LABORATORY ACTIVITY 8 Flashcards
Last stage of immature RBC
Stage between the
orthochromatophilic normoblast and a mature erythrocyte
maturation time in the bone marrow:
2-3 days
maturation time in the circulation:
1 day
First normal immature cell seen in the circulation at a very low value
Presence of other immature cell indicates
dysplasia, cytopenia, etc.
: with remnants/web/hairlike fragments; w/o nucleus
retic
youngest RBC with no nucleus
Retic
: very clear pink cytoplasm; w/o remnant; w/o nucleus
rbc
: w/ nucleus
orthochromatophilic normoblast
Cytoplasmic inclusions coprecipitate with the few remaining [?] to for visually stained [?] with a supravital stain
mitochondria and ferritin masses (also RNA remnants)
dark/blue clusters
: leaves RNA once precipitated
Mitochondria (+nucleus & ribosome)
: completely disappears with the nucleus
DNA
: slow to precipitate (left behind) = RNA remnants
RNA
: iron stores; precipitates in the cytoplasm and binds with the remnants (RNA + iron); used in Hb production
ferritin
cytoplasmic inc:
RNA precipitates (mostly) and Iron precipitates
- a dye that stains living cells and its inclusions; dark blue clusters
Supravital stain
: smear → air dry → stain (methylene blue, eosin, dist water, alcohol)
Preservation: (dies)
RBC
: EDTA-blood is mixed w/ stain → incubation → stain
No preservation: (living)
Retic
: remnants are not visible
Wright stain/Polychrome-stain
: remnants are visible
New methylene blue, Brilliant cresyl blue
Specimen:
WB mixed w/ any anticoagulant
Capillary (w/o anticoagulant)
– preferred; preserves size, shape; least contamination/fragmentation/precipitation
EDTA
Safer (allows multiple smears)
EDTA
o Collected on microtainer w/ graduations
Capillary (w/o anticoagulant)
o 1:1 (stain:blood)
Capillary (w/o anticoagulant)
o performed on babies
Capillary (w/o anticoagulant)
o requires smearing technique (limited to 3 smears)
Capillary (w/o anticoagulant)
SUPRAVITAL STAINS
New methylene blue and Brilliant cresyl blue
Must be filtered daily or before use
New methylene blue and Brilliant cresyl blue
may precipitate in amber bottle (confusion w/ remnant/contamination
New methylene blue and Brilliant cresyl blue
PROCEDURE:
DRY PREPARATION
- Mix equal amounts of [?] in a small test tube.
filtered stain and EDTA-anticoagulated blood or fresh capillary blood
blood : stain
1 ml : 1 ml
- Incubate mixture at [?]
room temperature for 10-15 minutes.
Rodaks: 3-10 mins; Minimum: 10 mins; Maximized for [?] in the lab to stain properly
15 mins
Inversion:
8x
Remix the tube after [?] (to prevent settling or rbc and stain at the bottom of the tube = no retics will be seen)
15 mins
- After incubation, mix thoroughly and prepare a [?]
wedge smear
Prepare at least [?] smears (1 for each stain)
Air dry
2
- Examine smear using [?]. Select an area where erythrocytes are close but not overlapping and reticulocytes appear to be well-stained.
100x objective
Criteria:
PASS (size, length, presence of bubble, dropping marks, consistency of the tail from thick to thin)
: scanning (to avoid rouleaux; tail)
10x
: confirm whether rbc or retic
40x
: counting
100x
- Count the number of reticulocytes in [?] red cells. Reticulocytes should also be counted as erythrocytes.
1000
10 fields with [?] rbc each in the lab
100
At least [?] is retic in the circ
1.5%
- Calculate as follows:
: no Hct and maturation day (count only)
Uncorrected
● measure of erythropoietic ability of the bone marrow in response to anemia
RETICULOCYTE COUNT
With retic:
normal rbc production
Indications:
BM transfusion
Anemia
Adult:
0.5 - 2.5% of total RBC
Newborn:
4.0 - 6.0% of total RBC
Circulation:
0.-5 to 1.5%
Decreased with aging
Adult: 0.5 - 2.5% of total RBC
Higher bm activity
Newborn: 4.0 - 6.0% of total RBC
: anemia = the retic prod is decreased
<0.5
: erythrocytosis = the retic prod is increased
> 1.5
37 retics are found when 1000 erythrocytes are examined (37 retics, 963 erythrocyte). What is the retic count?
