Download Complete Blood Count with 5-Part Differential in Whole Blood (NHANES 2013-2014) and more Study notes Hematology in PDF only on Docsity!
Laboratory Procedure Manual
Analyte: Complete Blood Count
Matrix: Whole Blood
Method: Complete Blood Count with 5-Part
Differential
Method No.:
Revised:
as performed by:
Contact:
Important Information for Users
CDC periodically refines these laboratory methods. It is the responsibility of the user to contact the person listed on the title page of each write-up before using the analytical method to find out whether any changes have been made and what revisions, if any, have been incorporated.
NHANES 2013-
Public Release Data Set Information
This document details the Lab Protocol for testing the items listed in the following table:
Lab Number Analyte^ SAS Label
CBC_H
LBXWBCSI White blood cell count (1000 cells/uL) LBXLYPCT Lymphocyte (%) LBXMOPCT Monocyte (%) LBXNEPCT Segmented neutrophils (%) LBXEOPCT Eosinophils (%) LBXBAPCT Basophils (%) LBDLYMNO Lymphocyte number (1000 cells/uL) LBDMONO Monocyte number (1000 cells/uL)
LBDNENO
Segmented neutrophils number ( cells/uL) LBDEONO Eosinophils number (1000 cells/uL) LBDBANO Basophils number (1000 cells/uL) LBXRBCSI Red cell count (million cells/uL) LBXHGB Hemoglobin (g/dL) LBXHCT Hematocrit (%) LBXMCVSI Mean cell volume (fL) LBXMCHSI Mean cell hemoglobin (pg) LBXMC MCHC (g/dL) LBXRDW Red cell distribution width (%) LBXPLTSI Platelet count ((1000 cells/uL)) LBXMPSI Mean platelet volume (fL)
NHANES 2013-
The UniCel DxH 800 Analyzer measures the following parameters:
WBC White Blood Cell count UWBC Uncorrected White Blood Cell count RBC Red Blood Cell count (for Whole Blood and Body Fluids) Hgb Hemoglobin Hct Hematocrit MCV Mean Corpuscular Volume MCH Mean Corpuscular Hemoglobin MCHC Mean Corpuscular Hemoglobin Concentration RDW Red Cell Distribution Width RDW-SD Red Cell Distribution Width Standard Deviation (SD) PLT Platelet count MPV Mean Platelet Volume NE Neutrophil percent LY Lymphocyte percent MO Monocyte percent EO Eosinophil percent BA Basophil percent NE# Neutrophil absolute number LY# Lymphocyte absolute number MO# Monocyte absolute number EO# Eosinophil absolute number BA# Basophil absolute number NRBC Nucleated Red Blood Cell percent NRBC# Nucleated Red Blood Cell absolute number RET Reticulocyte percent RET# Reticulocyte absolute number MRV Mean Reticulocyte Volume IRF Immature Reticulocyte Fraction TNC Total Nucleated Cell (Body Fluids)
NHANES 2013-
Coulter Method
In 1956, Wallace Coulter described the Coulter Principle as follows: A suspension of blood cells is
passed through a small orifice simultaneously with an electric current. The individual blood cells
passing through the orifice introduce an impedance change in the orifice determined by the size of
the cell.
Each cell suspended in a conductive liquid (diluent) acts as an insulator. As each cell passes through
the aperture, it momentarily increases the resistance of the electrical path between the submerged
electrodes on either side of the aperture. This causes a measurable electronic pulse. For purposes of
counting, the vacuum used to pull the diluted suspension of cells through the aperture must be at a
regulated volume. The number of recorded pulses indicates particle count, and the size of the
electrical pulse is proportional to the cell volume. In addition, the system counts the individual cells
and provides cell size distribution. The number of cells counted per sample is approximately 100
times greater than the usual microscope count which reduces the odds of statistical error
approximately 10 times.
VCSn Technology
Historically, Beckman Coulter analyzers housed a flow cell in a Triple Transducer Module (TTM),
which was first introduced commercially in the 1980’s. The TTM flow cell was the location for
detection of the processed samples. The TTM produced three measurement signals—volume,
conductivity, and light scatter.
The DxH 800 system replaces the TTM with the Multi-Transducer Module (MTM), which measures
additional multiple angles of light scatter, a major improvement over the single light scatter
measured by the TTM. All Diff, NRBC, and Retic analysis occurs in the VCSn module. The VCSn
module is responsible for controlled sample preparation and delivery of the prepared sample to the
flow cell for analysis of the WBC differential, reticulocyte, and NRBC. The VCSn module includes
the Air Mix and Temperature Control (AMTC) and the Multi-Transducer Module (MTM).
