Title page HEMA

Dr. Benjamin Q. Bengco III Medical & Surgical Clinic St. Jude Village Brgy Alfonso Concepcion,
Tarlac Clinical Laboratory

STANDARD OPERATING PROCEDURES

FOR

CLINICAL HEMATOLOGY
VERSION 2.0

Written by
RHENZ ALFRED D. GALDONEZ, RMT Medical technologist

Date:

March 2, 2020

Reviewed by

EMIL BRYAN M. GARCIA, MD Pathologist

Date:

March 2, 2020
Dr. Benjamin Q. Bengco III Medical & Surgical Clinic St. Jude Village Brgy Alfonso Concepcion,
Tarlac Clinical Laboratory

1.0 WHITE BLOOD CELL COUNT

       1.1 Principle:

             1.1.1 A sample of blood is diluted with solution which lyses non-nucleated red blood
cells. Following adequate mixing, the specimen is introduced into a counting chamber where the
white blood cells in a diluted volume are counted.
      
1.2 Reagents:
             1.2.1   2% Acetic acid. Add Glacial Acetic Acid to a 100 ml volumetric flask Dilute to the
mark with distilled water. Add 1 drop of 1% gentian violet and mix.
       
1.3 Procedure:
              1.3.1 Drew well-mixed capillary or venous blood exactly to the 0.5 mark in a white
blood cell diluting pipet. This blood column must be free of air bubbles.
              1.3.2 Wipe the excess blood from the outside of the pipet to avoid transfer of cells to
the diluting fluid. Take care not to touch the tip of the pipet with the gauze
              1.3.3 Immediately draw 2% acetic acid diluting fluid to the "11" mark while rotating the
pipet between the thumb and forefinger to mix the specimen and diluent. Hold the pipet upright
to prevent air bubbles in the bulb.
              1.3.4 Mix the contents of the pipet for 3-5 mins and dill the counting chamber.
1.3.5 Allow the cells to settle for about 3 minutes. Under low power magnification and
reduced light, focus on the ruled area and observe for even distribution of cells.
              1.3.6 Count the white cells in the four 1 sq. mm corner areas.
              1.3.7 Count all the white cells lying within the square and those touching the upper and
righthand center lines. The white cells which touch the left hand and bottom lines are not to be
counted. A variation of more than 10 cells between any of the four areas counted indicates
uneven distribution and requires that the procedure be repeated.
    
1.4 Calculation:
               1.4.1 WBC's counted x Dilution (20) = WBC's/cu.mm
       Volume (0.4)
      1.5 Normal Values:
            1.5.1 Adult: 5,000-10, 000/cu.mm.
            1.5.2 Birth: 9,000-30, 000/ cu.mm.

      1.6 Quality Control Procedures:

            1.6.1 Pre-Analytical
                  1.6.1.1 Patient Identification
                  1.6.1.2 Prepare materials for extraction
                  1.6.1.3 Sample Collection
                  1.6.1.4 Proper labelling

        1.6.2.Analytical
              1.6.2.1 Check machine to be used
              1.6.2.2 Testing process
         1.6.3 Post-Analytical
                1.6.3.1 Recording of results
                1.6.3.2 Releasing of results

2.0 RED BLOOD CELL COUNT

       2.1 Principle:
             2.1.1 A sample of blood is diluted with a special isotonic solution in a micro pipet. After
adequate mixing, a portion of the sample is introduced into the counting chamber. The red blood
cells in a known volume are then counted.

       2.2 Reagent:

            2.2.1 Gower's Diluting Fluid: Add 12.5 grams of sodium sulfate, anhydrous, and 33.3 ml
glacial acetic acid to 200 ml. Note: Add the water slowly and mix thoroughly.

