Week 1 Flashcards
What are the requirements for CBC test and PBF
-8-10 inversions
-must be filled 10% of fill volume
-under filling causes RBC/WBC shrinkage, crenated RBC and WBC membrane damage
-over filling would mean there isnt enough anticoag so it can cause platelet clumping
-sample needs to be analyzed in 5 hours or 4 in micotrainer
-if not done within the time frame then the following can happen
RBC morph-crenetation
WBC morph- vacuolization , necrosis
MCV/HCT = increased due to swelling
WBC- decreased due to dying cells
what do automated counter use to do the tests
-combination of hydraulics and pneumatic- vacuum and pressure
-light scatter, staining and conductivity
what is electronic impedance (resistance)
- detecting and measuring changes in electrical resistance that occurs when cells move through a small apeture
-the impedance is the current between two electrodes that produces voltage changes
-what the coulter principle is based off of
-counts WBCs, RBCs, and PLTs
what is optical scatter
-using light source interference to differentiate and enumerate cell types like in flow
what is the coulter principle
-when blood is diluted in isotonic saline and the cells pass through a narrow aperture displacing their own volume to increase resistance - or cause a voltage pulse
-the charge in the internal electrode is positive and the external electrode is positive
- each pulse is a cell, all the pulses together is the sum of all the cells present
-the height of pulse correlates to the cell size
-each pulse is categorized according to volume producing a histogram
-coutler principle does RBC count, PLT count and hematocrit
-however if you do a diff lysis with coulter principle you can do a WBC count, basophil percentage and basophil count
how is a WBC differential done
in a flow cyto or flow cell
-Reagent lyses RBCs and differentially stains Neuts, Lymphs, Monos and Eos
-basophils can be identified through the coulter principle
- cells are suspended in fluid and using hydrodynamic focusing they pass in front of the light source one by one through dual focused flow where they are categorized by size, complexity and staining
-the more scatter there is the large and granular the cell is
-the system uses lasers as a light source and fluorescence detectors- forward and side scatter detected
lymphs are small not complex
neuts - medium size and complex
Mono- large and low complex
Eos- medium volume and high complex
how is the scatter seen
-a diffplot is created with x axis= absorbance and y= volume
-different plot regions represent different WBC populations
what is the AcV technology from AcT5 does the diff and counts
A is Absorbance which needs a tungsten holegen lamp and forward scatter to detect cell volume, size and complexity
-new tech adds many angles of light scatter
C- cytochemistry which helps in diff staining of WBCs with formic or chlorazol black dyes
V- volume which has dual focused flow so it makes the cells line up one by one in the diluent .
Focused flow impedance- counts and measures the volume of the cell
how is the Sysmex XN Series different from the AcT5
WBC are tested by fluorescent staining not impedance like AcT5
HGB - sodium lauryl sulfate
HCT- cumulative RBC pulse height
have a 6 part differential
AcT5 reagents
diluent - counting and differentiating RBC made up of saline
-dilutes whole blood
-stabilizes cell membranes for accurate counting and sizing, rinses components between analyses
Act5diff fix- lyses RBC
-fixes leukocytes
-stains mono, neut, and eos granules NOT baso
Act5diff WBC lyse- lyses RBC for leuk count and used for differentiating basophils
Act5diff hbg lyses- Lyses RBC to determine HGB concentration
Act5diff rinse - enzymatic solution that has proteolytic action and can be used to rinse
how does the Act5 diff analyze samples
- need 30-53 ul for CBC or diff panel which is then divided into aliquots and mixed with saline.
