Reticulocytes Flashcards
Definition of Reticulocyte
Immature, non-nucleated RBC with >2 blue-stained granulofilamentous materials (reticulum) after supravital staining
Normal maturation time for reticulocytes in blood
1 day
Normal production of reticulocytes
50 x 10^9/L/day
Purpose of Reticulocyte Count
Effective assessment of RBC production by bone marrow; measure of effective erythropoiesis
Normal reference range for reticulocyte count in adults
0.5 to 1.5%
Normal reference range for reticulocyte count in newborns
1.8 to 5.8% (by 1-2 weeks of age, same as adults)
Increased reticulocyte count
First sign of accelerated erythropoiesis; observed in hemolytic anemias, iron deficiency anemia with therapy, thalassemia, sideroblastic anemia, acute and chronic blood loss
Decreased reticulocyte count
Observed in aplastic anemia and in conditions where bone marrow is not producing RBCs
Conditions associated with increased reticulocyte count
Hemolytic anemias, iron deficiency anemia with therapy, thalassemia, sideroblastic anemia, acute and chronic blood loss
Conditions associated with decreased reticulocyte count
Aplastic anemia, conditions with non-functioning bone marrow
Supravital Stains used for reticulocyte counting
New Methylene Blue, Brilliant Cresyl Blue
Purpose of Miller disk
Calibrated disk placed in the ocular of the microscope
Large Square (A) on Miller disk
Used for counting reticulocytes
Small Square (B) on Miller disk
Used for counting RBCs
Minimum number of cells to count for accurate reticulocyte count
112
Method for reticulocyte count
Routine Light Microscope Method, Calibrated Miller Disk Method, Flow Cytometry
Incubation time for Routine Light Microscope Method
3 to 10 minutes
Amount of blood and supravital stain in Routine Light Microscope Method
2 to 3 drops or around 50 μL each
Reticulocyte counting in Routine Light Microscope Method
Count 1000 RBCs under oil immersion (1000x magnification); reticulocytes counted as both RBC and reticulocyte
Calculation for Routine Light Microscope Method
Retic (%) = No. of Retics observed x 100 / 1,000 RBCs observed
Minimum RBCs to count in small square (B) for Calibrated Miller Disk Method
112 RBCs
Counting rule for reticulocytes in Square B (Calibrated Miller Disk Method)
A reticulocyte in square B is counted as both an erythrocyte and a reticulocyte
Total RBCs counted in Calibrated Miller Disk Method
1,008 RBCs (theoretically)
Computation for Calibrated Miller Disk Method
Retics(%) = (Total retics in Square A / (Total RBCs in Square B x 9)) x 100
Most rapid, accurate, and precise method for reticulocyte count
Flow Cytometry
Principle of Flow Cytometry for reticulocyte count
Counts reticulocytes based on optical scatter or fluorescence after treatment with fluorescent dyes or nucleic acid stains
Test values in Flow Cytometry
Reported in absolute and relative terms
Example of Flow Cytometry equipment
Sysmex R-3500
Sysmex R-3500 uses this fluorescent dye
Auramine O (supravital fluorescent dye)
How reticulocytes are categorized in Sysmex R-3500
Into low-fluorescence, middle-fluorescence, or high-fluorescence regions; less mature reticulocytes show higher fluorescence
Sum of middle-fluorescence and high-fluorescence ratios
“Immature Reticulocyte Fraction (IRF)”
Purpose of Immature Reticulocyte Fraction (IRF)
Indicates early erythropoiesis, a sensitive index of bone marrow erythropoietic activity
IRF reflects
Quantity of reticulocytes with the highest RNA content
Immature Reticulocyte Fraction (IRF) helps to indicate
Ratio of immature reticulocytes to total reticulocytes
To distinguish types of anemias, use of IRF together with _____
ARC
Brilliant cresyl blue in reticulocyte staining
Brilliant cresyl blue stains reticulocytes but shows too much unpredictability for routine use
Adjusting dye to blood ratio for anemia or polycythemia
Increase blood proportion for low hematocrit, decrease for high hematocrit
Time for reticulocyte staining
Staining should not be less than 10 minutes
not critical
Effect of increased blood glucose or heparin to reticulocyte
pale staining
Importance of mixing blood and stain well in reticulocytes
Reticulocytes settle on top of RBCs due to lower specific gravity
Deep purple using NMB, nuclear fragments (DNA)
Howell Jolly Bodies
Light blue-green, denatured and precipitated hemoglobin, usually at the peripheral edge of the RBC
Heinz Bodies
Purple, small cluster of granules, confirmed with Wright or Prussian blue stain, hemosiderin in mitochondria
Pappenheimer Bodies
Greenish-blue, multiple small dots, seen in alpha thalassemia
Hemoglobin H
Actual number of reticulocytes in 1 liter of whole blood
ABSOLUTE RETICULOCYTE COUNT
Corrects reticulocyte count to a normal Hct of 0.45 L/L for anemia degree
CORRECTED RETICULOCYTE COUNT
Refines CRC, indicating erythrocyte production increase in anemias
RETICULOCYTE PRODUCTION INDEX
Computation for ARC
ARC = (reticulocytes (%) x RBC count (x10^12/L))/100 x 1,000
Reference range for ARC
20 to 115 X 10^9/L
CRC formula
CRC = Retics (%) x (Hct in L/L / 0.45 L/L)
RPI formula
RPI = Corrected Reticulocyte Count / Maturation Time in peripheral blood
Hematocrit (%) 40 to 45 Maturation Time (Days)
1
Hematocrit (%) 35 to 39 Maturation Time (Days)
1.5
Hematocrit (%) 25 to 34 Maturation Time (Days)
2
Hematocrit (%) 15 to 24 Maturation Time (Days)
2.5
Hematocrit (%) <15 Maturation Time (Days)
3
RPI
Generally indicates adequate bone marrow response
“RPI >3”
RPI
Generally indicates inadequate bone marrow response
“RPI <2”