zambia hematology

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Automated Hematology: An Overview


Session Outline

Principles of Automation Initial Setup Calibration Quality Control Flagging Troubleshooting Case Studies2

Objectives Describe the electrical impedance and

light scatter principle for performing cell counts Explain the underlying causes of invalid automated hematology results. Utilize quality control procedures to determine if patient results are acceptable Recognize the significance of flagging and take appropriate actions3

Range of Technologies in ZambiaABX Micos 60


ABX Pentra 60 C+


Hematology Automation Two General Principles

Electronic resistance ( impedance) Light scattering


Cell counting Coulter Principle

Dilution Vacuum and pressure Electrical impedance Reagent systems


Electronic Resistance (Impedance) Utilizes non-conductive properties of bloodcells as blood cell passes through orifice of aperture it displaces its own volume increased resistance between electrodes results in an electrical pulse RBCs and Platelets counted together, separated by pulse heights hydrodynamic focusing forces cells to pass single file through sensing zone


Light scattering Cells counted as passed through focused beam of light(

LASER) Sum of diffraction(bending around corners), refraction (bending due to change in speed) and reflection (light rays turned back by obstruction). Multi angle polarized scatter separation (M.A.P.S.S)

0 : indicator of cell size 10 : indicator of cell structure and complexity 90 polarized: indicates nuclear lobularity 90 depolarized: differentiate eosinophils9

ABX Technologies



Initial Setup


Out of the Box

For the service Engineer Check instrument for visual damage Check for any loose parts or connections Make sure all computer boards are properly sealed Check the socket to verify proper voltage outlet Plug instrument power cord into (voltage stabilizer) electrical supply Confirm the correct voltage on instrument Main power supply Photometric voltage Any other voltage supply that is pertinent to instrument functions13

Out of the BoxFor the service Engineer

Permit instrument to stabilize/equilibrate

Let all components reach proper temperature Set in any parameters that may be required Ranges Temps


When to CalibrateYou should calibrate your instrument: At installation After the replacement of any component that involves dilution characteristics or the primary measurements (such as the apertures) When advised to do so by your service representative15

Calibration Calibration fine tunes your hematology analyzer

and provides the most accurate results possible. In the normal process of tracking data for an extended period of time, your laboratory can make a specific decision to recalibrate a given parameter. Never adjust to a specific value for an individual sample. For best performance, calibrate all the CBC parameters. The WBC differential is calibrated 16 at the factory. They do not require calibration in the laboratory.

Performing Reproducibility Check CBC (N=10)Sample Requirements - For reproducibility studies, ensure thepatient for the sample that is being tested:

Is receiving no medication Has normal hematologic parameters, with a WBC count of 5.0 1.0. Has normal erythrocyte, leukocyte, and platelet morphology and, if you want to check the Diff parameters, with Diff values Neutrophils 40 to 72% Lymphocytes 17 to 45% Monocytes 4 to 12% Eosinophils 0 to 10% Basophils 0 to 1% Ensure you have enough normal whole blood from a single donor for 11 cycles.


Daily startup procedures(Daily Maintenance)Daily cleaning Background counts Electronic checks Compare open and closed mode sampling (use a normal patient sample) Run controls

Must be within specified limits18

Ensure the Instrument is Functioning Properly

1. Check the reagent containers for: Sufficient quantity Not beyond expiration date No precipitates, turbidity, particulate matter, or unusual color Proper connections between the instrument and the reagent containers19

Ensure the Instrument is Functioning Properly2. Check the waste container for: Sufficient capacity Proper connections 3. Perform daily startup 4. In addition to verifying daily startup results, verify acceptable: Reproducibility Carryover Control Results20

Quality Control


Quality Control Purpose of QC

Assure proper functionality of instrumentation Means of assuring accuracy of unknowns Monitoring the Integrity of the Calibration-

When controls begin to show evidence of unusual trends When controls exceed the manufacturers defined acceptable limits


Quality Control Methods Assayed or Unassayed stabilized material

(Commercial) Previously analyzed patient samples Easily obtained Cost effective Results and samples readily available


QC Methods: Assayed or Unassayed Stabilized Material

Commercially available Known values (Assayed only) Analyze low, normal and high control Results stored in the instrument computer (Pentra only) Monitored with Levy-Jennings charts - Easily illustrates trends and shifts24

Quality Control OUT OF CONTROL!!!

