IMH Laboratory Manual

Name: Date:
Section: Group: Score:
EXPERIMENT 1 PREPARATION OF
RED BLOOD CELL SUSPENSION
Upon completion of this laboratory course in IMH 413, the student must be able to:
(1) Recall the procedures for each immunohematologic test and demonstrate the ability COURSE
to perform each of those test, (2) Indicate the significance of each test and deduce a OUTCOMES
correct interpretation of the results, (3) Develop the inherent qualities of being a
medical laboratory professional.
To achieve this unit, a learner must:
OUTCOMES
UNIT
1. Demonstrate how to compute for concentrations in preparing the red blood cell
suspensions.
2. Demonstrate how to prepare the cell suspension in various concentrations.
TEACHING AND LEARNING ACTIVITIES
Laboratory Experimentation
Washed Red Blood cells are the red cells remaining after washing with a volume of comparable
solution using a method known to remove almost all of the plasma.
Glossary: Plasma – liquid portion of whole blood containing water, electrolytes, glucose, fats,
proteins, and gases.
Washed cells - cells freed of plasma or serum by repeated centrifugation through fresh
volume of normal saline.
PRE-ANALYTICAL PHASE
A phlebotomist will collect blood from the patient following strict protocol for venipuncture. The
type of blood collecting tube/s to be used depend/s on the test/s requested by the physician.
Upon receiving in the laboratory a properly coagulated blood specimen of adequate volume, the
medical technologist checks for proper labeling and will duly log the information into the laboratory
information system or its alternative. Materials and devices appropriate for the test procedure requested are
then prepared.
1|IMMUNOH EM ATO LOGY L ABO RATO RY M ANU AL

OUTCOMES
UNIT 1. Demonstrate how to compute for concentrations in preparing the red blood cell
suspensions.
ANALYTICAL PHASE
Materials and Reagents
Lavender top evacuated tube Large beaker containing disinfectant

Test tube rack Laboratory tissues
Marking pen 0.1 mL Serological pipet
Monoject G-21 5.0 mL Serological pipet
Pasteur pipet (with bulb) Aspirator bulb
NSS (in a wash bottles) Parafilm
Serological centrifuge
Procedure
PART 1: PREPARING AN EXACT 5% WASHED RED CELL SUSPENSION
1. Label 3 test tubes as follows:

a. Plasma/Patient’s identification
b. Washed cells/Patient’s identification
c. 5% Red blood cell suspension/patient’s identification
NOTE: Labeling is a critical step in any laboratory procedure and should be done carefully.
2. Centrifuge the blood sample, if necessary. EDTA is the preferred anticoagulant during preparation
of red cell suspension.
3. Wearing gloves and goggles, use a transfer pipet and carefully remove the plasma to the labeled
tube.
NOTE: Be careful not to contaminate with red blood cells.
4. When most of the plasma has been removed, remove some red cells from the bottom of the
patient sample tube and place them in the tube labeled “Washed cells”.
NOTE: This tube should be no more than half full of cells.
5. Add NSS from the wash bottle to the “Washed Cells” tube until the liquid level is about ½” from
the top.
NOTE: Be careful not to contaminate the tip of the wash bottle dispenser with blood.
6. Cover the top of the tube with parafilm and invert to mix.
2|IMMUNOHEMATOLOGY LABORATORY M ANUAL

OUTCOMES
suspensions.
7. Centrifuge with a balancer tube for 1 minute at 3400 rpm.

NOTE: Never open the lid of the centrifuge before the spinning action has come to a complete
stop. The spin time may vary on individual centrifuges. The student should check the calibration
information for the centrifuge in use.
8. Discard parafilm into a biohazardous waste container.
9. Using a transfer/Pasteur pipet, carefully remove the supernatant and discard it into the large beaker
with disinfectant. Be certain not to splash any of the supernatant on top of the laboratory table.
10. Repeat steps 5 through 9 two or three more times. This makes a total of three or four washes.
11. Using the 5.0 ml pipet and a rubber aspirator, pipet 1.9 ml of saline from the small beaker into the
tube labeled “5% Red Cell Suspension”.
12. Using a 0.1 ml pipet and a rubber aspirator, add 0.1 ml of patient’s red cells to the saline in the “5%
Red Cell Suspension” tube.
13. Cover with parafilm and mix by several inversions. This is an exact 5% Red cell suspension.
14. Discard all biohazardous waste in a puncture proof biohazardous waste container.
Additional notes:
If other concentrations or volumes of red cell suspension are desired, the formula below can be used
to determine how much washed cells and NSS should be mixed together.
% Red Cell Suspension = amount of washed packed RBCs x 100

total volume
Where: Total Volume = amount of washed packed RBCs + amount of NSS to be added
* The desired total volume can be set by the medical technologist. For instance, in order to prepare
5 ml of 2% red cell suspension, the amount of washed packed RBC and amount of NSS to be
added can be determined by manipulating the formulas given above.

