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