In the flow cell, low-frequency direct current measures volume, while high-frequency (RF) current
senses cellular internal content through measuring changes in conductivity.
The MTM uses a flow cell to pass particles through a sensing zone, one particle at a time, and a
diode laser to illuminate the particles. The illuminated particles both scatter and absorb a portion of
NHANES 2013-
Parameter Measurement, Derivation, and Calculation
The following exhibit lists each reportable parameter and the method used to obtain the results. A
description of the method is also included.
Exhibit 6-1. Reportable parameters and analysis methods
Parameter (Reporting Units in US- Format) Method Description WBC Coulter White Blood Cell Count or Leukocyte Count Principle Measure directly, multiplied by the calibration factor. Corrected for interference if necessary. If no correction is required, then WBC = UWBC. WBC = N x 10^3 cells/μL. UWBC Coulter Uncorrected White Blood Cell Principle Measure directly, multiplied by the calibration factor. UWBC = N x 10^3 cells/μL. RBC Coulter Red Blood Cell Count or Erythrocyte Count Principle Measure directly, multiplied by the calibration factor. Corrected for very high white count if necessary. RBC = N X 10^6 cells/μL. Hgb Photometric Measurement
Hemoglobin or Hemoglobin Concentration Transmittance of light at 525 nm through a lysed WBC solution in the Hgb cuvette, compared to the transmittance of the same light through a reagent blank. The system converts this ratio to the Hgb value using a calibration factor. Weight (mass) of Hgb determined from the degree of absorbance found through photo current transmittance expressed in g/dL. Corrected for WBC interference. Hgb (g/dL) = [constant X log^10 (Reference %T/Sample %T)]. Exhibit 6-1. Reportable parameters and analysis methods (continued)
Parameter (Reporting Units in US- Format) Method Description Hct Calculated Hematocrit The relative volume of packed erythrocytes to whole blood. Hct (%) = RBC X MCVV^10 ). MCV Derived from Mean Corpuscular Volume RBC The average volume of individual erythrocytes derived from the Histogram RBC histogram. The system multiples the number of RBC in each channel by the size of the RBC in that channel. The products of each channel between 36 and 360 femtoliters (fL) are added. This sum is
NHANES 2013-
divided by the total number of RBC between 36 and 360 fL. The analyzer then multiplies by a calibration factor. Corrected for WBC interference. Expressed in fL.
The DxH 800 system includes a Specimen Processing Module (SPM), a Pneumatic Supply Module
(PSM), and a System Manager (SM), precisely integrated to provide process control, data
consolidation, and cassette or single-tube delivery of specimens.
Components of DxH 800
The Specimen Processing Module (SPM)
The SPM is made up of the following functional modules:
Specimen Transport Module (STM). The STM automatically transports specimens, delivering closed-vial and open-vial specimens, which accommodates a wide range of specimen tubes. The STM supports the following:
- Operator loading and unloading of specimens.
- Transport and queuing of specimens.
- Mixing and presentation of specimens.
- A Magnetic Transport System to move cassettes containing specimens.
NHANES 2013-
Pneumatic Supply Module (PSM)
The Pneumatic Supply Module supplies vacuum and pressure to the SPM.
II. Special Safety Precautions
All specimens may be potentially positive for infectious agents including HIV and Hepatitis B and C
viruses. Observe universal precautions. It is mandatory to wear gloves and a lab coat when handling
all human blood products and Coulter controls. Dispose of all biological samples in a biohazard
container and wipe down all work surfaces with a 10% bleach solution at the end of each session.
The MEC Working Safely with Hazardous Chemicals manual contains all Coulter material safety data
sheets (MSDS).
III. Computerization: Integrated Survey Information System
(ISIS)
The DxH 800 System Manager (SM) transmits individual study participant (SP) results to the MEC
automated ISIS system. All SP results are reviewed at the DxH 800 SM monitor.
The ISIS hematology module in the laboratory application automatically receives the results, or
results are transmitted manually to the hematology module. The laboratory application evaluates the
data for completeness and accuracy. The final decision to accept or reject a result is the
responsibility of the medical technologist.
All data are backed up and stored at Westat’s home office.
NHANES 2013-
IV. Specimen Collection and Preparation
Specimen Collection
The phlebotomist collects a 3 or 4-ml K3 EDTA (ethylene diamine-tetracetic acid) tube, used for a CBC on all SPs aged 1 year and older, following the established venipuncture protocol and procedures. (A 1-2% dilution effect occurs in this liquid EDTA tube.)