OUTCOMES
suspensions.
PART II: PREPARATION OF AN APPROXIMATE 5% WASHED RED CELL

SUSPENSION
1. Label 2 test tubes as follows:

a. Washed cells/patient’s identification
b. 5% Red cell suspension/patient’s identification
2. Wearing gloves and goggles, perform steps 4 through 10 from the procedure for preparing an exact
5% washed red cell suspension.
3. Using the tube labeled “5% red cell suspension”, fill the tube half full with NSS from the wash
bottle.
4. Using a transfer/Pasteur pipet, add cells from the “washed cell tube” until the color and
concentration of cells in the tube is approximately the same as the exact red cell suspension prepared
in the previous procedure.
NOTE: As the cells are added, use the transfer/pasteur pipet to mix the solution by rinsing the
pipet with the saline/cell mixture in the tube. This transfer pipet may then be used to transfer the
5% cell mixture into the tubes to be tested.
POST-ANALYTICAL PHASE
Observation and Illustration

OUTCOMES
suspensions.
Computation
Cleaning and Disinfection
All contaminated consumable devices/materials should be disinfected with 10% bleach or 10% Lysol
before disposal in trash bags for infective wastes. All reusable devices must be disinfected as well. The working
area should be cleansed with disinfectant after the experiment.
The PPEs must be removed properly and be disposed as appropriate. These cannot be exposed
outside the laboratory premises.

OUTCOMES
suspensions.
Critical Thinking
1. What is NSS chemically? Why should you use it in washing red cells?
2. What is the purpose of washing the red blood cells?
3. A Medical Technologist is going to prepare a 5 ml of 2% red cell suspension. Compute for the
volume of NSS solution and the amount of washed red blood cell needed. Show computation
below:
4. What is the purpose of preparing a red cell suspension in the laboratory?

Name: Date:
EXPERIMENT 2 DEMONSTRATION OF COMMON

IMMUNOHEMATOLOGIC REACTIONS
OUTCOMES
1. Demonstrate agglutination reaction macroscopically and microscopically

UNIT
2. Describe the different grades of hemagglutination reaction.

3. Differentiate the two types of hemolysis through examination of the cell button and
supernatant fluid from the reaction tube.
4. Understand the consequence of hemolysis.
Agglutination and Hemolysis are two common reactions that you can observe in performing
experiments in the blood bank laboratory. In some experiments, these are considered positive results and
therefore should always be emphasized. Agglutination refers to the clumping of particulate or cellular
antigens/ cells due to the presence of a specific antibody. Technically, if the reaction involves red blood cells,
this should be properly termed as Hemagglutination. Hemolysis, on the other hand, refers to the destruction
of red cells with subsequent release of hemoglobin to the surrounding medium.
A phlebotomist will collect blood from the patient following strict protocol for venipuncture. The
type of blood collecting tube/s to be used depend/s on the test/s requested by the physician.
Upon receiving in the laboratory a properly coagulated blood specimen of adequate volume, the
medical technologist checks for proper labeling and will duly log the information into the laboratory
information system or its alternative. Materials and devices appropriate for the test procedure requested are
then prepared.
I M M U N O H E M A T O L O G Y L A B O R A T O R Y M A N U A L | 10
1. Demonstrate agglutination reaction macroscopically and microscopically.
OUTCOMES
UNIT
3. Differentiate the two types of hemolysis through examination of the cell button and supernatant fluid from
the reaction tube.
ANALYTICAL PHASE
Materials
Microscope Test tubes

Pasteur pipet Test tube rack
Parafilm Centrifuge
Glass slides Distilled water
Gum label NSS (in a wash bottle)
Applicator sticks 5% RCS (any ABO-Rh positive blood)
Gloves Typing sera (Anti-D): undiluted and diluted (1:10)
Procedure
PART 1: DEMONSTRATION OF AGGLUTINATION REACTIONS
A. SLIDE METHOD
1. Divide your glass slide into 2 portions.

2. Label one as ‘U” (unknown) and the other as “NC” (negative control)
3. Place a drop of reagent in each portion (U: anti-D, NC: NSS )
4. Place a drop of blood sample in each portion of the glass slide.
5. Mix the reagent and the blood sample using an applicator stick.
6. Observe the result macroscopically and microscopically using the LPO.
7. Interpret the result within 2 minutes as positive or negative. There is no need to grade the reaction.
OUTCOMES
UNIT
the reaction tube.
B. TUBE METHOD
1. Prepare 2 test tubes and label them as U (undiluted) and D (diluted).

2. Place 2 drops of undiluted anti-D in tube labeled as U.
3. Place 2 drops of 1:10 diluted anti-D in tube labeled as D.
4. Place 1 drop of 5% RCS of an Rh (+) blood in each test tubes.
5. Mix and cover each test tube with parafilm.
6. Centrifuge for 15 seconds at 3400 rpm.
7. Gently dislodge the cell button and interpret the result.
8. Compare the 2 tubes and take note of the difference.
9. Grade the reactions accordingly.
PART 2: DEMONSTRATION OF HEMOLYSIS OR HEMOLYTIC REACTION

1. Prepare 2 test tubes and label them as P (expected positive) and N (expected negative)
2. Place 2 drops of 5% RCS of an Rh POSITIVE blood in each tube.
3. Place 2 drops of distilled water in tube labeled as P.
4. Place 2 drops of NSS in tube labeled as N.
5. Mix and cover each tube with parafilm.
6. Centrifuge for 15 seconds at 3400 rpm. Do not dislodge the cell button.
7. Interpret the result.
8. Compare the two tubes and take note of the difference.
Interpretation:
No Hemolysis: intact cell button with clear supernatant

Partial Hemolysis: presence of cell button with pink supernatant
Complete Hemolysis: absence of cell button with red supernatant.
OUTCOMES
UNIT
the reaction tube.
A. Agglutination Reaction (Slide Method)
U Nc
B. Agglutination Reaction (Tube Method)
U D
C. Hemolysis
P N
OUTCOMES
UNIT
the reaction tube.
Critical Thinking
1. Differentiate agglutination from hemolysis.
2. Why is it necessary to observe agglutinations microscopically?
3. Enumerate common causes of hemolysis.
OUTCOMES
UNIT
the reaction tube.
4. What is Rouleaux Formation? What causes it? How do we resolve this problem?
Name: Date:
EXPERIMENT 3 DIRECT ABO BLOOD GROUPING

(SLIDE METHOD)
OUTCOMES
UNIT
1. Perform the ABO blood grouping procedure accurately and precisely.

