Blood Smears, Automated Hematology, ans Manual Cell Counts Flashcards
Automated hematology analyzers can electronically (directly) measure (6)?
- RBC
- WBC
- PLT
- MPV
- Hgb
- MCV
From the directly measured paramaters, these quantities can be indirectly calculated (4)?
- Hct
- MCH
- MCHC
- RDW
What WBCs are in a Three-part WBC Differential?
- Granulocytes
- Lymphocytes
- Monocytes
What WBCs are in a Five-part WBC Differential?
- Neutrophils
- Lymphocytes
- Monocytes
- Eosinophils
- Basophils
Newer hematology analyzers may also include what other parameters? (4)
- Relative (%) WBC Count number
- Absolute WBC count number
- Reticulocyte analysis
- Enumeration of nucleated nRBCs
What is a Delta check?
Reviews past patient results
What are some of the instrument flags?
- R flags
- Population flags
- Suspect flags
- Definitive flags
- Quantitative flags
What is the Coulter Principle?
“electrical impedence principle” of counting cells
cells are sized and counted by detecting and measuring changes in electrical resistance when a particle passes through an aperture.
Describe the electrical impedence methodology?
- Blood sample diluted in saline (good conductor)
- Two electrodes: negative external electrode located in blood cell suspension. Posistive internal electrode located in glass hollow tube containing aperture.
- Low frequency DC applied between electrodes
- Electrical impedence (resistance) occurs as cells pass through sensing aperture causing a change in voltage that generates a pulse.
- Pulse is measured
In electrical impedence:
- The number of pulses = _________
- SIze of pulse = ____________
- Number of cells
- Cell volume
True/ False
Electrical Impedence - the number of pulses is proportional to the number of cells counted.
&
The size of the voltage is directly proportional to the volume/ Size of the cell.
True
What are the two counting chambers in the coulter counter?
How is Hgb measured?
- RBC bath - used for RBC and PLT count
- WBC bath - used for WBC and Hgb determination.
- RBCs are lysed and Hbg converted to cyanmethhemoglobin. Hgb measured colorimetrically at 540 nm.
What is Radio Frequency (RF)?
RF resistance is a high-volatage electromagetic current flowing between electrodes that detects cell size based in cell density.
Cell wall acts as a conductor, when exposed to high freq. current.
What information about the cell do conductivity or RF meansurements provide?
Internal characteristics of cells
What uses a high-frequency pulsating sine wave?
Radio Frequency
The cell interior, or nuclear volume is directly proportional to pulse size or change in _____ _____ resitance.
Radio Frequency
Describe Optical Light Scatter methodology?
- Blood sample diltuted w/ isotonic diluent.
- Cells passed through quartz flow cell on which a beam of light (laser) is focused.
- Light scatters as cells pass through sensing zone.
- Photodetectors sense and and collect scattered light and convert them into electrical pulse.
- Number of pulses are directly proportional to number of cells passing through sensing zone.
What cell information is determined from Optical light scatter? (5)
- Cell count
- Size
- Cell structure
- shape
- reflectivity
What are the advantages of automated lab methods?
- Reducted lab cost
- Faster turnaround time
- Improved accuracy and precision
- Fewer manual smears
- Measurements, counts and differentials done at same time
- Calculated corrections done by newer analyzers
- Delta checks
- Flagging system
- Numerical and graphical recording
What are the three types of histograms used?
- RBC
- WBC
- PLT
What do RBC Histograms show? What does this coincide with?
Normal size (anisocytocis) or any other particles in the RBC size range.
Coincides directly with MCV.
Particles with cell volumes greater than 36 fL are identified as?
RBCs
Whay is on the X - and Y - axis of an RBC Histogram?
- X - axis is relative size
- Y - axis is relative number
What does a normal RBC Histogram look like?
- Almost symmetrical
- Bell curve with single peak
Abnormal RBC Histogram:
Shift to the left?
Shift to the right?
Left shift = average RBC cell size is below normal (microcytic anemia)
Right shift = average RBC size is above normal (macrocytic anemia)
What is the normal (refrence) range for RBCs?
MCV = 80 - 100 fL
What does a WBC Histogram enable you to compare?
- A way to compare size and population of patients WBCs with normal population.
A WBC Histogram is used as a screening tool for what?
