Lecture 6 Flashcards
Reticulocyte:
- is an immature RBC
- cell has no nuclei
- RNA is still present (ribosomes and rough ER) in cytoplasm
- RNA is basophilic
- Retic count is a measure of RBC production (regenerative process) in the bone marrow.
Theory of • Supravital stain
• Supravital stain is a stain procedure that uses living cells.
• Supervital Stains such as New Methylene Blue Or (Brilliant Cresyl Blue)
Stain precipitates the cytoplasmic RNA in living cells
Recticulocytes stain bluish with network of darker granules called a ‘reticulum’
younger RBC have more RNA
Manual Method: -Supravital stain
- Mix equal parts of EDTA blood and stain ( diluted so the angle must be higher and move quicker when making smear)
- Let stand for 15 min
- At least two smear are made of stained cells
- Lay on a flat surface to air dry
- Total 1000 RBCs counted (500 /slide)
- # retics/1000 RBCs is recorded
Formula of Reticulocyte count
- reticulocyte count x100= % retics
total RBC counted - absolute reticulocyte count = % retics x RBCs (Necessary because the retic % does not account for the severity of the anemia)
- Correction of the retic count for anaemia
Corrected reticulocyte count = Patient’s retics % x patient’s Hct / 0.45 (normal Hct for male)
Automated Retic Count
- Done by several types of cell counters
- More precise than manual count (large # of cells are counted)
- Uses special fluro-chrome dye that bind to RNA and stains reticulocyte
- Cells pass through a flow cell
- Use laser light and optical sensor detects and count RNA (flow cytometric)
- Reticulocytes cells are detected by the degree of fluorescence
- Measurements are performed that characterize cell size, shape,and morphology.
New parameters Retic Count
- mean reticulocyte volume (MRV)- average volume of all retic events
- immature reticulocyte fraction (IRF) – retic /all retic events
- high light scatter reticulocyte (HLR) –retic event/all RBC events
Clinical Use for Retic count
- Investigation of anemia
- Monitoring the effect of therapy for anemia (eg iron,(iron deficiency) and vitamin B12 (megaloblastic or pernicious anemia) or folic acid
- Monitoring bone marrow response and the return of normal marrow function following chemotherapy, bone marrow transplant
Reference Value
Adults 0.5% – 1.5%
Newborns 2.5-6.5%
Absolute retic count 50x109/L
Increased Retic count
- Hemolytic anemia
- Acute blood loss
- Response to replacement therapy
Decreased Retic count
- Aplastic anemia
- Marrow suppression by drug, toxin, or viral infection
- Pure red cell aplasia
- Bone marrow replacement (leukemia, lymphoma, carcinoma)
Appearance of Reticulocytes on a Blood Film
- Reticulocytes are basophilic
- Reticulocytes stain blue with Wright stain and haemoglobin stains red
- Called polychromatic cells.(bluish)
Polychromasia
corresponds to aggregate retics in a retic stain
polychromasia can be used as a rough guide to estimate the reticulocyte count
Hemoglobin S
abnormal hemoglobin created by a point mutation at the sixth position on the beta globin chain that results in replacement of glutamic acid by valine. It causes sickling of red cells under conditions of reduced oxygen concentration**
very painful
KNOW THE AMINO ACID CHANGE FROM PREVIOUS LECTURE
Common RBC issues
sickle cell and thallesemia
Sickle Cell Solubility Screen (most common)
- A qualitative test based on the relative insolubility of hemoglobin S compared to other hemoglobin variants.
- Specimen requirement is one lavender top (EDTA) tube.