        2.3 Procedure:

               2.3.1 Draw well-mixed capillary or venous blood to the 0.5 mark on the red blood cell
diluting pipet. The blood column must be free of bubbles.         
              2.3.2 Wipe off the excess blood from the outside of the pipet to avoid transfer of cells
to the diluting fluid. When wiping, take care not to touch the end of the pipet with gauze.
              2.3.3 Immediately draw diluting fluid to the 101 mark at the same time rotating the pipet
between the thumb and forefinger to mix the specimen and diluent. Hold the pipet upright to
prevent air bubbles from flowing into the bulb.
               2.3.4 Place a finger over the tip of the pipet and remove the suction tubing.
               2.3.5 Mix the contents of the pipet for about 10 seconds by holding the pipet on
horizontal plane between the thumb and the forefinger of one hand and rotating in a figure of
eight. A mechanical shaker is used to further mix the contents.
               2.3.6 Place a clean hemocytometer cover glass on the counting chamber. Moistening
the shoulders on which the cover glass in place.
2.3.7 Mix the specimen in the pipet for about 3 minutes to assure even distribution of
cells.
       2.3.8 Expel the unmixed and relatively cell-free fluid the capillary portion of the pipet
(usually two to three drops).
       2.3.9 Place the forefinger over the top of the pipet tip to the junction cover glass and
the counting chamber.
      2.3.10 Allow the mixture to flow under the chamber when completely charged. Similarly
fill the opposite chamber of the hemocytometer. If overflowing into the moat or air bubbles
occur, clean and dry the chambers. Remix the contents of the pipet and refill both samples.
2.3.11 Allow the cells to settle for about 3 minutes under low-power magnification. Focus on the
ruled section of the counting chamber and locate the center one sq. mm area for counting the
red cells.
2.3.12 Observe for even distribution of cells.
2.3.13 Switch to high-power magnification and locate the square in the upper left-hand
corner.
       2.3.14 Count all the red cells living within this square and those touching the centerline
on the upper and right-hand triple lines. The red cells which touch the left hand and bottom
center lines are not counted.
       2.3.15 In the same manner, count the red cells in the remaining four small squares. A
variation of more than twenty five cells between any of the five
areas counted indicates uneven distribution.

2.4 Calculation:
2.4.1 RBC's counted x dilution (200) = RBC's per cu mm.
volume (0.02)

2.5 Normal values:

      2.5.1 Adult Male: 4.2-5.4 Million/cu mm
      2.5.2 Adult Female: 3.6-5.0 Million/cu mm
       2.5.3 Childhood: 3.8-5.4 Million/cu mm
      2.5.4 Birth: 4.7-7.1 Million/cu mm

2.6 Quality Control Procedures:

          2.6.1 Pre-Analytical
                2.6.1.1 Patient Identification
                2.6.1.2 Prepare materials for extraction
                2.6.1.3 Sample Collection
                2.6.1.4 Proper labelling

          2.6.2 Analytical

              2.6.2.1 Check machine to be used
2.6.2.2 Testing process

  2.6.3 Post-Analytical
        2.6.3.1 Recording of results
        2.6.3.2 Releasing of results

3.0 DIFFERENTIAL LEUKOCYTE COUNT

      3.1 Principle:

        3.1.1 The stained blood smear permits the study of the appearance and identification of
the different kinds of leukocytes and the appearance of erythrocytes and thrombocytes.
      3.2 Reagent:
        3.2.1 Wright and Giemsa stain
        3.2.2 Blood smear
      3.3 Procedure:
         3.3.1 Inspect the smear under low power magnification. Locate the thin end of the smear
where there is no overlapping of erythrocytes.
          3.3.2 Switch to oil immersion field. Identify and count 100 constructive leukocytes and
record each cell type separately on the differential counter. Begin at the thin end of the smear
and count the white cells observed as the slide moved in a vertical direction. When near the
edge of the smear, move the slide horizontally for a distance of about two fields, then proceed
vertically back across the smear. Continue this "snake-like" movement until 100 leukocytes
have been counted and classified.
         3.3.3 If the WBC count is between 20,000 and 50,000 per cu mm of blood count and
classify 300 leukocytes. When the count is greater than 50, 000 cu mm of blood classify 500
leukocytes.
         3.3.4 The number of each type of leukocyte is expressed as a percent of the total
number of white blood cells counted. Absolute values may be calculated by multiplying the
percent value by the total leukocyte count.
    