2. Determine and interpret the results of the test correctly.
3. Understand the clinical significance of blood grouping.
Anti-A, Anti-B and Anti-A, B are prepared from the sera of appropriate people who lack other atypical
antibodies. These reagents are used to test for the presence of the A and B antigens on the surface of
erythrocytes. The test is routinely performed at room temperature and must not be heated. The manufacturer's
directions always must be meticulously followed. Some manufacturer's specify the use of whole blood,
whereas others specify different concentrations of RBCs in saline or in autologous serum or plasma.
Glossary: Agglutination – the clumping together of red blood cells or any particulate matter
resulting from the interaction of antibody and its corresponding antigen.
Agglutinin – an antibody that agglutinates cell
Anti-A – an agglutinin found in the plasma of group “B” individuals and which reacts
specifically with “A” agglutinogens
Anti-B – an agglutinin found in the plasma of group “A” individuals and which reacts
specifically with “B” agglutinogens
Antibody – a protein substance secreted by plasma calls that is developed in response to,
and interacting specifically with, an antigen.
Collection of blood by capillary puncture
19 | I M M U N O H E M A T O L O G Y L A B O R A T O R Y M A N U A L
OUTCOMES
UNIT 1. Perform the ABO blood grouping procedure accurately and precisely.
ANALYTICAL PHASE
Materials
Applicator sticks Typing Sera A (anti-A antisera)

Disposable blood lancet or pricker Typing Sera B (anti-B antisera)
Glass Marker or labeling material
Procedure
1. Place 1 drop of anti-A and 1 drop of Anti-B reagent separately on a labeled slide.
2. Add 1 drop of 20% test red cell suspension or a drop of whole blood from a capillary puncture to
each drop of the typing antiserum (the suspension may be prepared by adding 20 parts of red cells
to 80 part of normal saline).
3. Mix the cells and reagent using a clean applicator stick. Spread each mixture evenly on the slide
over an area of 10-15 mm diameter.
4. Tilt the slide for 2 minutes at room temperature (22°C – 24°C) and observe for agglutination. Do
not read results after 2 minutes.
5. Read and Record the result.
6. Report as “+” for agglutination and “0” for no agglutination.
7. Cal the instructor to check results.
8. Dispose all biohazardous waste in a puncture-proof waste container.
OUTCOMES
Name of patient:
Anti-A Anti-B Anti-A, B BLOOD

GROUP
Antigens detected:
Critical Thinking
1. Enumerate the common sources of error in cell typing.
OUTCOMES
2. Give the purpose of Blood typing.
3. Name some causes of false positive and false negative reactions in the slide method of ABO grouping
4. Complete the table below:
BLOOD TYPE Anti-A Anti-B

A
B
AB
O
(+) with agglutination

(0) no agglutination
Name: Date:
EXPERIMENT 4 DIRECT / FORWARD

ABO BLOOD GROUPING (TUBE METHOD)
OUTCOMES
UNIT

Determination of blood groups is made with Anti-A and Anti-B typing sera to demonstrate the
presence of the antigen on the red cells. Blood grouping may be performed on the slide or in the tube using
saline or serum suspensions of the red cells. However, the tube method is preferred over the slide method.
Drying of the specimen is faster if slide method is used, and may be mistaken for false positive reaction. If
methods described below differ from the recommended procedures given by the manufacturer of the
antiserum is used, the manufacturer's instructions should be used.
Collection of blood through venipuncture

Preparation of 2% to 5% red blood cell suspension
OUTCOMES
ANALYTICAL PHASE
Materials
Test tube Typing sera A (anti-A antisera)

Centrifuge Typing sera B (anti-B antisera)
Glass marker or an alternative labeling device Group “O” type serum
Procedure
1. Prepare three clean test tubes and label as follows: anti-A, anti-B, and anti-A,B
NOTE: Labeling should be done with care since clerical errors are the most frequent errors in the
blood bank.
2. Place two drops of the appropriate reagent (anti-A, Anti-B and O serum).
NOTE: Use a free falling drop. Do not touch the dropper to the side of the tube. Always add antisera
before cells. Always check for the clarity and expiration of the antisera.
3. Add one drop of 2% to 5% suspension of red blood cells to be tested to each tube.
NOTE: Use a free falling drop. Do not touch the dropper to the side of the tube.
4. Mix the reagent and RBC suspension and centrifuge at 3400 rpm for 15 seconds.
NOTE: Time may vary with each centrifuge. Check the calibration information for each individual
centrifuge. Never open the lid of the centrifuge before the spinning motion stops.
5. Gently resuspend the RBC button and then observe for agglutination or hemolysis macroscopically.
6. Observe suspected weakly reacting results by viewing the mixture under low power objective of the
compound microscope.
7. Grade reactions and record the results.
8. Have the instructor check your work.
9. Dispose all the biohazardous waste in the puncture –proof container.
OUTCOMES
Draw and color your results. Label the tubes completely then interpret and grade the reactions.
Determine the Blood Type/Group.
Name of patient:
Anti-A Anti-B Anti-A, B

