Retic analysis and Validation Flashcards
What does a pronormoblast have
Highn: C ration
Round nucleus with nucleoli
high concentration of ribosomes
lots of RNA
able to divide
-dark blue cytoplasm
-found in bone marrow
-biggest
What does a basophilc normoblast have
high N:C ratio
very dark blue cytoplasm
-able to divide
-found in the marrow
dectectable hgb synthesis
-lots of RNA and ribosomes
-dna synthesis
Polychromatic Normoblast what does it have
-N:C ratio starts to even out
-pink cytoplasm that reflects complete hgb production
-last stage of cell able to undergo mitosis
-found in the bone marrow
-increased hgb synthesis
Orthochromic normoblast
-low N:C ratio
-chromatin is condensed
-pink cytoplasm
-residual ribosomes present and RNA/Organelles are degrading
-start of cells not being able to divide
-loses nucleaus
Polychromatic erythrocyte
retic
-no nucleus
-no division
-moves from marrow to circulation
-residual ribosomes and organelles
-increased acidophilia
-no DNA because there is no nucleus
mature erythrocyte
-no nucleus
-no division
- found in circulation for 120 days until removed by spleen
-delivers O2 to tissues
-its biconcave shape helps for optimal O2 exchange
-flexibility allows it to enter small capillaries
Increased Retic count means what
hemolytic anemia
hemorrhage
treated IDA and megaloblastic Anemia
Uremia
-proper BM response
DECREASED RETICULOCYTE COUNT
Ineffective Erythropoiesis as seen in:
pernicious anemia
sideroblastic anaemia
Thalassemia
-Bone marrow failure so there are fewer precursors being released
Insufficient Erythropoiesis
Aplastic crisis
Aplastic anemia
decreased RBC production
Reticulocyte Staining
Wright’s Stain: Reflects Hgb present, residual ribosomes and RNA
Polychromatic erythrocyte
Supravital Stain:
Reticulocyte
MANUAL RETICULOCYTE COUNT
with supravital stain
-cells are alive
-New Methylene Blue or Brilliant Cresol Blue
-Reticulum is precipitated as a dye-protein complex
-The reticulum is RNA in ribosomes looks like a rope with knots you need more than 2 blue/black granules to call it a retic
Reporting retic count
report both relative in SI units and absolute in retics/L whole blood
SOURCES OF ERROR in retic count
refractive artefacts in RBCs caused by moisture in the air and poor drying
Poor mixing – retics float
Poor counting – missing or double counts
Blood and stain are not mixed before being made
Other RBC inclusions that stain supravitally can be mistaken for retics:
Howell-Jolly Bodies
Heinz bodies (stain at periphery)
Pappenheimer Bodies
Reticulocyte & Basophilic Stippling
both have RNA filaments
Reticulocytes:
“Rope with multiple tied knots” formation
the granules are uneven
Basophilic Stippling:
Fine or coarse granule evenly distributed throughout the cytoplasm
-unstable RNA that precipitated evenly
Corrected Retic Count
-in low Hct, the % retic may be falsely ↑ because whole blood contains fewer RBCs.
-A correction is made using the average normal Hct of 0.45 L/L
Assumption: Average HCT is 0.45 L/L
-dependent on anemia
- Patients with 0.35 L/L to have elevated corrected reticulocyte count of 2-3% - That will tell us they’re compensating for their mild anemia
- Patients with <0.25 L/L the count should increase to 3-5% to compensate.
Reticulocyte Production Index (RPI)
-Normal ‘retics’ mature in PB within 1 day
-Retics pushed to PB early due to a hemolytic anemia are called “shift” retics.
