IMHM321 LABORATORY

RH TYPING (SLIDE METHOD)

Objective: To detect the presence of Rh (D) antigen on the red cells using the slide method.

Introduction:

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.

Pre-lab Activity: Collection of blood through capillary puncture

Materials:
Glass slides Rh viewbox Applicator sticks
Reagents:
Rh typing sera (anti-D)
Patient's blood sample
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.
 ILLUSTRATIONS:

Name of patient: ___________________________________________

D C c

Reaction: ___________ ___________ ___________

E e CTRL

Reaction: ___________ ___________ ___________

Antigen/s detected: C Ag, e Ag

Rh Blood Group: Rh NEGATIVE (initially) proceed to test for weak

D/Du variant (IAT)
 IMHM321 LABORATORY

RH TYPING (TUBE METHOD)

Objective: To detect the presence of Rh (D) antigen on the red cells using the tube method.

Introduction:

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:

* Hemolysis 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.

Pre-lab Activity:

Collection of blood through venipuncture

Preparation of 2% to 5% Red blood cell suspension
Materials:

Test tubes
Centrifuge

Reagents:
Rh typing sera Normal Saline Solution
Patient's 2-5% red cell suspension Rh Control

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 ( 10x 75mm).

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.

NOTE: Run Rh control to validate results.

ILLUSTRATION:

NAME OF PATIENT: ________________________________________

Anti-D

Reaction Grade: 0 0

Antigen detected: no D Antigen detected

Rh type: Rh NEGATIVE (initially) (proceed to test for weak D (IAT)

 IMHM321 LABORATORY

DETECTION OF Du VARIANT
(INDIRECT COOMBS' TEST)
Objective: To determine the presence or absence of Du factor on red cells giving negative or
weak reactions with Rh typing method.

Introduction: 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 – Test to ascertain the presence or absence of red cell coating by
immunoglobulin G (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.

 Pre-lab Activity:

Collection of blood through venipuncture

Preparation of 2% to 5% Red blood cell suspension

Materials:
Test tube
Water bath at 37 °C
Centrifuge

Reagents:
Anti-D antisera
Antihuman Globulin reagent/Coomb’s reagent

PROCEDURE:

1. If the results of the Rh typing tube method is negative, proceed to the testing for the presence
of weak D. Incubate patient tube and Rh control tube for 15 minutes at 37 degrees Celsius water
bath.
2. Wash the cells 3-4 times with NSS.
Washing: Add NSS to both tubes up to ½ inch from the top of the tube. Centrifuge for 60
seconds at 3400 rpm. Decant the supernatant making sure that the red cell button is still intact at
the bottom of the tube. Resuspend the red cell button. Do this washing step 3-4x.
3. Decant the saline completely after the final washing.
4. Add 2 drops of Anti-Human Globulin reagent to all tubes and mix gently. Cover with
parafilm.
5. Centrifuge the tubes for 30 seconds* at 3400 rpm
6. Gently dislodge each cell button and examine for hemolysis and agglutination.
7. 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).
8. For each negative tube, add 1 drop of well mixed check cells. Centrifuge for 30 seconds* at
3400 rpm. At least 2+ agglutination must be observed in order for results to be valid as true
negative.
NOTE: Confirming negative results under a microscope is a good practice to ensure absence of
agglutination.

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)
+ Rh POSITIVE Positive reaction
(with agglutination) indicates presence of D
Antigen
0 Rh NEGATIVE (initial) Negative reaction
(no agglutination) should be further tested
for the presence of weak
D with the use of AHG.
INDIRECT AHG TEST for WEAK D: (Indirect Anti-Human Globulin Test)
Du Reaction Manner of Reporting Additional Comment
0 Rh(-) Du (-): Negative
(no agglutination) Du NEGATIVE reactions confirm that
the person is Rh
NEGATIVE. (patient or
donor)
+ Rh (-) Du (+):should be
(with agglutination) Du POSITIVE given proper
classification:
If recipient: Rh
NEGATIVE
If donor: Rh
POSITIVE
ILLUSTRATION :

Draw and color your results. Label the tubes completely then interpret and grade the reactions.
Determine the Rh Blood Type/Group.

NAME OF PATIENT: ________________________________________

FINAL Rh GROUPING (IAT):

U Rh Control

Reaction Grade: 0 0

Antigen detected: NONE

Du reaction: NEGATIVE

FINAL RH BLOOD GROUP: RH NEGATIVE (CONFIRMED)

Rh type : As donor: RH NEGATIVE

As recipient: RH NEGATIVE