Automated hematology
# of Pulses
# of Cells
Radio Frequency (RF)
*Cell density* measurement determined by *changes in electrical resistance*.
Platelet Histogram: Fitted Curve
0 - 70 fL must be obtained.
RDW Reference Range
11.5 - 14.5%
Controls run at least _ levels.
2
Most labs perform _ control levels.
3
Particles with cell volumes greater than _ fL are identified as RBCs.
36
Hemoglobin is measured colorimetrically.
540 nm
MPV Reference Range
7.8 - 11.0fL
MCV Normal Range
80 - 100 fL
PDW Reference Range
> 20%
If the fitted curve is not obtained...
A manual platelet determination must be done.
Commercially Preserved RBCs
Adequate only for several days at room temperature and about 4-6 weeks when refrigerated.
Multivariate Check
An interrelation between different tests and the measurements within that test are compared to established limits.
Shift to the Right
Average size of the patient's red cells are above normal range; Macrocytic.
Shift to the Left
Average size of the patient's red cells are below normal range; Microcytic.
Electrical Impedence
Based on the detection and measurement of the changes in electrical resistance produced when cells pass through a small *aperature*.
Synthetic RBC Controls
Being researched for future applications.
Scattergram: Reticulocyte Counting
Blood cells are stained with fluorescent dyes such as *Thioflavin* (T) and *Pyronin* (Y).
Hematology Analyzers
Calibrated most commonly for initial setup, major repairs, and as specified by local SOP.
RF Pulse Size
Can determine N:C ratio, nuclear density, and cellular granulation.
RF Pulse Size
Cell *Interior Density*
Optical Light Scatter
Cell countng and differentiations by the *detection and measurement of interference* (scattered light) in a *laser beam* caused when a cell passes through a *sensing zone*.
Instrument Limitation
Cell fragments may be counted as platelets.
Forward-angle light scatter (0°)
Cell volume/size
Optical Light Scatter
Cells flow in a single stream through a *sensing zone* and unterrupt a beam of light *scattering* in all directions.
Granulocytes
Cells measuring between *160 - 450 fL*.
Lymphocytes
Cells measuring between *35 - 90 fL*.
Mononuclear Cells
Cells measuring between *90 - 160 fL*.
Electrical Impedence
Cells suspended in an electrically conductive *diluent* (saline) are pulled through an *aperature* in a glass tube within a counting chamber.
Size of Pulses
Cellular Volume
Orthogonal ligh scatter (90°)
Cellular complexity
If a Flag Occurs:
Check for unusual cell distributions.
Interfering Substance
Cold Agglutinins
Multivariate Check
Correlation of erythrocyte indices with automated blood count
Electrical Impedence
Coulter Principle
Platelet Histogram Criteria for a Fitted Curve
Count above 20,000/µL
Leukocyte Differentiation
Counts and classifies neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
RBC Histogram
Created by plotting MCV on the X axis of the graph.
RBC Histogram
Created by plotting the relative number of RBCs on the Y axis.
RBCs are lysed in the coulter cell counter. Hemoglobin is converted to...
Cyanmethemoglobin
Hemoglobin Reference Method
Cyanmethemoglobin Standard
Platelet Satellitism
Decreased PLT
Giant Platelets
Decreased PLT, Increased WBC
Platelet Clumps
Decreased PLT, Increased WBC
Agglutinated RBCs
Decreased RBC
Hemolysis
Decreased RBC, Decreased HCT
Cold Agglutinins
Decreased RBC, Increase MCV, Increase MCH, Increase MCHC
Flag Type: Definitive
Defines the *limit* of normal values. Increase or decrease for all CBC parameters based on numeric limits set by lab.
Forward-angle light scatter (0°)
Dependent on the size of the cell.
Indirect Platelet Calculations
Derives Mean Platelet Volume (MPV)
Indirect Platelet Calculations
Derives Platelet Distribution Width (PDW)
Idirect RBC Calculations
Derives Red Cell Distribution Width (RDW)
Amount of light scattered...
Determined by intermal complexity of each WBC.
Electrical Impedence: Hemoglobin Concentration
Determined by measuring the optical density of cyanmethemoglobin.
Cyanmmethemoglobin
Directly proportional to the concentration of hemoglobin found in the sample.
Principle of Automated Cell Counting
Electrical Impedence
If a Flag Occurs:
Examine blood smear.
In a counting, a low frequency electrical current is applied between two electrodes. This electrode is located in the blood cell suspension.
External (Negative)
Flag Type: Suspect
Flags abnormal cell populations or distributions.
Optical Light Scatter
Flow Cytometer
For Duplicate Specimen Testing, use...
Fresh, whole patient blood less than 1 hour old.
Indirect Platelet Calculations
Gives information about platelet production in your bone marrow.
Flag
Has an upper/lower threshold (preset by the user).
Platelet Histogram
Helps identify *Giant Platelets* and *Thrombocytopenia*
Reference Method for RBC, WBC, and PLT counts
Hemacytometer Chamber Counts
Electrical Impedence: Indirect Calculations
Hematocrit, MCH, MCHC, RDW, and MCV.
Electrical Impedence results reported through...
Histograms
Interfering Substance
Icterus
Advantage(s) of Control Material
Ideally should be similar to fresh blood in all aspects. Increased shelf-life.
Flagging System
Identifies Abnormalities in WBCs, RBCs, and PLTs.
Background Checks
Important to identify interference caused by extraneous particles that may be counted as platelets.
Patient Controls
Impractical for use as a QC product to monitor shifts and trends.