3.4 Normal Values:
           3.4.1 Neutrophils: 55-65%
           3.4.2 Lymphocytes: 20-35%
           3.4.3 Monocytes: 2-6%
           3.4.4 Eosinophils: 2-4%
           3.4.5 Basophils: 0-1%

3.5 Quality Control Procedures:

       3.5.1 Pre-Analytical 
3.5.1.1 Patient Identification
     3.5.1.2 Prepare materials for extraction
     3.5.1.3 Sample Collection
     3.5.1.4 Proper labelling
  3.5.2 Analytical
      3.5.2.1 Check machine to be used
      3.5.2.2 Testing process
   3.5.3 Post-Analytical
       3.5.3.1 Recording of results
       3.5.3.2 Releasing of results

4.0 HEMOGLOBIN DETERMINATION

    
4.1 Principle:
           4.1.1 Blood is diluted with a dilute solution of potassium ferricyanide and potassium
cyanide at a slightly alkaline pH. The ferricyanide converts hemoglobin to methemoglobin. The
cyanide then reacts with the methemoglobin to form the stable cyanmethemoglobin. The
hemoglobin content is then determined in a photometer.
     
4.2 Reagent:
          4.2.1 Hemoglobin Reagent Concentrate
   
   4.3 Procedure:
           4.3.1 Preparation of working hemoglobin standard reagent. Add 1 ml of hemoglobin
standard reagent to 4 volume of reagent grade water. Mix and store at room temperature and
use within 1 month.
            4.3.2 Transfer 5.0 ml of working hemoglobin standard reagent into vials labeled:
Unknown, Calibrator, Blank.
            4.3.3 Add 20 ul of well mixed whole blood sample to its respective vial and mix
vigorously.       
            4.3.4 Let stand at room temperature for at least 30 mins.
           4.3.5 Zero the photometer at 540 nm using the blank. Measure the absorbance of the
samples within 60 minutes.

      4.4 Calculation:

       4.4.1 Where A = absorbance, U=unknown, S = Standard C= concentration
     A(U) / A(S) X C(S) = C(U)

    4.5 Normal Values:

           4.5.1 Adult Male: 14-17 grams/dl
          4.5.2 Adult Female: 12-16 grams/dl
          4.5.3 Childhood: 10-14 grams/dl
          4.5.4 Birth: 18-27 grams/dl

    4.6 Quality Control Procedures:  

4.6.1 Pre-Analytical
       4.6.1.1 Patient identification
       4.6.1.2 Prepare materials for extraction
       4.6.1.3 Sample Collection
       4.6.1.4 Proper labelling