-Shift retics are released prematurely from the marrow to compensate for anemia itll take 2-3 for them to lose their reticulum
When assessing the BM response we check Retic count daily because retic count can be falsely increased when there is polychromasia
maturation time is based on a persons HCT
An RPI > 3 indicates a good marrow response
An RPI < 2 indicates an inadequate response
-indicates how good someones bone marrow response is
AUTOMATED RETICULOCYTE COUNT
-whole blood analyzed on a cell counter
-retics measured by flow cytometery; blood mixed with dyes or NA stain and the analyzer measure optical scatter or fluorescence
-auramine O is added to blood
-the more RNA the more fluorescence
-measures forward scatter which corresponds to cell volume/size and RNA content
-retics have high forward scatter and high fluorescence (high RNA count)
Automated Retic Parameters
Immature Reticulocyte Fraction or IRF is replacing RPI
-ratio of immature reticulocytes to total reticulocytes in a sample
-the anaylzer can differentiate between immature retics that were released early and older ones
-newer ones have more RNA - BM response
-can be seperated into low, middle and high fluorescent pops
-so IRF is the sum of the middle and high pop since they have higher RNA wereas low pops are most developed since they have less RNA
-early indication of erythropoiesis and detect early therapy
how can IRF Absolute Retic Count be Used to distinguish diff types of anemias
- Hemolytic anemias have high retic count, increased IRF
-Chronic Renal Disease have decreased retic count, decreased IRF
-Response to Nutritional Anemias have normal retic count, increased IRF
Reticulocyte Hemoglobin Concentration
To assess response to IDA treatment
Reticulocyte Indices
mean reticulocyte volume and distribution width
-early detection and diagnosis of iron deficient erythropoiesis in children
Quality Assurance in the Hematology Lab
Daily validation of methods & equipment
Detailed SOPs with sources of error
Reflex / confirmation testing
Validation of Operator
Licensed MLT
Member in good standing at CMLTO
Trained at an accredited MLS program
Michener
Properly trained on analyzer and proficient in the methods used for testing
Continuing Education
Validation of Analyzer
Run & assess QC regularly
Manufacturer’s QC 1 / shift
Patient QC samples every ? Samples
Westgard rules – check system
Calibrate periodically
Preventive maintenance
Alerts for reagents & electronics
Validation of Specimen:
Volume – Within 10% of stated draw volume
-Underfilled Tubes – FALSELY low RBC counts, HCT, morphological changes
-Overfilled Tubes – Impropoer mixing of anticoagulants . Resulting in PLT clumping or clots
Anticoagulant – Usually EDTA
-Acts as Ca2+ chelator (inhibits clotting), Preserves cellular components and morphology for assessment
Age/ Storage:
EDTA Blood Samples should be analyzed within 6hours of collection and kept at RT
-RBCs, PLTs swell as sample ages. WBCs degenerate
Mixing too aggressively may lead to hemolysis of RBCs»_space; Messed up RBC parameters
Validation of Results:
Rule of 3 – Normal HCT should be 3X of the Hgb +/- 3
-Failure to meet this may indicate sample integrity issues or abnormal RBCs
Linearity Limits – Results proportional to the calculated concentration/activity of the analyte
-Samples above linear range – Sample must be diluted and results need to be multiplied by dilution factor
Analyzer flags – Due to analyzer, specimen or patient abnormalities. Results must be investigated before release
Nonsense Results – Results are so out of range it’s seen as incompatible with life (sometimes due to interfering substances) MCV > 130, MCH/MCHC greatly increased
Which parameters are directly measured?
Which parameters are calculated?
Which parameters are directly measured?
WBC Total Count and DIFF, RBC, PLT, Hgb
PLT volume/ MCV on new analyzers
Which parameters are calculated?