-a sequential dilution system SDS is used to make the dilution series
-each dilution is sent to a “bath” for analysis (Rince, hgb bath, diff bath, RBC/PLT bath, WBC/Baso bath)
-counts are timed and done in duplicate
-system uses flags to notify of off results prior to reporting
what happens in the first Dilution / HGB bath
-first an aliquot is removed and sent to the RBC/PLT bath
-remaining is for HGB determination
-RBCs diluted and lysed with HB lyse reagent, HBG is released and converted into cyanmethemoglobin and measured by spec
what happens in the first RBC and PLT bath
- diluted again with diluent reagent
-the RBC and PLT are counted by the coulter principle
-plt up to 18 fL and RBC upto 30-300 fL
-this dilution makes up the PLT and RBC histogram that helps to get the HCT, MCV, RDW and MPV results
what happens in the WBC/BASO bath
-total WBC is performed as reference
-the whole blood is mixed with WBC lyse reagent which lyses the RBC and able to differentiate specifically between basophils and leukocytes by volume
-the basophils are then counted
-because the basophils are resistant to acidic lytic reagent they can be differentiated from other leukocytes
-WBC/BASO histogram created to determine total leukocyte count and basophil percentage
-reference WBC count
what happens in the DIFF bath
2nd WBC count
- whole blood is mixed in the DIFF bath with FIX reagent and diluent
-RBCs get lysed, WBC get fixed and are stained differentially
-measured via AcV with dual focused flow where the primary is impedance (volume) and secondary is for optical detection (absorbance +cytochem)
-the wbc/baso bath and DIFF bath are compared before reporting and if the counts dont match they wouldnt be reported
-this is where the 5 part diff comes from the combination of both
when will the machine flag
-abnormal size and chromasia
-high/low counts
-clumping
-NRBCs
Parameter (Instrument) Flags
R (Region)
V (Vote out)
(WBC)*
(Diff)*
++++ (result above linearity)
NOT RELIABLE - HOLD
what are patient range flags
H-Result is above the patient limit Reportable Result
L -Result is below the patient limit Reportable Result
what are action range flags
HH Result is above the action limit Exceeds ACTION LIMIT
LL -Result is below the action limit Exceeds ACTION LIMIT
what is the rule of 3
-when you get a patient result you check HGB and HCT where the HCT should be 3x the HGB
-only for normochromic/normocytic erythrocytes
-if the rule stands then go forward with validation
-if it fails then you investigate . Check sample integrity (hemolysis, lipemia), if the sample is valid (hypo or micro present)
after checking the rule of 3 what is the next step
-look at the parameters, check the RI, action limits, delta checks, linear limits, if there are any flags present
-if there are critical values , prepare a smear and inform the physician based on findings
after checking the rule of 3 , and parameters what is the next step
-look at the histograms and diffplot
-the histogram should start and end at baseline with normal curve
-the DIFFplot should show distinct populations and only 4 pop not baso
CBC – HGB, MCH & MCHC are all ↓
what is happening
-low HGB due to decreased production
-increased central pallor -hypo chromasia
-the decreased internal volume can cause shape changes
HGB is Low & RBC is Low MCH & MCHC
are Normal
Error in BM production
Early destruction
Loss of blood
Normochromic / Normocytic
the RBCs appear normal (size and colour) on the PBF,
‘Hypochromic / Microcytic
RBCs appear small and with exaggerated central pallor on PBF,
RBC Distribution Width (RDW) increased conditions
-increases are important but decreases are not
-even if MCV is normal the difference in RDW can let us know about aniso
‘Normocytes’ & Microcytes MCV Decreased
‘Normocytes’ & Macrocytes MCV Increased
Microcytes & Macrocytes MCV Normal
Microcytes & Reticulocytes MCV Normal
CBC & PBF Correlation
-you must compare auto to the manual and if there is over a 10% difference you report the manual
-take the +/- 10% with the auto and then see if your manual falls within the range
Manual Diff Neut 45% - Auto Diff Neut 55%
55/10 = ± 5.5
55 – 5.5 = 49.5
55 + 5.5 = 60.5
what is the Relative Differential
the ratio of each type of WBC
what is the Absolute Differential
when the relative is coverted to a total number of that SPECIFIC leukocyte and then you multiply by the total WBC count
Relative Neutrophil 0.50 ratio
WBC count of 20.0 x 109/L
Relative x WBC = 0.50 x 20.0 = 10.0
10.0 x 109/L
