DEMONSTRATION OF

COMMON
IMMUNOHEMATOLOGIC
REACTION

Laboratory Discussion 3
 INTRODUCTION

Agglutination and hemolysis are two common reactions that you can
observe in performing experiments involving blood specimen. In some
experiments, these are considered positive results and therefore should
always be emphasized. Agglutination refers to clumping of cells due to
antigen- interaction. Technically, since it involves red cells, this should be
properly termed as hemagglutination. Hemolysis, on the other hand
refers to destruction of red cells with subsequent release of hemoglobin
to the surrounding medium.
REAGENT

Normal Saline Solution: NSS (0.85- 0.9% NaCl) in a wash bottle

5% Red Cell Suspension (any ABO Rh Positive blood)
Typing Sera (Anti-D): undiluted and diluted 1:10 (1mL anti-D + 9mL NSS)
PROCEDURE:
DEMONSTRATION OF AGGLUTINATION REACTIONS

1. Slide Method
a. Divide your glass slide intro two portions
b. Label one as U (Unknown) and the other as NC (Negative control)
c. Place a drop of reagent in each portion. (U: Anti-D, NC:NSS)
d. Place a drop of blood sample in each portion of the glass slide.
e. Mix the reagent and blood sample using an applicator stick
f. Observe the result macroscopically and microscopically using the Low power
Objectives
g. Interpret the result within 2 minutes as positive or negative. There is no need to
grade the reaction
PROCEDURE:
DEMONSTRATION OF AGGLUTINATION REACTIONS

2. Tube Method
a. Prepare two test tube and label them as U (undiluted) and D (diluted)
b. Place two drops of undiluted anti-D in tube labeled as U
c. Place two drops of 1:10 diluted anti-D in tube labeled as D.
d. Place one drop of 5% red cell suspension of an Rh (+) blood in each test tubes.
e. Mix and cover each test tube with nescofilm
f. Centrifuge for 15 seconds at 3,400 rpm
g. Gently dislodge the cell bottom and interpret the result
h. Compare the two tubes and take note of the differences
i. Grade the reactions accordingly
PROCEDURE:
DEMONSTRATION OF HEMOLYSIS OR HEMOLYTIC REACTION

1. Prepare two test tube and label them as P (expected positive) and N (expected
Negative)
2. Place 2 drops of 5% red cell suspension of an Rh (+) 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. Mix and cover each tube with nescofilm.
5. Centrifuge for 15 seconds at 3,400 rpm. Do not dislodge the cell button.
6. Interpret the result
7. Compare the two tube 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