Reaction grade:
Antigen/s detected:
ABO blood group:
OUTCOMES
Critical Thinking
1. What are the potency requirements to be met by an antiserum? Explain each.
2. What is the principle of Direct / Forward typing?
3. Name some causes of false positive and false negative reactions in the tube method of ABO
grouping.
Name: Date:
EXPERIMENT 5 INDIRECT / BACKWARD

ABO BLOOD GROUPING (TUBE METHOD)
OUTCOMES
UNIT

This method is frequently used as a check on the accuracy of the blood grouping performed on the
red cells. The test is performed by examining the serum of the blood under test against known A1 red cells, B
red cells and O red cells for the presence of anti-A and/or anti-B. The principle follows that whenever a blood
group antigen is present on the red cell the opposite antibody is present on the serum.
NOTE: Reverse grouping should be performed in the test tube only.
Glossary: Agglutinin – an antibody that agglutinates cells.
Agglutinogen – a substance that stimulates the production of an agglutinin,

thereby acting as an antigen.
Backward grouping – testing patient's serum with commercial or reagent A and B

blood cells to determine which ABO are present.
Collection of blood through venipuncture separating the serum to be tested.

Preparation of Known A (KA), Known B (KB), Known O (KO) and Known AB (KAB).
OUTCOMES
UNIT
ANALYTICAL PHASE
Materials
Test tube Glass marker or an alternative labeling device

Droppers Normal Saline Solution (NSS)
Centrifuge A, B, AB and O red blood cell suspensions
Procedure
1. Prepare 2-5% suspensions of A1 red cells, B red cells and O red cells in saline.
2. Label 4 tubes. : A, B, O, AB
3. Place 2 drops of serum into each of three tubes identified as specimen and labeled “A”, “B” , “O”
and “AB”.
4. Add 1 drop of the suspension of A cells to tube “A”, 1 drop of the suspension of B cells to the tube
“B”, 1 drop of suspension of O cells to tube “O” and 1 drop of suspension of AB cells to tube “AB”.
5. Mix by shaking gently and centrifuge for 15 seconds at 3400 rpm.
6. Gently dislodge the cell button and examine for hemolysis or agglutination.
7. Grade each reaction and record the results.
8. Have the instructor check your work.
9. Dispose all the biohazardous waste in the puncture-proof container.
OUTCOMES
Determine the ABO Blood Type/Group.
Name of patient:
A B O AB
Reaction grade:
Antibody/-ies detected:
ABO blood group:
OUTCOMES
Critical Thinking
1. What is the purpose of performing a reverse typing? What is the Principle of Reverse / Backward
blood typing?
2. What are the common causes of discrepancies between Cell grouping and reverse serum grouping?
How do you remedy them?
3. Complete the table below
Blood AGGLUTINOGEN AGGLUTININS

Type/Group
A
B
AB
O
Name: Date:
EXPERIMENT 6 RH TYPING (SLIDE METHOD)
OUTCOMES
UNIT
1. Perform the Rh blood typing procedure accurately and precisely.

2. Determine and interpret the results of the test.
3. Understand the clinical significance of the Rh blood group.
Human erythrocytes are classified as Rh positive or Rh negative depending solely on the presence or
absence of the D antigen. Agglutination will be observed if reagent antiserum containing anti-D is mixed with
erythrocytes expressing the D antigen. The Rh slide test should be done with a high concentration of both
protein and red cells. Therefore, the typing serum must have a protein concentration of 6% or more,
preferably 25-30%, and red cell suspension should have a 40-50% concentration in plasma or serum.
Glossary: Antiserum – a reagent source of antibody; as in commercial antiserum.
Collection of blood through capillary puncture
OUTCOMES
UNIT
ANALYTICAL PHASE
Materials
Glass slides Rh typing sera (anti-D)

Applicator sticks Patient's blood sample
Rh viewbox
Procedure
1. Prepare two glass slides.

2. Divide each slide into 3 portions by using a marking pen.
3. Label the three portions of the first slide as D, C, and c. Label the three portions of the second slide
as E, e and CTRL.
4. Place the glass slides on Rh viewbox (surface temperature: 45-50 degrees Celsius)for 5 minutes.
5. Remove the pre-warmed slides and deliver 1 drop of anti-D, anti-C, anti-c, anti-E, anti-e and Rh/Hr
Control on the appropriately marked portion of the glass slide.
6. Perform skin puncture by prick method and add 1 drop of whole blood to each portion.
7. Mix well with separate applicator sticks.
8. Tilt the slides back and forth for 2 minutes.
9. Observe for agglutination.
NOTE: Reading of the results should be completed within 2 minutes, otherwise, drying could be falsely
interpreted as a positive reaction. Results that show no agglutination within 2 minutes are considered to
be negative and therefore suggest absence of the specific antigen.
OUTCOMES
UNIT 1. Perform the Rh blood typing procedure accurately and precisely.
Name of patient:
D C c
Reaction:
E e CTRL
Reaction:
Antigen/s detected:
Rh Blood Group:
OUTCOMES
UNIT
Critical Thinking
1. Give the importance of performing Rh typing.
2. Why it is necessary to pre-warm the slide prior to use?
3. How is Rh typing serum prepared?
Name: Date:
EXPERIMENT 7 RH TYPING (TUBE METHOD)
OUTCOMES
UNIT