3.7%
Interpretation: Increased production of reticulocyte.
● Traditional way of counting that reduces the cells to count
MILLER OCULAR DISC
A disc inserted into the eyepiece
MILLER OCULAR DISC
Permits a less labor-intensive surveying of RBCs
MILLER OCULAR DISC
Count at least 112 RBCs in successive fields (CAP)
MILLER OCULAR DISC
MILLER OCULAR DISC Determine the retic ct using this formula:
MILLER OCULAR DISC
2 squares:
o A (large square) - for counting retics
o B (small square) - for counting RBC
INCREASED
Blood loss
Crisis associated with HA
Subsequent treatment for PA
Folate and iron deficiency
Hypoxia
DECREASED
Aplastic anemia
Aplastic crisis of HA
Chemotherapeutic radiation and induced hypoproliferation
Pernicious anemia
Dec erythropoiesis
: retics are released early from the bm = ↑ retic to compensate for the loss
Blood loss
: rbc is destroyed from the circulation due to and antigen causing hemolysis = ↑ retic
Crisis associated with HA
: treatment of intrinsic factor = = ↑ retic
Subsequent treatment for PA
: lack of oxygen
Hypoxia
hiking in very high altitude: dec oxygen intake due to dec production of rbc
Hypoxia
↓ rbc ct, ↓oxygen, ↓ rbc production = ↓ EPO
Hypoxia
: no rbc
Aplastic anemia
: hemolysis of both rbc and retic
Aplastic crisis of HA
: bm cannot produce rbc and rectic; blast cell is also affected
Chemotherapeutic radiation and induced hypoproliferation
: intrinsic factor deficiency in hydrochloric acid produced by the stomach
Pernicious anemia
absorbs Vit B 12; helps in absorption of Vit B 12 and Folic acid for the maturation of rbc
Intrinsic factor
: bm is not produced efficiently = dec retics
Dec erythropoiesis
Retic differentiates
anemias
Causes of anemia:
- Problem in the bm
- Increased destruction
defective production = anemia
- Problem in the bm
cause: hormone, genetic, chemotherapy
- Problem in the bm
abnormal rbc = decreased retic
- Problem in the bm
normal bm but destructed in circ
- Increased destruction
cause: antigen, viral, para, bacte infection
- Increased destruction
decreased survival of rbc
- Increased destruction
normal rbc = increased retic
- Increased destruction
Hallmark of anemia:
increased retic
SORCES OF ERROR
Interobserver variation in the definition of a retic
Accuracy of counting of the observer (±20%)
Refractile bodies due to poor drying
Precipitates
Others:
-Severe anemia
-Failure to re-mix after incubation, prior to smear preparation
-Bias in the use of Miller Disc grid
o atmospheric moisture may confuse remnant w/ refractile bodies
Refractile bodies due to poor drying
o may resembke dark blue clusters in retic filter stain
Precipitates
o confuse remnant w/ contaminats
Precipitates
o solution: filter the stain before use
Precipitates
o Proportion of dye-to-blood must be adjusted accordingly
Severe anemia
1 ml of blood : 0.5 ml stain
Severe anemia
use the similar inverted L rule applied in hemocytometers
Bias in the use of Miller Disc grids
INCLUSIONS CONFUSED WITH RETICS
Howell-Jolly bodies
Heinz bodies
Pappenheimer bodies
: 1-2 deep purple-colored, dense structures
Howell-Jolly bodies
irregular circular inclusion (not web-like)
Howell-Jolly bodies
: light-blue green (not blue)
Heinz bodies
usually on the periphery of the rbc
Heinz bodiesv
: small iron clusters
Pappenheimer bodies
most common confusion
Pappenheimer bodies
o Solution: prepare 2 smears (retic and wright-stained)
Pappenheimer bodies
o use prussian cytochemical stain blue (preferred) over wright-giemsa
Pappenheimer bodies
: more effective to differentiate Pappenheimer from retic remnant
o use prussian cytochemical stain blue (preferred) over wright-giemsa
FALSELY DECREASED
Understaining (cause: improper incubation)
High glucose levels (can be mask the appearance of RNA remnants)
(cause: improper incubation)
Understaining
(can be mask the appearance of RNA remnants)
High glucose levels
Absolute Reticulocyte Count (ARC) FORMULA
Absolute Reticulocyte Count (ARC) RV
20-115x109 /L
Corrected Reticulocyte Count (CRC) FORMULA
Corrected Reticulocyte Count (CRC) RV
