6. Reticulocytes Flashcards
These are blue-stained, granulomatous materials that are made of residual RNA present in immature non-nucleated RBCs
Reticulum
Immature non-nucleated RBCs which contain >2 reticulum after staining with supravital stain
Reticulocytes
Last stage in RBC development that still produces hemoglobin
Reticulocyte
If Wright stain is used to stain reticulocytes, no reticulum are seen and therefore reticulocytes are called (2)
diffusely basophilic erythrocyte
polychromatophilic erythrocyte
Normal maturation time for reticulocytes in blood
1 day
usual number of reticulocytes produced per day
50 x 10^9/L per day
This is a measure of effective erythropoiesis because it permits effective assessment of RBC production by the bone marrow
Reticulocyte count
Normal reference range for Reticulocyte count in adults
0.5-1.5%
Normal reference range for Reticulocyte count in newborns
1.5-5.8%
The reference age for reticulocyte count in newborns (1-2 weeks of age) are
the same as adults
Terms for increased reticulocyte count
reticulocytosis
polychromasia
polychromatophilia
This is considered as the first sign of accelerated erythropoiesis
Reticulocytosis/Polychromasia/Polychromatophilia
Accelerated RBC production results to polychromasia and is observed in (5)
hemolytic anemia
IDA receiving Fe therapy
sideroblastic anemia
thalassemia
chronic and acute blood loss
A term for decreased reticulocyte count
Reticulocytopenia
This is observed in aplastic anemia and in conditions in which the bone marrow is not producing RBCs
reticulocytopenia
T/F
New Methylene blue is chemically different from Methylene blue.
True
Supravital stains used in staining reticulocytes
New Methylene Blue
Brilliant Cresyl Blue
2 components of New methylene blue and functions
sodium oxalate: prevents coagulation
sodium chloride: provides isotonicity
components of BCB and functions
sodium citrate: anticoagulant
sodium chloride: provides isotonicity
The supravital stain that is more preferred in staining reticulocytes
new methylene blue
T/F
Medical technologists can still count reticulocytes even without using Miller disk
True
What anticoagulant is used in the collecting tube for reticulocyte count spx
EDTA
A calibrated disk placed in the ocular of the microscope
Miller disk
Large square/Square A is used for
counting reticulocytes
Small square/Square B is used for
counting RBCs
Square B’s size is _____ of Square A
1/9
Minimum number of RBCs that should be counted in Small square (B)
112
Square B’s size is 1/9th of Square A and the minimum number of RBCs that should be counted in SB is 112. Therefore, 112 is multiplied by
9
112 multiplied by 9 is
1008
Methods of counting reticulocytes (3)
Routine light microscope method
calibrated miller disk method
flow cytometry
Procedure of routine light microscope method in reticulocyte counting
- Equal parts blood + supravital stain (2-3 drops/50 uL) in a tube.
- Incubate for 3-10 mins at room temp
- Remix
- Prepare 2 blood smears
- Have another MT check the other smear.
- Count 1000 RBCs in the field where RBCs are near each other without overlapping. Also count reticulocytes as RBCs (included in the 1000).
In routine light microscope method for reticulocyte count, 2 blood smears are prepared and examined by 2 MTs. The Retic counts should agree within
20%
Formula for routine light microscope method for reticulocyte count
RC = (# of retics counted/1000) x 100
Formula for routine light microscope method for reticulocyte count
RC = (retics counted/1000) x 100
RC formula for calibrated miller disk method
RC= (total retics SA / total RBCs in SB x 9) x 100
Most rapid, accurate, and precise method for reticulocyte count
flow cytometry
In flow cytometry, reticulocytes are counted on the basis of _______ after treatment with fluorescent dyes or nucleic acid stains
optical scatter or flourescence
In flow cytometry, the test values are reported in
absolute and relative terms
Example of a machine that uses flow cytometry to count reticulocytes
Sysmex-3500
Sysmex-3500 uses what supravital fluorescent dye
auramine O
In flow cytometry, reticulocytes fall into — regions
low-, middle-, high-fluorescence
In optical scatter, less mature reticulocytes fall into
high-fluorescence regions
IRF means
Immature reticulocyte fraction
the sum of the middle-fluorescence and high-fluorescence ratios
IRF
T/F
IRF is the most sensitive index of bone marrow erythropoietic activity. However, it does not demonstrate an early indication of erythropoiesis.
