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1

Hemoglobin Electrophoresis

Haemoglobin electrophoresis test is done to check the different types of haemoglobin in the blood.
It is the most important diagnostic test for Hgb S. (confirmatory test)
An electrical current separates normal and abnormal types of hemoglobin in the blood.
All Hemoglobin have a negative charge and migrate towards the anode (positively charged).
Hemoglobin types have different electrical charges and move at different speeds. The amount of each hemoglobin type in the current is measured.

2

Procedure - Hemoglobin Electrophoresis

• EDTA or Heparin whole blood- RBCs are lysed (deionized water and K Cyanide)
• Very small samples of the hemolysates is applied to the Cellulose Acetate Plate.
• Electric current draws the hemoglobin across the cellulose acetate or gel stip.
• The hemoglobin protein in the sample are separated by electrophoresis using an alkaline buffer (pH8.2-8.6)
• The cellulose acetate strip (Hgb) is stained with a Red Stain.
• The patterns are scanned on a scanning densitometer, and the relative percent of each band determined

3

• hemoglobin F (newborn):

50 to 80 percent

4

• hemoglobin F (6 months):

8 percent

5

• hemoglobin F (6 months+) and adults :

0.8 to 2 percent

6

• hemoglobin A (adults)

95 to 98 percent

7

• hemoglobin A2 (adults)

2 to 3 percent (if high then thalessemis)

8

• hemoglobin S and C (adults)

0 percent

9

Systemic Lupus Erythematous (SLE)

• Systemic lupus erythematosus (SLE) is chronic inflammatory autoimmune disease in which the body's immune system mistakenly attacks your own tissues and organs.
• Affects skin, joints, blood cells, brain, kidneys, heart and lungs
• SLE is much more common in women than men.
• The most distinctive sign of lupus — a facial rash that resembles the wings of a butterfly across both cheeks
- arthritis, heart problems, raynauds phenomenon

10

LE Cell Prep (using Buffy Coat Prep)

-rarely performed and has been replaced by ANA test which is a better tests to help diagnose lupus.
--- Heparinized blood is mixed with glass beads to damage WBCs
--- Buffy coat prep is used to make smears
--- Examine smear for lupus neutrophils engulfing damaged nuclei.

11

Anti-nuclear antibody test (ANA)

ANA are a group of autoantibodies produced by a person's immune system when it fails to distinguish between "self" and "nonself."
Antinuclear antibodies attack your body's own tissues — specifically targeting each cell's nucleus and cause organ and tissue damage.
In most cases, a positive ANA test indicates that your immune system has launched a misdirected attack on your own tissue

12

Method: Immunoassay and Indirect fluorescent antibody (IFA)

• Anti-dsDNA antibody test

target the genetic material in DNA

13

Method: Immunoassay and Indirect fluorescent antibody (IFA)
• Anti-Smith (Sm)

ribonucleoprotein found in the nucleus of a cell, is found almost exclusively in people with lupus
specifically target the genetic material

14

Osmotic Fragility Test

RBC fragility in blood samples in which cells have decreased surface area to volume ratio.
Used to assists with the diagnosis of two hereditary conditions:
• Thalassemia
• Hereditary spherocytosis.

15

Thalassemia

causes your body to make an abnormal form of hemoglobin. Hemoglobin is the protein that allows red blood cells to carry oxygen. If you have thalassemia, your red blood cells are more likely to be destroyed. This can lead to anemia.
Target cells and tear drops

16

Hereditary spherocytosis

causes problems with the outer layer of your red blood cells, distorting their shape. This can also lead to anemia.
looks like filled in red dot

17

Blood Sample for Osmotic Fragility Test

---- Heparinized blood used (green top tube)

--- Blood is mixed with solutions of isotonic saline 0% to 1.0 % concentration and examined for haemolysis by spectrophotometer

--- If red blood cells are more fragile than normal, the test is considered positive

Most normal RBCs remain intact until the % saline reaches about 50%. As the % saline decreases further the amount of leakage or lysis increases. (Positive test)

0.85% lysis starts - fragility whereas normal dont lyse until .66%. if sick destroyed earlier - check the picture