Human erythrocytes are classified as Rh positive or Rh negative depending solely on the presence or
absence of the D antigen. Agglutination will be observed if reagent antiserum containing anti-D is mixed with
erythrocytes expressing the D antigen. Determination of the D antigen status of patients is important in pre-
transfusion and pre-paternal testing because of the immunogenecity of the D antigen. If a D negative person
is exposed to the D antigen through transfusion or pregnancy, sensitization is likely to occur. This could
subsequently result in incompatible crossmatches or Hemolytic Disease of the Newborn.
Glossary: Hemolytic Disease of the Newborn – A disease characterized by anemia, jaundice,

enlargement of the liver and spleen, and generalized edema (hydrops fetalis)
that is caused by maternal IgG antibodies crossing the placenta and attacking
fetal red cells when there is a feto-maternal blood group incompatibility
(usually ABO or Rh antibodies). Synonym is Erythroblastosis fetalis.
OUTCOMES

Preparation of 2% to 5% Red blood cell suspension
ANALYTICAL PHASE
Materials
Test tubes Normal Saline Solution

Centrifuge Patient's 2-5% red cell suspension
Rh typing sera
Procedure
1. Prepare a 2-5% red blood cell suspension from a freshly drawn whole blood. Label it with patient’s
identification.
2. Place 2 drops of anti-D antiserum to a small test tube.
3. Add 1 drop of 2-5% patient’s red cell suspension.
4. Mix and centrifuge at 3400 rpm for 15 seconds.
5. Examine for agglutination. If desired, readings may be confirmed with the aid of a small hand lens of
magnifying mirror.
OUTCOMES
Determine the Blood Type/Group.
Name of patient:
Anti-D
Reaction grade:
Antigen detected:
Rh type:
OUTCOMES
Critical Thinking
1. What is the importance of knowing the Rh type of an expectant mother, of a donor, and of a
patient?
2. Enumerate the similarities and differences of Rh antigens compared to ABO antigens.
3. Give some causes of False Positive reactions.
4. Give some causes of False Negative reactions.
Name: Date:
EXPERIMENT 8 DETERMINATION OF THE DU VARIANT

(I NDI RECT COOMB’S
TEST)
OUTCOMES
UNIT
1. Perform the Coomb’s test accurately and precisely.

2. Read and interpret the results correctly.
3. Understand the practical significance of the test in blood banking protocols.
The weak D ( Du ) antigen is a variant of the D antigen. The presence of weak D can be demonstrated
by incubating the red cell suspension with anti-D followed by antiglobulin testing. This test used to detect
bound antibody indirectly is known as the indirect antiglobulin test or IAT.
Glossary: Antihuman globulin test – used to ascertain the presence or absence of red cell coating
by immunoglobulin F (IgG) or complement, or both.
Indirect antihuman globulin test – used to detect antigen-antibody reactions that occur
in vitro.
In vitro – within a glass; observable in a test tube.
In vivo – within the living body.
OUTCOMES
UNIT 1. Perform the Coomb’s test accurately and precisely.

ANALYTICAL PHASE
Materials
Test tube Anti-D antisera

Water bath at 37 °C Antihuman globulin reagent/Coomb’s reagent
Centrifuge
Procedure
1. Prepare 2-5% red cell suspensions to be tested.

2. Label another set of tubes as U and NC.
3. Follow the table below:
CONTENT UNKNOWN NEGATIVE
CONTROL
Anti-D 1 drop -
22% Bovine Serum - 1 drop
Albumin
2-5% RCS 1 drop 1 drop
4. Mix gently and cover all tubes with parafilm. Incubate both tubes for 15 minutes at 37 degrees Celsius
water bath.
5. Centifuge tubes at 3400 rpm for 15 seconds. Unknown tube with agglutination is regarded as Rh
POSITIVE. Unknown tube without agglutination goes to the next procedure.
6. Wash the cells 3 times with NSS.
OUTCOMES
7. Decant the saline completely after the final washing.

8. Add 2 drops of Anti-Human Globulin reagent to all tubes and mix gently. Cover with parafilm.
9. Gently dislodge each cell button and examine for agglutination.
10. Interpret and record results.
NOTE: Absence of agglutination confirms the blood group to be Rh NEGATIVE. Presence of
agglutination means presence of weakly reacting D antigen (Du Ag).
11. For each negative tube, add 1 drop of well mixed check cells.
NOTE: Check cells or Coomb’s cells are RBCs coated with IgG. A positive result is expected after
the addition of check cells. This implies that the test has been properly performed. Negative result
with these cells indicates an improperly performed test and the test should be repeated.
GUIDE IN THE INTERPRETATION OF TEST FOR WEAK D ANTIGEN

Anti-D (slide or tube Manner of Reporting Additional Comment
method)
+ Positive reaction indicates
presence of D Antigen
(with agglutination) Rh POSITIVE
0 Negative reaction should be
further tested for the
(no agglutination) Rh NEGATIVE (initial) presence of weak D with the
use of AHG.
AHG TEST for WEAK D: (Indirect Anti-Human Globulin Test)

Du Reaction Manner of Reporting Additional Comment
Rh (-) Du (-): negative

reactions confirm that the
0 Du NEGATIVE person is Rh NEGATIVE.
(Patient or donor.)
(no agglutination)
Rh (-) Du (+): should be
given proper classification:
+ Du POSITIVE
If patient: Rh NEGATIVE
(with agglutination)
If donor: Rh POSITIVE
OUTCOMES
Determine the Rh Blood Type/Group.
Name of patient:
Initial Rh grouping:
U NC
Reaction Grade:
Antigen detected:
Rh type:
OUTCOMES
Final Rh grouping (IAT):
Reaction Grade:
Antigen detected:
Du reaction:
Rh type: As donor:
As recipient:
Critical Thinking
1. What is Du?
OUTCOMES
2. Explain the mechanism behind the existence of weak D antigen.
3. What is required to demonstrate the presence of cells carrying a weak D antigen?
4. What are the factors affecting the indirect antiglobulin test?
5. Give the purpose of performing indirect Coombs' test. Give the Principle of the test.
Name: Date:
EXPERIMENT 9 ANTIGLOBULIN TEST

(DI RECT COOMB’S TEST)
OUTCOMES
UNIT
1. Perform the Coomb’s test accurately and precisely.