0.5-1.5% (adults) 4.0-0.6% (newborn)
If Hct = 35%, CRC is typically at
25%
If Hct = <25%, CRC is typically at
3-5%
Reticulocyte Production Index (RPI) FORMULA
In anemic patients, RPI should be
> 3
If RPI [?] , adequate bm response is seen
> 3
If RPI [?], an inadequate bm response is seen
<2
40-45
1
35-39
1.5
25-34
2
15-24
2.5
Expression of retic count out of 1000 rbc
Uncorrected/Relative Reticulocyte Count (URC/RRC)
Bias for patients with very low Hct (packed rbc)
Uncorrected/Relative Reticulocyte Count (URC/RRC)
Actual number of reticulocytes in a 1 L of blood
Absolute Reticulocyte Count (ARC)
More preferred method of reporting reticulocyte counts
Absolute Reticulocyte Count (ARC)
More accurate and reliable than URC
Absolute Reticulocyte Count (ARC)
Actual counting of number of retic expressed through the rbc
Absolute Reticulocyte Count (ARC)
Opposite of URC
Corrected Reticulocyte Count (CRC)
Calculated to account for the degree of anemia by using a standard normal Hct of 45% (0.46 L/L)
Corrected Reticulocyte Count (CRC)
URC cannot be reported alone (may be normal out of 1000 rbc, but prbc may be low in actual)
Corrected Reticulocyte Count (CRC)
Correction factor: 45% Hct (to correct the degree of anemia using hematocrit)
Corrected Reticulocyte Count (CRC)
are often present during compensation of anemia
Shift reticulocytes
(w/ more RNA remnants)
Shift reticulocytes
Reticulocytes that have transitioned or shifted from the bone marrow to the circulation earlier than usual
Shift reticulocytes
If [?] is seen in a stained PBS, corrections should be made to account for shift retics.
polychromasia
indication of shift retics
polychromasia
gray-pink, salmon pink, gray, very red
Retics w/ polychromasia
Px hematocrit %, Correction factor (?)
maturation time or days
during cases of anemia, shift retics increase tocompensate for [?] (↓ rbc = no oxygen)
hypoxia
maturation: 2-3 days (bm) ; 1 day (circulation)
Anemia: release from the bm earlier than 2-3 days
To correct the presence of shift reticulocytes
Reticulocyte Production Index (RPI)
shift retics often mature loger than normal usually takes 3 days
Reticulocyte Production Index (RPI)
CF must be with reference to the Hct value of the patient
Reticulocyte Production Index (RPI)
Example: Retic ct: 7.8%, Hct (%) = 30%; Ans: 2.6%
Reticulocyte Production Index (RPI)
Purpose: To determine an adequate bm response in cases of anemia
Reticulocyte Production Index (RPI)
Solution: transplant the blood or transplant the bm
Reticulocyte Production Index (RPI)
OTHER METHODS
had been widely used nowadays
- Flow cytometry
automated: provides absolute and relative counts (unlike in CRC and RPI)
- Flow cytometry
can be assessed using automated methods
- Immature Reticulocytes Fraction (IRF)
Counts retics with more RNA (immature/shift)
- Immature Reticulocytes Fraction (IRF)
Provides CRC and RPI
- Immature Reticulocytes Fraction (IRF)
assessment of erythropoietic activity after chemotherapy or stem cell transplantation (to asses bm function)
- Immature Reticulocytes Fraction (IRF)
is analogous to the RBC MCH
- Reticulated Hemoglobin Content (CHr)
o measurement of Hb in retic
- Reticulated Hemoglobin Content (CHr)
can be used to detect early cases of iron deficiency
- Reticulated Hemoglobin Content (CHr)
o MCH: content Hb of the cell (hypochromic)
- Reticulated Hemoglobin Content (CHr)
Reticulocyte count is very important in determining whether the anemia is caused by
(1) defect in erythropoiesis, (2) hemolysis/ shortened survival
there is an adequate release of the bone marrow
defect in erythropoiesis
there is a normal production of RBC but RBCs die in the circulation
hemolysis
In this case, napipilitan si bone marrow na labas ng labas ng reticulocyte. Thus, mas nag compensate yung bone marrow kay (?) in defect in erythropoiesis.
hemolysis/ shortened survival
a hallmark of anemia.
increased reticulocyte
● Same goes with blood loss when injured, there is also an (?) because the bone marrow needs to compensate for the RBC that was lost.
increased reticulocyte
