1) Draw the blood up to the 0.5 mark in the Thomas Pipet for WBC’s and RBC’s count respectively.

2) Holding the pipet almost vertical. Place the tip into the punctured site with blood.

3) Draw desired diluting fluid into respective Thomas pipet slowly. Until mixture reaches the 11 mark
of WBC and 101 mark for RBC.

4) Gently rotating the pipet to ensure proper amount of mixing.

5) Place the pipet in a horizontal position and firmly hold the index finger of either hand over the
opening in the tip of the pipet. Detach the aspirator from the other end of pipet.

6) Mix the Thomas pipet for approximately 3 minutes to ensure hemolysis of the red blood cells for
WBC diluting fluid.

7) Fill the Counting Chamber

 Make sure the Improved Neubauer Hemocytometer is clean with cover glass on the top of ruled
area of about 0.01 millimeter.

8) Counting WBC’s and RBC’s.

 Discard the first four drops of the mixture onto a piece of gauze.
 Place the tip of the Thomas pipet on the edge of the ruled area of the counting chamber.
 Allow mixture to stand to be absorbed under the cover glass approximately 1 minute, gradually
and exactly fill the area.

 COUNTING WBC’S AND RBC’S

- Allow to stand at least 1 minute for WBC and RBC cells to settle.

- Count the cells, Using the Low power (10X) objective.

a) Scan the four large corner squares marked “W”, there should be an even distribution of
white blood cells in all four large squares. Count all WBC in the four large corner
squares beginning with the upper left squares.

b) Counting the red blood cells with the large middle square containing 25 smaller squares
of equal size. The 5 small squares labeled “R” are the areas to be counted.

 COMPUTATION:

- Add results together to obtain the total number of cells.

a. CALCULATION OF WBC :

Total Number x Correction for Volume x Correction for dilution

WBC count (2.5) (20) = WBC/uL

 b. CALCULATION OF RBC :

Total Number x Correction for Volume x Correction for dilution

RBC count (50) (200) = RBC/uL

D. HEMOGLOBIN DETERMINATION:

a) ACID HEMATIN METHOD BY SAHLI HELLIGE:

a. Add N/10 HCl to the hemoglobinmeter tube up to its lowest mark.

b. Take blood up to 20 cu mm mark on the pipette and transfer it to the hemoglobinometer

tube containing N/10 HCl.

c. Leave the solution for 10 minutes (for the conversion of hemoglobin to acid hematin.

d. After 10 minutes add distilled water drop by drop and mix it by stirrer until the color
matches with the color of comparator. While matching the color glass rod should be
removed from the solution.

e. The lower meniscus of the solution should be taken as the result which expresses
hemoglobin content as g%.

f. If the hemoglobin is too low (less than 3g/dl) then 40µl blood is added to HCl up to 20
marks in the tube. Color is matched and result is halved.

2. CYANMETHEMOGLOBIN METHOD:

a) Into a clean test tube pipet 5 ml Drabkin’s reagent.

b) Add 20 ul of whole blood sample either thru finger puncture or EDTA.

c) Mix and allow to stand at room temperature for 10 minutes.

d) Read absorbance at 540 nm against water blank, followed by hemoglobin standard in a 1

cm cuvette.

- Optical density is converted into the concentration of hemoglobin by using a constant

determined from optical densities of series of standards of known concentrations.

E. HEMATOCRIT DETERMINATION
 The hematocrit is the amount of packed cells in a sample of whole blood expressed in
percent.

a) MACRO METHOD

 Anticoagulated blood (EDTA, Heparin or Oxalate) is added to a Wintrobe sedimentation rate

tube to the 0 (zero) mark and centrifuge for 30 minutes at 2500 G. After centrifugation, the
volume of packed cells is read at the appropriate mm graduation on the mm scale of the tube.
The number of mm equal to the % (percent) of packed cells.
b) MICRO METHOD

 Whole blood is collected into a heparinized capillary tube by finger puncture, should be at least
two-thirds (2/3) full. One end of the tube is sealed with clay or other suitable material. The tubes
are centrifuged in a micro-hematocrit centrifuge for five minutes at a minimum of 12000 G.
 After centrifugation, the capillary tubes are read on a microhematocrit reading device.