MCV on Michener AcT5diff AL analyzers
HCT L/L=[RBC(1012/L)x MCV (10-15L)]
MCH (pg)= [Hb (g/L)/RBC (x1012/L]
MCHC (g/L)= [Hb (g/L)/Hct L/L]
RDW-CV: derived from RBC histogram
WBC Absolute counts: derived from DIFF & Total WBC counts
Impedance principle:
how the act5 works
How the analyzer counts cells
) Cells enter a conductive diluent
2) The counting chamber has a current applied between the external and internal electrode
3) When cell passes through aperture, this disturbs flow between internal and external electrode – Creates measurable voltage pulse
4) Oscilloscope displays pulses generated by the cells interrupting the current
Number of Pulses is PROPORTIONAL to number of cells counted
Size of Pulses is PROPORTIONAL to size of the cell
RDW – Histogram showing size distribution of cells counted
AcV technology
Absorbance
AcT5 - Tungsten halogen lamp
New Technology- laser optical scatter
AcT5-
Forward scatter (FS): 0°to detect cell volume or size
Side scatter (SS): 90° to detect internal complexity (granules, vacuoles etc.)
Cytochemistry
Diff Fix contains Chlorazole black
Stains granules of Neutrophils, Lymphocytes, Monocytes & Eosinophils
Volume
Focused flow impedance- measures volume of cell (change in resistance as cell passes through flow cell aperture)
Dual focused flow - where cells line up
Has a 5 part differential
Cytochemical staining
Differential Lysis
Technologies of Absorbance
Technologies of Cytochemistry
Coulter Impedance Principle
WBC Diffplot signaling
Based on Cell Size (Volume) and Complexity (Absorbance)
Lymphocytes
Small volume, low complexity/absorbance
Neutrophils
Medium volume, moderate complexity
Monocytes
Large volume, low complexity
Eosinophils
Medium volume, high complexity
X-axis= Absorbance
Y-axis= Volume
Technologies
again in the act 5
Electronic Impedance – Counting WBCs, RBCs and PLTs
Cyanmethemoglobin Method– Hemoglobin Determination
AcV Technology – WBC Diff Determination
Reagent in the act 5
AcT5diff Diluent
counting and differentiating blood cells
-composed of saline
-stabilizes cell membranes for counting and sizing
AcT5diff Fix
Lyses erythrocytes.
Fixes leukocytes.
Differentially stains granules of monocytes, neutrophils, and eosinophils
AcT5diff
WBC Lyse
Lyses red blood cells for the leukocyte count.
Used to differentiate basophils.
AcT5diff
Hgb Lyse
Lyses red blood cells.
Used to determine hemoglobin concentration.
AcT5diff
Rinse
Enzymatic solution with proteolytic action.
Used as a rinsing agent.
First dilution/Hgb bath:
what could go wrong
A dilution is made, and a certain amount is removed and sent for dilution in RBC/PLT bath
Remaining dilu’n is used for Hemoglobin Determination
RBCs are diluted again and lysed with HB Lyse reagent
Hbg released and is converted to cyanmethemoglobin and measured at 540 nm
What could go wrong?
-Lipemia or High WBC Count – Both increase turbidity which affects spectrophotometric reading of Hgb
RBC/PLT Bath
Amount removed from First dilution/Hgb bath is then:
Further diluted using Diluent reagent
RBCs and PLTs are counted and discriminated by electrical impedance (Coulter principle)
RBC count = All blips between 30-300 fL
PLT Count = All blips up to 18 fL
RBC and PLT Count
Develop the RBC histogram, which is needed to obtain the HCT, MCV, and RDW results
MCH calculated from RBC count and HGB value
MCHC calculated from HGB value and HCT
Develop the PLT histogram, which is needed to obtain MPV results
WBC/BASO Bath
False cell counts?
Total WBC count is performed
-lyses RBCs and specifically differentiates between basophils and other leukocytes by volume
Next basophils are counted
Resistance of the basophils to the acidic lytic reagent is the basis for the differentiation of the basophils from the other leukocytes
False cell counts?
-nRBCs – resistant to lyse and can be counted as WBCs
100-400 fL – WBCs
300-400 fL – Likely basophils
RBCs were lysed, PLTs are too small and won’t register on histogram (>20 fL)
Falsely increased RBC count?