  4.6.2 Analytical
      4.6.2.1 Check machine to be used
      4.6.2.2 Testing process

  4.6.3 Post-Analytical
      4.6.3.1 Recording of results
      4.6.3.2   Releasing of results

5.0 HEMATOCRIT DETERMINATION

     
5.1 Principle:
          5.1.1 Whole blood is obtained (either venous or capillary) and a heparinized
capillary tube is filled by gravity to within 1 or 2 cm if the end. The unfilled end of the tube is then
sealed. Following the centrifugation, the capillary tube is placed in a reading device and the
hematocrit value determined.
     5.2 Materials:
           5.2.1 Lancet
           5.2.2 Heparinized capillary tube
           5.2.3 Microhematocrit Centrifuge
           5.2.4 Microhematocrit reader
      5.3 Procedure:
           5.3.1 Make a puncture into the finger which has been properly cleansed with 70%
alcohol.
           5.3.2 With a sterile cotton, wipe off first drop of blood as it wells up on the finger.
           5.3.3 Fill the heparinized tube with the second drop of blood until 1-2 cm of the end.
Note: Blood will enter the capillary tube by gravity.
           5.3.4 Seal the unfilled end of the capillary tube using a clay and wax. This is done by
holding the capillary tube upright onto the clay to fill it up, seal it finally using the prepared wax.
           5.3.5 Place the capillary tube in one of the numbered slots in the centrifuge head in
such a manner that the sealed end is in contact with the peripheral rim of the centrifuge head. 
           5.3.6 Screw the flat centrifuge head cover in place.
           5.3.7 Centrifuge the capillary tube at 10,000 rpm for 5 minutes.
           5.3.8 Determine the hematocrit value with the aid of a microhematocrit reader.
Note: Since there are a variety of readers available it is necessary that the technician should
carefully follow the directions of the particular device at hand.  

5.4 Normal Values:

      5.4.1 Adult Male: 40-54%
      5.4.2 Adult Female: 37-47 %
      5.4.3 Childhood: 34-41%
        5.4.4 Birth: 44-64%

  5.5 Quality Control Values:

  5.5.1 Pre-Analytical
          5.5.1.1 Patient Identification
           5.5.1.2 Prepare materials for extraction
           5.5.1.3 Sample Collection
           5.5.1.4 Proper labelling

   5.5.2 Analytical
         5.5.2.1 Check machine to be used
         5.5.2.2  Testing process
 
    5.5.3 Post-Analytical
         5.5.3.1 Recording of results
         5.5.3.2  Releasing of results

6.0 PLATELET COUNT

      6.1 Principle:

           6.1.1 A platelet count is a diagnostic test that determines the number of platelets in
the patient's blood. Platelets, which are also called thrombocytes are small disk-shaped blood
cells produced in the bone marrow and involved in the process of blood clotting. There are
normally between 150,000-450,000 platelets in each microliter of blood.
     6.2 Materials and Reagents:
         6.2.1 Red blood cell diluting pipette
         6.2.2 Hemocytometer (Neubauer Counter)
         6.2.3 Petri dish into Wet house chamber
         6.2.4 Rees and Ecker diluting fluid

      6.3 Procedure:

          6.3.1 Gently mix blood with EDTA for approximately 2 minutes.
          6.3.2 Draw blood to exactly 0.5 mark of the red blood cell pipette and dilute to the
101 mark (with dilution 1:200). From this point, the platelet count should be completed within 30
minutes to avoid platelet disintegration.
           6.3.3 Gently mix the blood and diluting fluid in the pipette for 3-5 minutes.  
6.3.4 Clean the hemocytometer thoroughly and make certain it is completely free of
dirt and lint. The use of 95 % ethyl alcohol and a lint-free cloth is recommender.
    6.3.5 Prepare a wet house chamber as follows:
              6.3.5.1 Get hold of a petri dish and a piece of filter paper of approximately the
same size of the petri dish.
              6.3.5.2 Moisten the filter paper and Make sure that the filter paper will adhere
onto the petri dish.
      6.3.6 Prepare the first 3 drops from the red cell diluting pipette and fill the 2nd place it
on top of the petri dish ruled areas of the hemocytometer.
     6.3.7 Place the charged hemocytometer inside the wet house chamber and allow
preparation to stand for 15 minutes.
     6.3.8 Read under the microscope using HPO. Count all the platelets in 10 of the 25
intermediate squares of the central large square.
     6.3.9 Calculate the number of platelets per cu mm as shown below:

PLT count/cu mm = number of cells counted x correction for volume x correction for
dilution (200)

7.0 BLOOD TYPING (TUBE METHOD)

      7.1 Forward Group Tube Method

          
7.1.1 Principle
                   A person's blood group is determined by testing the red blood cells with Anti-A and
Anti-B agglutination of the test cells indicates the presence of the relevant antigen, while no
agglutination indicates its absence.
         