The Direct Antiglobulin Test (DAT) is used to detect and identify in vivo bound proteins, particularly,
it is important to detect bound IgG and C3d on selected red cell samples.
Glossary: Direct antihuman globulin test – used to detect antigen-antibody reactions that occur
in vivo.
Autoantibody – antibodies reactive against one's own red cell antigens.

OUTCOMES
ANALYTICAL PHASE
Materials
Test tube Blood sample

Microscope Antihuman globulin reagent
Centrifuge
Procedure
1. Prepare a test tube and label it with patient’s name.

2. Deliver 2 drops of well-mixed anticoagulated blood into the tube.
3. Wash the anticoagulated blood three times with NSS.
4. Decant completely the supernatant after last washing.
5. Add 2 drops of AHG sera to the tube.
7. Gently dislodge the cell button and examine for hemolysis or agglutination.
8. Grade each reaction and record the results.
9. If agglutination is not observed, 1 drop of Coomb’s check cells should be added.
NOTE: Coomb’s check cells are also added to confirm if AHG was previously added. If AHG was
not added, no agglutination will be observed after the addition of check cells. It is suggested that the
procedure should be repeated.
Interpretation:
With agglutination: DAT POSITIVE
No agglutination: DAT NEGATIVE
OUTCOMES
Draw and color your results. Label tubes completely then interpret and grade the reactions.
Name of patient:
Reaction grade:
Result:
OUTCOMES
Critical Thinking
1. Give the principle of Direct Coombs' test.
2. Why should cells be washed with NSS carefully before the addition of Coombs' reagent?
3. What are some causes of False Positive and False Negative results in this test?
Name: Date:
EXPERIMENT 10 DETERMINATION OF THE

SECRETOR STATUS
OUTCOMES
UNIT
1. Perform the determination of the secretor status accurately and precisely.

3. Understand the application of the test in blood banking protocols.
Certain blood group substances occur in soluble form in a large proportion (78%) of individuals in
secretions such as saliva and gastric juice. These individuals are termed “secretors” (they possess the Se gene)
and secrete ABH-soluble antigens. These water-soluble blood group substances are readily detected in very
minute quantities because they have the property of reacting with their corresponding antibodies and thereby
neutralizing or inhibiting the capacity of the antibody to agglutinate erythrocytes possessing the corresponding
antigen.
Glossary: Secretors – an individual who is capable of secreting soluble glycoprotein ABH-soluble

substances into saliva and other body fluids.
Collection and preparation of saliva sample
OUTCOMES
UNIT 1. Perform the determination of the secretor status accurately and precisely.
ANALYTICAL PHASE
Materials
Test tubes Microscope

Paraffin wax Anti-A serum
Pasteur pipette Anti-B serum
Centrifuge Normal saline solution (NSS)
Water bath 2-5% A and B cells suspension
Rubber stopper for test tubes
Procedure
1. Chew a piece of parrafin wax to stimulate secretion of saliva.

2. Collect about 2 to 3 mL of saliva in a test tube.
3. Place stoppered tube of saliva in a boiling water bath for 10 minutes. This inactivates enzymes that
might otherwise destroy blood group substances.
4. Centrifuge at 3400 rpm for 10 minutes.
5. Collect supernatant into a clean tube. The supernatant should be clear or slightly opalescent.
6. Dilute saliva 1:2 with NSS (undiluted saliva contains nonspecific glycoproteins that can inhibit
antisera and lead to incorrect results).
NOTE: Mix 1 ml of saliva with 1 ml of NSS.
7. Dilute Anti-A and Anti-B antisera 1:4. (Mix 1 ml antisera with 3 ml NSS.)
8. Prepare two test tubes and label as A and B.
OUTCOMES

CONTENT A B
Diluted saliva 1 drop 1 drop
Diluted anti-A 1 drop -
Diluted anti-B - 1 drop
Mix and cover with parafilm. Incubate all tubes at room temperature for 10
minutes.
5% Known A cells 1 drop -
5% Known B cells - 1 drop
10. Cover and mix parafilm. Incubate at room temperature for 30-60 minutes or for 15 minutes at 37
degrees Celsius water bath.
12. Gently dislodge the cell button and examine for agglutination.
13. Grade each reaction and record the result.
Interpretation:
Non-secretor: agglutination of red cells by antiserum-saliva mixture

Secretor: no agglutination of red cells by antiserum and saliva mixture.
OUTCOMES
Draw and color your results. Label tubes completely then interpret and grade the reactions. Indicate the
blood type and determine the secretor status.
Name of patient:
A B
Reaction Grade:
Blood soluble substance/s detected:
Secretor status (Secretor or Non-secretor):
OUTCOMES
Critical Thinking
1. What is the principle involved in this test?
2. Why is saliva be boiled prior to use?
3. What other substances beside A and B can be found in saliva?
Name: Date:
EXPERIMENT 11 BROAD SPECTRUM COMPATIBILITY

TESTING (CROSSMATCHING)
OUTCOMES
1. Perform the crossmatching accurately and precisely.