F
IRC is used together with ____ to distinguish types of anemias
absolute reticulocyte count
IRF indicates the ratio of immature reticulocytes to —- in a blood sample
total reticulocytes
BCB also stains reticulocytes. State the disadvantage of BCB in staining retics
Shows too much unpredictability in staining for routine use
If a px is very anemic or polycythemic, the proportion of dye to blood should be adjusted. How?
anemic/low Hct: larger portion of blood to dye
polycythemic/high Hct: smaller amount of blood to dye
T/F
The time allowed for staining of the reticulocyte is critical. It should not be more than 10 minutes.
First statement is false. Second statement is true.
2 factors that can cause the reticulocytes to appear pale
use of heparin as anticoagulant
increased blood glucose
2 factors that can cause the reticulocytes to appear pale
use of heparin as anticoagulant
increased blood glucose
The blood and the stain should be mixed well prior to smearing. Why?
reticulocytes have lower SG compared to RBCs, therefore they settle on top of RBCs in the mixture
Color of Howell-Jolly bodies when using NMB
deep purple
color of heinz bodies when using NMB
light blue green
color of pappenheimer bodies when using NMB
purple
color of hemoglobin H bodies when using NMB
greenish-blue
Other RBC inclusions stained by NMB (4)
howell-jolly bodies
heinz bodies
pappenheimer bodies
hemoglobin h bodies
This RBC inclusion still has nuclear fragments (DNA) present
howell-jolly bodies
rbc inclusion that is usually present at the peripheral edge of the red cell, and contains denatured and precipitated hemoglobin
heinz bodies
Generally appear as several granules in a small cluster. Hemosiderin in mitochondria.
Pappenheimer bodies
Stains used to confirm the presence of pappenheimer bodies
wright stain
prussian blue
multiple dots in rbc
hemoglobin h
hemoglobin h is seen in what condition
alpha thalassemia
3 methods of reporting reticulocyte count
absolute reticulocyte count
corrected reticulocyte count
reticulocyte production index
This is the actual number of reticulocytes in 1 liter of whole blood
ARC
ARC formula
ARC= [retic (%) x rbc count (x10^12) / 100] x 1000
Reference range for ARC
25-75x10^9/L
ARC and IRF of px with hemolytic anemia
both increased
ARC and IRF of px with hemorrhage
both increased
ARC and IRF of px with chronic renal disease
both decreased
ARC and IRF of px with chronic renal disease are both decreased. why?
damaged kidneys cannot produce EPO to stimulate erythropoiesis in the BM
ARC of px’s early response to tx in nutritional anemias. why?
normal to low;
ARC is not a sensitive index
IRF of px’s early response to tx in nutritional anemias
increased
3 other terms for CRC
Reticulocyte Index
Hematocrit Correction
Poor Man’s Bone Marrow Aspirate
There is a need for CRC since the percentage of retics may falsely increase due to (2)
early release of reticulocytes into the bloodstream
the number of mature rbcs in the circulation is decreased
The CRC corrects the observed retic count to a normal Hct of _____ to allow correction for the degree of the px’s anemia
0.45 L/L
the average normal Hct for both male and female
0.45 L/L or 45%
CRC formula
CRC= Retic (%) x (px Hct / 0.45 L/L)
Reticulocyte production index is also known as
shift correction
Provides a further refinement of the CRC
RPI
A general indicator of the rate of rbc production increase above normal in anemias
shift correction
RPI formula
RPI= CRC/maturation time in peripheral blood
maturation time for px with Hct of 40-45%
1.0 day
Hct (%); Maturation time (days)
40-45 ; 1.0
35-39; —–
——–; 2.0
15-24: —–
——-; 3.0
1.5
25-34
2.5
<15
If RPI is >3,
adequate BM response
If RPI <2,
inadequate BM response
Case:
29/M px
Hct: 28%
RC: 5.0%
Calculate CRC and RPI
CRC: 3.11%
RPI: 1.6 –> inadequate BM response