Used as screening tool to detect hematologic diseases
A normal WBC Histrogram will show how many distinct poulations of WBCs?
3
- Lymphocytes
- Mononuclear cells
- Granulocytes
What are the reference ranges on normal WBC Histrogram for the distinct poulations of WBCs?
- Lymphocytes 35 - 90 fL
- Mononuclear cells 90 - 160 fL
- Granulocytes 160 - 450 fL
What other cells may be included in a WBC histogram?
- Blasts
- other immature cells - promyelocytes and myelocytes
What information is obtained from a PLT Histogram?
- Size of PLTs
- Helps identify giant PLTs and thrombocytopenia
What particles are 2 - 20 fL?
PLTs
What must be obtained for a valid count in a PLT Histogram?
What are the criteria for a fitted curve?
a fitted curve, 0 - 70 fL
- PLT count above 20,000 microliter
- Log-normal distribution
- No PLT count vote-out
What must be done when a fitted curve is not obtainted for a PLT Histogram?
A manual PLT determination.
What is the reference range for RDW?
11.5 - 14.5 %
What is and how do Cold Agglutinins affect automated cell counting?
- antibodies produced by a person’s immune system that mistakenly target red blood cells (RBCs). They cause RBCs to clump together in cold temperatures.
- Falsely decrease RBC count and increase MCH (average weight)
What is and how does Lipemia affect automated cell counting?
- Excess fat in blood
- Causes turbidity and falsely elevated Hgb and MCH (average weight)
What is and how does Icterus affect automated cell counting?
- Yellow pigentation of body fluids caused by jaundice
- Causes turbidity and falsely elevated Hgb and MCH (average weight)
Where are instrument limitations defined?
operations manuals and SOPs
What is a common method limitation?
Inability to distinguish cells from particles or cells fragments of the same size.
Interfering subtances: Variant Hgb / Lysis resistant RBCs
(Variant Hgb can make RBCs resistant to lysis and counted as WBCs)
Affected results?
Indicator?
Corrective action?
- Increase WBC and decrease Hgb
- Interference with WBC count on histogram
- Manual dilutions allowing incubation to assist lysis
Interfering subtances: Microcytes and schistocytes
What results are affected?
Indicator?
Corrective action?
- Increase PLTs and decrease RBCs
- Left shift on RBC histogram
- Examine peripheral blood smear for prescence of abnormal cells and determine correct counts.
Interfering subtances: Cold Agglutinins
Affected results?
Indicator?
Corrective action?
- Decrease RBC, Increase MCV, MCH, MCHC
- Right Shift on RBC histogram
- Warm blood to 37 degrees if cold agglutinin present
Interfering subtances: Significantly increased WBCs (leukemia)
Affected results?
Indicators?
Corrective action?
- Increase Hgb and RBC. HCT incorrect
- Rule of three violated. WBC count above normal
- Dilute and do manual cell count
Interfering subtances: PLT Clumps
Affected results?
Indicators?
Corrective actions?
- Decrease PLTs and Increase RBCs
- PLT flag, abnormal histogram
- Review slide to confirm clumps, warm and agitate specimen to disperse PLTs. Recollect w/ sodium citrate (correct for diltion for multiplying by 1.1)
Interfering subtances: Agglutinated RBCs
Affected results?
Corrective action?
- Decrease RBC
- Review slide for confirmation of giant PLTs. perform manual cell count.
Interfering subtances: Giant PLTs
Larger than normal PLT counted as WBC
Results affected?
Indicator?
- Decrease PLT and increase WBC
- PLT Flag, abnormal histogram
Interfering subtances: PLT Satelitetism
PLTs adhere to and surround neutrophils. Caused by PLT cold agglutinins in prescence of EDTA.
Affected results?
Indicator?
Corrective action?
- Decreased PLTs
- PLT Flag, abnotma histogram.
- Recollect w/ sodium citrate to rule out reactivity to EDTA
Interfering subtances: Increased or abnormal plasma proteins
Lysing reagens precipitate plasma proteins. Precipitates may be counted as WBCs
- Affected results?
- Corrective action?
- Increased WBC
- Hgb x 3 NOT equal th Hct +/- 3
- Replace plasma with normal saline or perform manual cell count.
