LABORATORY ACTIVITY 11 Flashcards
Normal hemoglobin function carries oxygen around the body so that the tissues could breathe. What happens is there is a production of abnormal hemoglobin called
hemoglobin S.
● caused by amino acid substitution at the 6th position of the B chain
HEMOGLOBIN S (HBS)
● Glutamate/Glutamine should be in the 6th position but it is replaced by valine
HEMOGLOBIN S (HBS)
● RBC takes on sickle-like-shape when the oxygen supply is decreased
HEMOGLOBIN S (HBS)
● Causes sickle cell anemia and sickle cell trait
HEMOGLOBIN S (HBS)
There is lower HbS (?).
20-40%
METHODS USED
Sodium Metabisulfite Method
Sodium Dithionite (Solubility) Test/Solubility Test
● Deoxygenated hemoglobin
Sodium Metabisulfite Method
● Uses saponin
Sodium Dithionite (Solubility) Test/Solubility Test
○ Immediately lyse RBCs
saponin
● Cannot differentiate between sickle cell anemia and trait
Sodium Metabisulfite Method
● HbS and other sickling hemoglobins are positive
Sodium Dithionite (Solubility) Test/Solubility Test
● Sickle cells/holly leaf form is considered positive
○ There is abnormal Hbs
Sodium Metabisulfite Method
Problem: cannot differentiate sickle cell anemia and sickle cell trait. It cannot measure the degree of deoxygenation
Sodium Metabisulfite Method
● Forms liquid crystals and turbid appearance is a positive result
Sodium Dithionite (Solubility) Test/Solubility Test
PROCEDURES
A. SCRIVER AND WAUGH METHOD
B. SICKLE CELL SLIDE TEST
C. TUBE SOLUBILITY TEST
A. SCRIVER AND WAUGH METHOD
1. Place a rubber band around the base of the middle finger and allow staying in place for [?]
2. Make a finger puncture on the ball of the finger and place a drop of [?] on a slide
3. Immediately cover with a coverslip and seal edges with [?]
4. Incubate the preparation at room temperature. Observe for red cell sickling at hourly intervals for [?] if desired.
5. Microscopic examination (400x): if more than [?] of the cells are sickled, the result is positive
5 minutes
capillary blood
petroleum jelly
2, 3 hours, or after 24 hours
10%
● The finger may appear bluish/purplish and you may feel numb. This is due to [?] of the finger
deoxygenation
● Because oxygen from the environment can [?] the blood
oxygenate
● In the absence of the HbS solubility test, the [?] is useful in detecting sickle cells in patients who have either sickle cell disease or sickle cell trait.
sickle cell slide test
- Weigh [?] and transfer to a test tube capable of holding [?].
- Add [?], stopper, and mix until the chemical is fully dissolved. The chemical can only be used within the day it was suspended (?)
- Deliver one drop of patient’s [?] on a slide and add an equal volume of freshly made reagent, mix and cover with a cover glass. Exclude any air bubbles.
- Place the slide in a plastic box or Petri dish with damp piece of [?] at the bottom to prevent drying of the preparation (moist chamber). Leave at room temperature.
- After [?], examine the preparation microscopically for sickle cells. Focus the cells first with the [?] and examine for sickling using the [?]. Examine several fields. Sickling usually takes place in one part of the preparation rather than the other.
- Sickle cells usually appear in [?]. Report as “sickle cell test positive” when crescent shape cells are seen, or ‘sickle cell test negative” when cells appear rounded or oval shape.
0.1 g of sodium metabisulphite; 15 ml of water
5 ml of distilled or deionized water; within 8 hrs
capillary blood or well mix venous blood
blotting paper or tissue
10-20 minutes; 10x objective; 40x objective
crescent shape with pointed ends or holly leaf shape
The principle was based on turbidity created when Hb S is incubated with sodium dithionate.
solubility method
C. TUBE SOLUBILITY TEST
1. Add [?] of blood with [?] of the reagent prepared in procedure B (?)
2. Mix and stand at room temperature for [?]
3. Examine the tubes using a [?] as the background in ambient light settings.
4. Interpret results as positive if the [?] are not visible.
20 uL; 2 ml; 2% w/v sodium metabisulphite or sodium dithionite
5 minutes
white board with black lines
black lines
- sickling readily occurs when oxygen is reduced/slightly decreased
● In sickle cell anemia
- Sickling ofRBCs (HbS): 80-90% (severe: causes stroke to the brain or death)
● In sickle cell anemia
Some hemoglobins that aggregate and have (?) are capable of polymerizing and crystallizing within the red cell causing a distortion of cell shape (sickle shape).
reduced solubility
Hb S (Sickling Hb), when fully
oxygenated is (?).
fully soluble
Polymerization and formation into tactoid crystals occur only when (?) at tissue level.
oxygen is decreased
Laboratory Activity 11: SICKLE CELL EXAMINATION
Specimen needed:
Capillary blood