Repeat the assay ( One time occurrence ) Check for trends (from Levy Jennings) Check integrity of material Troubleshoot Verify instrumentation


Remedial Action to Take When a Control is Outside its Expected Range1. Ensure the control Material was mixed properly. If not, mix it according to the package insert. Identification information was entered correctly. If using the Numeric Keypad, ensure you typed the correct information. Setup information (assigned values and expected ranges) matches the control package insert for the current lot number being used. If they do not match, change the controls information to match the package insert.26

Remedial Action to Take When a Control is Outside its Expected Range


If any of the problems still exist, rerun the control; otherwise, proceed to the next step Rerun the control to ensure the problem was not a statistical outlier. Ensure the control material was not contaminated by running another vial or level of control.27



Automated HematologyProblem Solving Troubleshooting Specimen Related OR Instrument

Data Review A review of instrument data, such as

background, control, and blood sample results, is helpful in detecting problems. Sometimes a questionable blood sample

result is the only symptom of subtle reagent or pneumatic problems.


Specimen-Related Problems An instrument problem is differentiated

from a specimen-related problem by running a control.

If the control results are acceptable, the

problem is probably specimen-related. Check for: clots hemolysis lipemia


Instrument Problems If the control shows similar problems, it

indicates an instrument problem. Electronic? Pneumatic / Hydraulic? Reagent? Because it is easiest to detect a problem in the electronic subsystem and hardest to detect a problem in the reagent subsystem, the subsystems are usually checked in the following order: electronic, pneumatic / 31 hydraulic, reagent.

Electronic Troubleshooting Detecting a problem in the electronic

subsystem or eliminating the electronic subsystem as the source of the problem is simplified by indicators and electronic tests. Correcting Electronic Problems:

minor problems,such as loose cables most electronic problems require the assistance of your instrument service representative.32

Pneumatic / Hydraulic Troubleshooting Most pneumatic / hydraulic problems are

detected by observing the Diluter section in operation. When you identify a symptom of a malfunction, try to isolate the malfunction to the specific part of the cycle, for example, during preparation, counting, or cleanup. Then, try to isolate the malfunction to the specific components and tubing. Next, look for one of four possible problems pinched tubing, plugs, leaks, or defective 33 components.

Correcting Pneumatic/Hydraulic ProblemsYou can correct most pneumatic / hydraulic problems, including defective components. Tubing, (Need other examples) Follow manufacturers instructions Sticking float in moisture chamber34

Reagent Troubleshooting A reagent problem can be as obvious as

precipitate in the reagent tubing. In the less obvious cases, the most effective way of detecting a problem is by keeping a log of the lot numbers with the opening and expiration dates of the reagents in use, and knowing how each reagent affects the data. Refer to the labeling information with your reagents for details.35

Correcting Reagent Problems You can correct most reagent problems

by: changing the container of reagent priming the instrument with the new reagent.


Flagging Condition flags Describes cell population normal abnormal WBC Suspect flags Blasts Imm Grans/Bands 1 Imm Grans/Bands 2 Variant lymphs Review Slide

Check li e


More Flagging RBC Suspect flags

Slide Check

NRBCs Macrocytic RBCs Dimorphic RBC population Micro RBCs/RBC fragments RBC agglutination Definitive Flagging Based on predetermined lab limits Provide information for review


Review of Histograms as a Quality Control Test39

Histograms RBC, PLT, and WBC

plotted on histogram X-Axis Cell size in femtoliters (fL) Y-Axis # of cells


Histogram W