Interfering subtances: Lipemia and Icterus
Tubidity causes interference with colorimetric reading
- Affected results?
- Indicator?
- Corrective action?
- Increased WBC
- Hgb x 3 NOT equal th Hct +/- 3, Abnormal histogram
- Replace plasma with normal saline or perform manual cell count. Cyanmethemoglobin analysis with patient’s plasma as blank.
Interfering subtances: Hemolysis
Red blood cells lysed
- Affected results?
- Indicator?
- Corrective action?
- Decreased RBC and decreased Hct
- Hgb x 3 NOT equal th Hct +/- 3, Abnormal histogram
- Obtain new specimen
What is Reflex testing?
- Machine flags abnormal results
- Additional testing may be required
What are Multivariate checks?
What does it include?
- Interrelations between different tests and the measurements within a test are compared to established limits
- Review of peripheral blood smear
- Rule of three
- correlate erythrocyte indices with automated blood count.
Pre-analytical factors?
- specimen identification
- proper collection methods
- time
- specimen integrity
Analytical factors?
- Reagents
- Equipment
Post-analytical factors?
- Documentations
- calculations
What are Standards / calibrators?
Solutions with a known amount of analyte and used to calibrate a method.
Ex: Hgb standars is 12g/100mL
What are Controls used for?
Monitor the performance of a method after calibration
Who provides performance goals and standards for hematology analyzers?
Clinical Laboratory Standards Institute
What is Calibration?
Established by manufacturer to ensure accuracy and precision.
When is an analyzer calibrated?
- Installation
- Every 6 months
- Verification:
- After part replacement
- After instrument repair
True or false: All laboratories must establish/ verify the manufacturer means or “true value” before using a new lot of controls?
True
How are manufacturer (“true values”) verfied for a new lot of controls?
Running 3 control levels each shift, every 8 hours, and/or daily before patient results are reported.
Controls must be evaluated daily for? (3)
- Acceptability
- Ensuring instrument is in good working condition
- Validating patient results before being reported
True or False:
If control is not within acceptable range, patient results cannot be reported?
True
When would you rerun a control?
To verify an error has been resolved. So patient resuls can be run and reported.
What are the important components of QC? (3)
- Standards/ calibrators
- control materials
- statistics
What other times would you run controls? (4)
- New reagent lot #
- after calibration
- unusual shift/trend in patient results
- after maintenance
When should a Microcontainer be processed?
Within 1 hour of collection
How must all microcontainers be checked for clots?
With an applicator stick
Delays in speciment processing may lead to? (2)
- Falsely macrocytic RBCs
- Degeneration of WBCs and platelets
Prolonged storage in EDTA can cause what in WBCs?
Small evenly distributed vacuoles
What is the most common method for slide preparation?
What is another method?
- Push-Wedge (most common)
- Spun
What angle is the spreader slide held at for the Push-Wedge Method?
30 - 45 degree angle
What stain is used in slide preparation?
Wright Stain
What objective is used to conduct an initial scan prior to performing any specific microscopic examination?
10X objective
During the initial scan what must the slide be checked/observed for? (4)
- Properly stained
- even cell distribution
- platelet clumps
- Rouleaux formation of RBCs
How many fields are scanned of how many cells for RBC morphology?
10 oil immersion fields of 200 - 250 RBCs
RBC Morphology is evaluated for? (4)
Size - Anisocytosis
Shape - Poikilocytosis
Hgb conc.
immature cells (if present)
What are the componenents of a WBC differential count? (4)
- WBC Morphology
- RBC Morphology
- PLT estimate
- WBC estimate
How many cells are counted in a WBC differential?
100
When would you correct the Total WBC count?
When more than 6 nRBCs are seen in a 100 cell differential.
Platelet Estimate: Each PLT seen represents approximately how many PLTs?
20,000 PLTs/uL
Platelet Estimate (PE) =
(total # PLTs counted in 10 fields / 10) x 20,000
What objective is used for PLT estimates?
100X
When comparing PE results with automated PLT, if results are significantly different what should you check for?
How is this corrected?
- PLT clumps - invalidate automated count
- Request new speciment to repeat test
What objective is used for WBC Estimates?
40X dry
50X oil, if available
How many fields are counted in a WBC estimate?
What is the WBC estimate formula?
10
WBC Est = average # WBCs x 2000