UNIT
2. Differentiate major crossmatching from minor crossmatching.

3. Determine the appropriate crossmatching procedure suited for a given blood product.
The test between a prospective recipient of a blood transfusion and his proposed donor (or donors)
is known as ths crossmatch or compatibility test. This test is performed to show any possible incompatibility
between the recipient's serum and the donor's red cells (the major crossmatch); infrequently, the plasma of
the donor is also tested against the red cells of the recipient (the minor crossmatch).
Glossary: Compatible blood – blood containing erythrocytes which have been tested in vitro against
the patient's serum and, which is thought, will survive normally if
administered to that patient.
Compatibilty test – a test carried out between serum and erythrocytes to ensure that
they are not antagonistic. Usually this term refers to the direct
crossmatch between the patient's serum and donor's red cells.
Crossmatch – to test a patient and a prospective donor for compatibility.
Major Crossmatch – recipient's serum tested with donor cells.
Minor Crossmatch – recipient's cells tested with donor's serum.
IMMUNOHEMATOLOGY L A B O R A T O R Y M A N U A L | 78
OUTCOMES
UNIT
Set-up of waterbath at 37 °C
ANALYTICAL PHASE
Materials
Water bath Patient's serum

Test tubes Anti-human globulin reagent
5% cell suspension of donor's cells Normal saline solution (NSS)
22% bovine albumin
Procedure
A. Protein / Albumin / Room Temperature Phase (Immediate Spin Phase)

1. Label 3 test tubes: M, N, AC
Legend: M: Major Crossmatch
N: Minor Crossmatch
AC: Auto-control
IMMUNOHEMATOLOGY L A B O R A T O R Y M A N U A L | 79
OUTCOMES
UNIT

CONTENT M N AC
Patient’s serum 2 drops - 2 drops
Donor’s serum - 2 drops -
2-5% donor’s RCS 1 drop - -
2-5% patient’s RCS - 1 drop 1 drop
3. Mix the contents and cover with parafilm.

5. Gently dislodge the cell button and observe for agglutination or hemolysis.
6. If no agglutination is observed, proceed to the THERMO PHASE.
Interpretation of a positive reaction in the protein phase:

1. Incompatibility in the ABO system will be detected at this point. Hemolysis especially may indicate
the presence of an immune anti-A and/or anti-B;
2. An incompatibility due to cold antibodies such as anti-M, anti-P1, or anti-Lea will be detected; the
latter may hemolyze incompatible red cells.
B. Thermo Phase / Incubation Phase (370 C)

1. Add 2 drops of 22% Bovine Serum Albumin on all tubes. (M, N, and AC).
2. Incubate all of the tubes for 30 minutes at 370C water bath.
NOTE: LISS can be used instead of albumin, in this case, incubate the tubes for 15 minutes only.
4. Gently dislodge the cell button and observe for agglutination or hemolysis.
5. If no agglutination or hemolysis is observed in all of the three tubes, proceed to Antihuman Globulin
Phase.
OUTCOMES
UNIT
Interpretation of a positive reaction in the thermo phase:

1. Incompatibility in this phase is usually due to the presence of a lower-titered anti-Rh antibody that
does not react on immediate centrifugation.
2. Certain Rh antibodies (anti-c, anti-E, and some anti-D) occasionally react only in an albumin medium
and are non-reactive in the antiglobulin test
C. Antihuman Globulin Phase / Coomb’s Phase (AHG Phase)

1. Wash cells of all the three tubes with NSS 3 times.
2. Decant NSS COMPLETELY after the last washing.
3. Add 2 drops of AHG antisera.
5. Dislodge cell button and examine for agglutination or hemolysis.
6. Add 1 drop of Coomb’s check cells to tubes showing no agglutination.
Interpretation of a positive reaction in the AHG phase:

1. Antibodies are detected here which usually react only in this test, e.g., anti-Fya, anti-Jka , anti-K etc.
2. Those antibodies in the Rh system which react only in the antiglobulin test are so-called “third order”
or “cryoagglutinoid antibodies”.
3. Antibodies present in acquired hemolytic anemia will be found.
NOTE:
All the three phases must be free from agglutination and/or hemolysis before the donor's blood could
be transfused to the recipient. Agglutination or hemolysis in any or all of the three phases will mean an
incompatible blood and donor's blood cannot be transfused to the recipient.
Interpretation:
COMPATIBLE: if there’s no agglutination or hemolysis in all tubes in all phases
INCOMPATIBLE: if there’s agglutination or hemolysis in all tubes in all phases
OUTCOMES
UNIT
Observation and Results
PROCEDURAL PHASE TUBE REACTIONS INTERPRETATION
A. Protein Phase Major XM:

Minor XM:
Autocontrol:
B. Thermo Phase Major XM:

Minor XM:
Autocontrol:
C. AHG Phase Major XM:

Minor XM:
Autocontrol:
(+) = with agglutination (0) = no agglutination
Check cells reactions (if performed):
OUTCOMES
UNIT
Critical Thinking
1. Give the purpose of doing Broad-Spectrum compatibility testing.
2. Enumerate factors or conditions that will result to:
A. False Negative results B. False positive results
3. In extreme emergency cases when crossmatching is impossible, when can a group O blood be
administered to recipient of any group?
Name: Date:
EXPERIMENT 12 PRE-WARMING TECHNIQUE FOR TESTING

SERUM CONTAINING COLD AGGLUTININS
OUTCOMES
UNIT
1. Perform the pre-warming technique accurately and precisely.

