# Presentation: 313809

## Automated Hematology:

**Automated Hematology:**
**An Overview**

## Session Outline

- Principles of Automation
- Initial Setup
- Calibration
- Quality Control
- Flagging
- Troubleshooting
- Case Studies

## 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 actions

## Range of Technologies in Zambia

**ABX Micos 60**

**Range of Technologies in Zambia**

## ABX Pentra 60 C+

**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

**Notes:**
When automation was first introduced, the Coulter principle was the first mechanism to be put into widespread use.  The Coulter principle is based on the following:
Particles suspended in an isotonic diluent, when drawn through an aperture  which has an electric current flowing through it will cause a measurable drop in voltage which is proportional to the size of the particle passing through the aperture.  The pulses produced can then be counted and if the flow through the aperture is constant the particles can be quantified per unit volume.  This is also called electrical impedance.  This is the mechanism employed in most hematology analyzers in one way or another.  Most analyzers use a combination of high and low vacuum and pneumatic pressure to push and pull the sample through the analyzer.  

## Electronic Resistance (Impedance)

-  Utilizes non-conductive properties of blood cells
- 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 eosinophils

## Technologies

**Technologies**

**ABX**

## Slide 11

## 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 functions

## Out of the BoxFor 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 Calibrate

- You 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 representative

## 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 at the factory.  They do not require calibration in the laboratory.

## Performing Reproducibility Check – CBC (N=10)

- Sample Requirements - For reproducibility studies, ensure the patient 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 limits**

## 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 containers

## 2.  Check the waste container for:
Sufficient capacity
 Proper connections
Perform daily startup
In addition to verifying daily startup results, verify acceptable:
Reproducibility
Carryover
Control Results

- Sufficient capacity
-  Proper connections
- Perform daily startup
- In addition to verifying daily startup results, verify acceptable:
- Reproducibility
- Carryover
- Control Results

**Ensure the Instrument is Functioning Properly**

## Quality Control

## 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 manufacturer’s defined acceptable limits

**Notes:**
Quality control is an integral part of all laboratory assays and automated assays are no exception.  A properly designed QC program enable s the operator to be assured that all instrumentation is functioning properly and results obtained for patient samples are precise and accurate.

There are several methods of QC that can be utilized by a laboratory.  They are XB analysis, which uses patient samples and target values which are determined locally and when used properly can usually predict impending instrument problems
Assayed stabilized materials can be used.  These are products bought from a manufacturer that have values and acceptable ranges pre-established
Previously analyzed patient samples can also be analyzed at various intervals to determine the functionality of the analyzer

## 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 shifts

## Quality Control

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

**Notes:**
In the event QC is unacceptable there are a number of things that should or could be done.  Firstly, if this is an abhorrent result and all other QC has been ok, repeat the offending control.  Prior to doing this however, you should analyze previously recovered control data and determine if there have been trends or shifts.
	If previous QC looks ok, verify the integrity of the vial that yielded the value in question and discard the vial and open and repeat the control.  
	If the problem is still occurring, perform any necessary troubleshooting.  Once the performance of the analyzer has been verified, it may be necessary to recalibrate the analyzer in order to re-establish control

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

- 1.  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 control’s information to match the package insert.

## 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.

## Automated Hematology

- Problem 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.

## 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

- If the control results are acceptable, the problem is probably specimen-related. Check for:
- clots
- hemolysis
- lipemia

**Specimen-Related Problems**

## 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 / 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.

## 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 components.

## Correcting Pneumatic/Hydraulic Problems

- You can correct most pneumatic / hydraulic problems, including defective components.
-  Tubing, *(Need other examples)*
- 	Follow manufacturer’s instructions
-  Sticking float in moisture chamber

## 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.

## 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

## More Flagging

**More Flagging**

- RBC Suspect flags
- 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Test

## Histograms

- RBC, PLT, and WBC  plotted on histogram
- X-Axis
- Cell size in femtoliters (fL)
- Y-Axis
- # of cells