-Very high WBC – They’re included in counts from 100-300 fL
-Giant PLTs – Counted as over 30 fL
Falsely increased MCV? MCV calculations req. HCT and RBC
Falsely decreased MCV?
Very high WBC – may affect HCT and RBC – interfering substances
-Very small RBCs (ex. Fragments >20 fL) – They aren’t counted as RBCs. So this can FALSELY INCREASE PLT counts
-RBC agglutination – Increases RBC size so that it’s excluded from RBC count
Falsely decreased MCV?
-Giant PLTs – counted as small RBCs
Red Cell Distribution Width
Tight RDW, most cells are a uniform size. Decreased anisocytosis = 11.4%
Large RDW indicates a large difference between smallest and largest cells therefore increased anisocytosis = 27
Mean Cell Volume (MCV)
MCV RI = 80-100 fL
Right Shift – Expect macrocytes
Left shift – Expect microcytes
Two Distinct Peaks – 2 populations of cells
DIFF Bath
Whole blood aliquot is mixed in DIFF bath with AcT5diff Fix reagent and Diluent
-cells are lysed
-Differentially stains Lymphocytes, Monocytes, Neutrophils, Eosinophils
-Primary focused flow for impedance (volume)
Second focused flow for optical detection (absorbance + cytochem)
Where does the 5-part WBC Differential come from
Combined results obtained from WBC/BASO bath + DIFF bath
Interfering substances
Lipemia
Turbidity affects spectrophotometric reading
- Falsely Increased – Hgb, MCH
-the instrument will show abnormal histrogram
-Corrective Action – Plasma replacement
Interfering substances
High WBC
Increased turbidity affects spectrophotometric reading
-Falsely Increased – Hgb, RBC count,
-Incorrect HCT
-Rule of 3 will not match
-Corrective Action – Manual HCT, Correct RBC count, Recalculate RBC indices, Dilute the sample
Interfering substances
cold agglutinins
clumps exclude it from being counted as RBC
Falsely Decreases –RBCs, MCV
Falsely increases – MCHC
-has a grainy appearance
-instrument can show right shift on RBC histrogram
Corrective Action – Warm specimen to 37C + rerun
Interfering substances
PLT Clumps
Falsely Decrease –PLT count
Falsely Increases – WBC count
-large clumps are counted as platelets
Corrective Action
IF PATHOLOGICAL - Redraw specimen in sodium citrate and multiply result by 1.1
Corrective Action IF COLLECTION ISSUE – Redraw sample in EDTA, the and do 8-10 inversions
Interfering substances
Nucleated RBCs
Lyse resistant so it gets counted with WBCs
Falsely Increase – WBC counts because nucleated RBCs are large so they are counted at WBC
Corrective Action – Make PBF and count number of NRBCs per WBCs to correct WBC count
Microspherocytes/fragments
These are counted with PLTs bc so small
Falsely Decreased – RBC count
Falsely Increased – PLT count
May see LEFT shift in RBC histogram
Corrective Action – Look at PBF and confirm PLT estimate, then possible a manual PLT count
Rule of 3
- Hct should be 3x the value of the Hgb (± 3)
-3 x Hgb/1000 = Hct ± 0.03 L/L
-for Normocytic/Normochromic erythrocytes
-indicate abnormal RBCs or may be 1st indicator of error (sample integrity)
Delta Checks
used more in clinical
-compares current with most recent
-values can stay the same unless there was major intervention
-if the delta check fails this can indicate analytical error or mislabeled sample
-must investigate , sample held until cause is found
Test and what poik should be noticed
Reticulocyte count
Iron studies
Sickle screen
Hemoglobin Electrophoresis
Osmotic fragility
DAT
G6PD Assay
Reticulocyte count-Increased polychromasia
Iron studies - hypo/micro
Sickle screen -Sickle cells
Hbel - N/N anemia with targets, folded cells crystals +/- sickles
Osmotic fragility -Spherocytes
DAT-Spherocytes & polychromasia
G6PD Assay-Bite/Blister cells