The reactivity of IgM cold auto-antibodies can be reduced or eliminated by performing pre-warmed
tests. Most problems encountered in compatibility testing, antibody detection, and identification tests that are
caused by cold agglutinins can be resolved with the use of this technique. By preventing the reaction between
the cold agglutinin and the red cell at room temperature (during centrifugation, and so on), one prevents
complement activation. The anti-C3 in polyspecific antihuman globulin reagents with the remaining C3 makes
the detection of significant AHG-reactive antibodies difficult. Pre-warming of the serum, cells and additive
will hinder the binding of the cold autoantibody and the resulting complement activation.
Glossary: Agglutinin – an antibody that agglutinates cell.

Autoantibody – an antibody reactive against one's own red cell antigens.
Cold agglutinin – an antibody reactive at temperatures below 37 °C.
OUTCOMES
UNIT 1. Perform the pre-warming technique accurately and precisely.
Set-up of water bath at 37 °C

Preparation of patient's serum suspected of cold agglutinins
ANALYTICAL PHASE
Materials and Reagents
Water bath Additive solution (bovine albumin, LISS,

Test tubes polyethylene glycol, etc.)
Normal saline solution (warmed at 37 °C) Cell suspension to be tested
Procedure
1. Label 1 tube for each reagent or donor cell sample to be tested.

2. Add 1 drop of the appropriate 2 to 4% red cell suspension to each tube.
3. Place the tubes containing the red cells, a tube containing small amount of patient's serum, and a tube
containing the additive solution, if any, at 37 °C for 5-10 minutes.
4. Transfer 2 drops of pre-warmed serum into each tube containing pre-warmed red cells. Mix without
removing the tubes in the incubator or water bath.
5. Incubate at 37 °C for at least 30 minutes with no additive, or the appropriate time for the additive
used.
6. Without removing the tubes from the water bath, fill all tubes with pre-warmed saline (37 °C).
Centrifuge and wash 2-3 times with warm saline.
7. Add anti-IgG antiglobulin reagent, and record reactions.
OUTCOMES
UNIT 1. Perform the pre-warming technique accurately and precisely.
Critical Thinking
1. What are the considerations in the performance of the pre-warmed technique?
2. What is the significance of detecting cold agglutinins in transfusion therapy?

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Section: Group: Score:

TEACHING AND LEARNING ACTIVITIES

1|IMMUNOH EM ATO LOGY L ABO RATO RY M ANU AL

Materials and Reagents

Lavender top evacuated tube Large beaker containing disinfectant

PART 1: PREPARING AN EXACT 5% WASHED RED CELL SUSPENSION

1. Label 3 test tubes as follows:

2|IMMUNOHEMATOLOGY LABORATORY M ANUAL

7. Centrifuge with a balancer tube for 1 minute at 3400 rpm.

8. Discard parafilm into a biohazardous waste container.

% Red Cell Suspension = amount of washed packed RBCs x 100

3|IMMUNOHEMATOLOGY LABORATORY M ANUAL

PART II: PREPARATION OF AN APPROXIMATE 5% WASHED RED CELL

1. Label 2 test tubes as follows:

Observation and Illustration

4|IMMUNOHEMATOLOGY LABORATORY M ANUAL

Cleaning and Disinfection

5|IMMUNOHEMATOLOGY LABORATORY M ANUAL

2. What is the purpose of washing the red blood cells?

4. What is the purpose of preparing a red cell suspension in the laboratory?

6|IMMUNOHEMATOLOGY LABORATORY M ANUAL

EXPERIMENT 2 DEMONSTRATION OF COMMON

1. Demonstrate agglutination reaction macroscopically and microscopically

2. Describe the different grades of hemagglutination reaction.

TEACHING AND LEARNING ACTIVITIES

Microscope Test tubes

PART 1: DEMONSTRATION OF AGGLUTINATION REACTIONS

1. Divide your glass slide into 2 portions.

1. Prepare 2 test tubes and label them as U (undiluted) and D (diluted).

PART 2: DEMONSTRATION OF HEMOLYSIS OR HEMOLYTIC REACTION

No Hemolysis: intact cell button with clear supernatant

Observation and Illustration

A. Agglutination Reaction (Slide Method)

B. Agglutination Reaction (Tube Method)

Cleaning and Disinfection

1. Differentiate agglutination from hemolysis.

2. Why is it necessary to observe agglutinations microscopically?

3. Enumerate common causes of hemolysis.

EXPERIMENT 3 DIRECT ABO BLOOD GROUPING

1. Perform the ABO blood grouping procedure accurately and precisely.

TEACHING AND LEARNING ACTIVITIES

Collection of blood by capillary puncture

Applicator sticks Typing Sera A (anti-A antisera)

Observation and Illustration

Anti-A Anti-B Anti-A, B BLOOD

Cleaning and Disinfection

1. Enumerate the common sources of error in cell typing.

2. Give the purpose of Blood typing.

4. Complete the table below:

BLOOD TYPE Anti-A Anti-B

(+) with agglutination

EXPERIMENT 4 DIRECT / FORWARD

1. Perform the ABO blood grouping procedure accurately and precisely.

TEACHING AND LEARNING ACTIVITIES

Collection of blood through venipuncture

Test tube Typing sera A (anti-A antisera)

Observation and Illustration

Anti-A Anti-B Anti-A, B

Cleaning and Disinfection

1. What are the potency requirements to be met by an antiserum? Explain each.

2. What is the principle of Direct / Forward typing?

EXPERIMENT 5 INDIRECT / BACKWARD