7.1.2 Reagents
                7.1.2.1 Anti A anti sera
                7.1.2.2 Anti B anti sera
                7.1.2.3 Isotonic Saline Solution
         7.1.3 Specimen
                7.1.3.1 Blood sample collected in EDTA anticoagulant; or
7.1.3.2 Venous or capillary blood sample from neonates.
7.1.4 Procedure
   7.1.4.1 Place 1 drop of anti-A in a clean, labeled test tube;
   7.1.4.2 1 drop of anti-B in a separate, clean, labeled tube;
   7.1.4.3 Add 1 drop of a two (2) % to five (5) % patient cell suspension to each
tube;
    
7.1.4.3.1 Preparation of Red Cell Suspension
       7.1.4.3.1.1 Place 1 to 2 ml of blood in large tube.
       7.1.4.3.1.2 Fill with saline and centrifuge.
       7.1.4.3.1.3 Aspirate/decant the remaining saline away from the
red blood cells.
       7.1.4.3.1.4 Repeat washing (steps two and three) until
supernatant is clear. The remaining red blood cells are called a packed red blood cell
button. Pipette 10 ml of saline into a clean test tube.
      7.1.4.3.1.5 Add 0.2 ml (two drops) of the packed red blood cell
button to the 10 ml of saline. After the red blood cells settle to the bottom of the test
tube, rinse the pipette with the surface layer of the saline to remove any red blood cell
residue.

  7.1.4.4 Mix the contents of the tubes gently;

  7.1.4.5 Gently resuspend the cell buttons, and examine agglutination, and
  7.1.4.6 Read, interpret, and record the test results.    
     7.1.4.6.1 Agglutination in any tube of RBC tests them for and
agglutination or hemolysis in serum test constitutes a positive test result. The expected
agglutination reaction for positive tests are 3+ to 4+.
     7.1.4.6.2 A smooth suspension of RBCs after resuspension of RBC
button is a negative test result. All negative results must be verified under microscope.
Cells should be separate without any clumping.
     7.1.4.6.3 The interpretation of ABO group is as follows:
7.2 Rh Typing (Tube Method)
    
7.2.1 Principle
       7.2.1.1 Rh (D) typing is based on the principle of agglutination. Normal human red
blood cells, possessing antigens, will clump in the presence of antibody directed toward the
antigens. Agglutination of patient or control red blood cells with anti-D serum and no
agglutination with the control reagent is a positive test result, which indicates the presence of
the D antigen on the red blood cells. Absence of agglutination is a negative test result, which
indicates the D antigen is not
demonstrable.
   
7.2.2 Reagents
         7.2.2.1 Anti-D Reagent
         7.2.2.2 Isotonic Saline Solution
 
      7.2.3 Specimen
          7.2.3.1   Blood sample collected in EDTA anticoagulant
 
    7.2.4 Procedure
       7.2.4.1 Label a tube with the recipient's name and "D".
       7.2.4.2 Add 1 drop of anti-D reagent to the appropriate labeled tube.
       7.2.4.3 Prepare a 3-5% recipient red cell suspension.
       7.2.4.4 Add 1 drop of the recipient's red cell suspension to the appropriate tube.
      7.2.4.5 Mix the contents of the tube.
      7.2.4.6 Centrifuge the tube (speed and time as recommended by manufacturer's
instructions).
      7.2.4.7 Remove the tubes from the centrifuge. Verify that the recipient's identification on
the tubes, specimen and request form correspond. If more than one recipient is being tested,
read and record results on one recipient at a time.
      7.2.4.8 Re-suspend the cell button. Read macroscopically.
     7.2.4.9 Presence of agglutination is a positive result

7.2.5 Reporting of Result

     7.2.5.1 It should be reported as: "Blood Type" Positive/Negative