## Histogram

- WBC: Distribution with three individual peaks and valleys at specific regions representing the lymphocytes, monocytes, and granulocytes.
- All curves normally start and end at baseline

## RBC Histogram As A Quality Control Tool

| ABN / INDICATOR | PROBABLE CAUSE | COMMENT |
| --- | --- | --- |
| Left of curve does not touch baseline | Schistocytes and extremely small red cells | Review smear CBC and Platelet histogram |
| Bimodal peak | Transfused cells, therapeutic response | Review Smear |
| Right portion of curve extended | Red cell autoagglutination | Review CBC & Smear |
| Left shift of curve | Microcytes | Review smear & CBC |
| Right shift of curve | Macrocytes | Review smear & CBC |

## WBC Histogram As A Quality Control Tool

| ABN / INDICATOR | PROBABLE CAUSE | COMMENT |
| --- | --- | --- |
| Trail extending downward at extreme left, or lymph peak not starting at baseline | NRBC, Plt clumping, unlysed RBC, cryoproteins, parasites | Review smear and correct WBC for NRBC |
| Peak to the left of lymph peak or widening of lymph peak towards left | NRBC | Review smear & correct WBC for NRBC |
| Widening of lymph peak to right | Atypical lymphs, blasts, plasma cells, hairy cells, eosinophilia, basophilia | Review smear |
| Wider mono peak | Monocytosis, plasma cells, eosinophilia, basophilia, blasts | Review smear |

## WBC Histogram As A Quality Control Tool

| ABN / INDICATOR | PROBABLE CAUSE | COMMENT |
| --- | --- | --- |
| WBC histogram (lymph peak) does not start at baseline | Giant platelets, NRBC, Plt clumping | Review smear, correct WBC for NRBC |
| Elevation of left portion of granulocyte | Left Shift | Review smear |
| Elevation of right portion of granulocyte peak | Neutrophilia | Review smear |

## Platelet Histogram As A Quality Control Tool

| ABN / INDICATOR | PROBABLE CAUSE | COMMENT |
| --- | --- | --- |
| Peak or spike at left end of histogram (2-8 fl) | Cytoplasmic fragments | Review smear |
| Spike towards right end of histogram | Schistocytes, microcytes, giant platelets | Review smear + CBC
( MCV &  RDW)
( MPV &  PDW) |
| Bimodal peak | Cytoplasmic fragments | Review smear |

## Interpreting Test Results 

- Misleading results can occur if the specimen was not properly collected, stored, or transported.  The following situations can also yield misleading results for the parameters listed:

## Interfering substances

- Cold Agglutinins
- Cryoglobulins
- Lipemia
- Platelet Clumps
- RBC  fragments
- NRBCs

## Interferences  Cont.

- All Parameters:
- WBC:

- Clotted specimen.  Inspect every specimen for clots
- Unusual RBC abnormalities that resist lysing
- malarial parasites
- giant platelets, platelet clumps, NRBCs
- fragmented white cells, agglutinated white cells,
- lyse-resistant red cells,
- cryoglobulin, some extremely elevate proteins,
- unlysed particles greater than 35 fL in size

## Interferences

- RBC:  	Very high WBC count, high concentration of very 		large platelets, auto-agglutination
- Hgb:  	Very high WBC count, severe lipemia, heparin, 		certain unusual RBC abnormalities that resist 		lysing
- MCV: 	Very high WBC count, high concentration 			of very large platelets, auto-agglutination
- RDW: 	Very high WBC count, high concentration 			of very large platelets, auto-agglutination
- Plt:	 	Giant platelets, platelet clumps, white cell 			fragments, electronic noise, very small red 			cells, red cell fragments
- Hct: 	Known references related to RBC and  MCV

## Interferences

- MCH:  	Known interferences related to Hgb and 		RBC
- MCHC: 	 Known interferences related to Hgb, RBC, 		and MCV
- Troubleshooting Flagged Results:
- Refer to the Instrument Manual to troubleshoot codes, flags, and messages displayed with patient results