Benefit of Automated Counting Systems
Improved accuracy and precision
Benefit of Automated Counting Systems
Improved turnaround times
Scattergram: Monitoring T-Helper Cells
In HIV patients.
Instrument Limitation
Inability to distinguish cells from particles or cell fragments.
The most common cause of error when using automated cell counters.
Inadequate mixing of samples prior to testing.
Lipemia and Icterus
Increased Hgb, Increased MCH
Significantly Increased WBCs (leukemia)
Increased Hgb, Increased RBC, Incorrect HCT
Microcytes, Schistocytes
Increased PLT, Decreased RBC
Increased or Abnormal Plasma Proteins
Increased WBC
Abnormal Hemoglobins/Lysis Resistant RBCs
Increased WBC, Increased Hgb
Flag Type: Condition
Indicates whether WBC, RBC, or PLT populations are normal or abnormal.
Idirect RBC Calculations
Indication of the degree of anisocytosis.
Disadvantage(s) of Control Material
Instability of RBC components due to swelling and the lysis of cells. Failure of Control RBCs to mimic properties of fresh RBCs.
In a counting, a low frequency electrical current is applied between two electrodes. This electrode is located inside a hollow glass tube.
Internal (Positive)
Interfering Substance
Lipemia
Platelet Histogram Criteria for a Fitted Curve
Log-normal distribution
Scattergram: Screening for Blood Parasites
Malarial RBCs contain DNA and will fluoresce.
Hematocrit Reference Method
Manual Microhematocrit
Scattergram: Screening for Blood Parasites
Mature erythrocytes containing no DNA do not fluoresce.
Manual Hematocrit
May be *1.3-3.0%* Higher
If a Flag Occurs:
May indicate an instrument problem.
Non-Unimodel Curve
May reveal the presence of an underlying disease of condition (e.g., cold agglutinin disease, erythrocyte fragments, recent transfusion).
Manual Hematocrit
Measurement includes *trapped plasma* between the RBCs.
Scattergram/Scatterplot
Measures *Size and Complexity* of the nucleus and granules.
Platelet Histogram Criteria for a Fitted Curve
No platelet count vote-out
Idirect RBC Calculations
Numeric expression of the variation in red blood cell volume distribution.
Principle of Automated Cell Counting -
Optical Light Scatter
Commercial Controls
Overlap use of old and new control lots. New lot should be validated before old lot becomes outdated.
Platelets
Particles between *2 - 20 fL*.
Background Checks
Performed to check for any particle or substance interfering with the sample, diluent, or the instrument.
Multivariate Check
Performing the "Rule of Three"
QC Measure
Personnel competency assessment - evaluation of replicate slides
Reference Method for PLT count
Phase Microscopy
Histograms
Plots the distribution of cell types based on size.
QC Measure
Proficiency testing (e.g., CAP surveys)
When examining the Patient Specimen for Acceptability, check for...
Properly-filled Tubes
Histograms
Provide a means of comparing the sizes of a patient's cells with a normal population.
Hematocrit Calculation
RBC Count × MCV ÷ 10
Counting Chambers in Coulter cell counter
RBC bath: Used for RBC and platelet count; WBC bath: Used for WBC and hemoglobin determination.
Electrical Impedence: Direct Measurements
RBC, WBC, and PLT counts.
Scattergram: Screening for Blood Parasites
RBCs are stained with acridine orange.
Principle of Automated Cell Counting
Radiofrequency
Benefit of Automated Counting Systems
Reduced lab costs
RBC Histogram
Reflects the normal size of anisocytosis of erythrocytes or any other particles in the erythrocyte size range.
Duplicate Specimen Testing - Day-to-day Monitoring
Repeat after 24 hours.
Duplicate Specimen Testing - Within-day Monitoring
Repeat throughout the day to check reproducibility.
Leukocyte Differentiation
Reported on WBC, RBC, and platelet scattergrams or scatterplots.
QC Measure
Result verification performed to confirm validity of lab results
Multivariate Check
Review of peripheral blood smear and correlation with automated blood count.
Controls
Run periodically throughout the day to make sure calibration is maintained.
When erythrocytes call outside normal range.
Shift
Histograms
Shows the presence of subpopulations.
Reference Method for WBC and RBC counts
Single-channel Semi-automated Electronic Counters
Commercially Preserved RBCs
Sometimes avian nucleated RBCs are used as WBCs. Cells are suspended in a glucose-containing preservative.
Commercially Fixed RBCs
Stable for 3-4 months with minimal change in MCV. Large discrepancy in results obtained between methods.
Cyanmethemoglobin Standard
The only commercially-available standard used in routine hematology. It is used to prepare a standard curve for hemoglobin measurement.
Commercial Controls
Used for day-to-day and longer-term monitoring. Available in preserved and fixed cells.
Patient Controls
Used for reproducibility and monitoring within-the-day performance. Stable only for a few hours at room temperature and about 24 hours at 4°C.
Optical Light Scatter
Used in multiparameter hematology analyzer.
Radio Frequency (RF)
Used simultaneously with electrical impedance.
Radio Frequency (RF)
Uses an *electromagnetic current* to measure the conductivity of the cell.
Scattergram/Scatterplot
Uses two separate variables to plot positions on a *Cartesian coordinate system* (X and Y axis).
Commercial Calibrators
Values are supplied by manufacturer of the instrument.
RBC Histogram
Will coincide directly with the Mean Corpuscular Volume (MCV).
When checking for clots, use...
Wooden Applicator Sticks
QC Measure
Written grading criteria should be a part of SOP