Sample volume is 165μL of whole blood in the closed-vial mode or the single tube presentation mode. The minimum sample volume per tube in the closed-vial mode is 1-ml with the proper proportion of blood to anticoagulant.
Specimen Preparation
For SPs 1-5 years old, the medical technologist working in the blood specimen-processing workstation initially processes the tube by first taking off whole blood required for various tests in the SP’s profile. The medical technologist working in the blood specimen-processing workstation places the specimen on a rocker until the medical technologist working in the hematology workstation can perform the CBC. The CBC should be run as soon as possible; there is no requirement to wait any length of time between drawing the blood and running the CBC.
There is a dedicated tube drawn for the CBC on SPs aged 6 years and older.
V. Procedure for Microscopic Examination
Not Applicable. Do not prepare differential microscopic slides.
VI. Preparation of Reagents, Calibrators (Standards), Controls, and Other
Materials; Equipment and Instrumentation
Reagents and Supplies
DxH reagents, including storage and handling requirements, are listed below:
Coulter DxH diluent (store at 2 - 40° C).
- Coulter DxH diluent is shipped in ready-to-use containers.
- Opened containers are stable for 60 days. Do not use this product beyond the expiration date.
NHANES 2013-
NOTE: If any Coulter DxH reagent has been partially or completely frozen, allow the product to
warm to room temperature. Mix product by gentle inversion prior to placement on the instrument.
DxH controls, including storage and handling requirements, are listed below:
Coulter 6C cell control (store at 2 – 8 ° C).
- The Coulter 6C cell control is an integrated control that enables monitoring of system performance and calibration status for all directly measured and calculated CBC, Diff, and NRBC parameters. Instructions for use are as follows:
- Remove 6C cell control vials from refrigerator and warm at ambient temperature for 10 to 15 minutes.
- Roll the tube slowly between the palms of the hands eight times in an upright position. Invert the tube and slowly roll between the palms of the hands eight times. Then gently invert the tube eight times. Repeat.
- Run the controls in the cassette presentation on the instrument
- Return the control tubes to the refrigerator within 30 minutes.
The Coulter LIN-X Linearity control is recommended for verification of the reportable range and linearity, and for calibration assessment of the WBC, RBC, HGB, and PLT parameters.
- Remove Coulter Lin-X Cell control vials from refrigerator and warm at ambient temperature for 15 minutes.
- Roll the tube slowly between the palms of the hands eight times in an upright position. Invert the tube and slowly roll between the palms of the hands eight times. Then gently invert the tube eight times. Repeat mixing procedure three times.
- Run the Lin-X Cell controls in consecutive order beginning with Level 0 and ending with Level 11. Run controls in cassette presentation on the instrument
- Level 11 is used to clean the system. The values for this level are not recorded.\
Coulter Latron CP-X control (store at 2 to 30° C) is recommended for the verification of the system calibration status of the VCSn parameters. Coulter Latron CP-X control checks the alignment of the laser and the flow cell on the DxH 800.
- Perform daily checks prior to running Latron control.
- Gently mix Latron CP-X control by inversion five to eight times. Avoid foaming.
NHANES 2013-
- Run the controls in the cassette presentation on the instrument.
Coulter S-CAL® Calibrator is traceable to reference methods and recommended for determining adjustment factors to ensure accurate SPM measurements of directly measured CBC parameters. Calibrator results should be monitored with Coulter 6C Cell control.
- Remove S-CAL from the refrigerator and warm at ambient temperature for 15 minutes.
- Roll the tube slowly between the palms of the hands eight times in an upright position.
- Invert the tube and slowly roll between the palms of the hands eight times. Then gently invert the tube eight times. Repeat.
- Inspect the tube contents to determine if all cells have been uniformly distributed. Repeat the mixing procedure if contents have not been uniformly distributed.
- Complete entire procedure within 1 hour.
DxH 800 Supplies
3-ml K 2 EDTA BD Hemogard Vacutainer® tube (367856)
4-ml K 2 EDTA BD Hemogard Vacutainer® tube (367861)
Tube rocker
Bleach, 5.25% sodium hypochlorite
Bottled deionized water
Plastic squirt bottle
Cotton gauze pads
Three-hole paper punch
Notebook
Flashlight
Precision screwdriver set
CD-RW
VII. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES
NHANES 2013-
Power Off the computer. Powering off the computer also powers off the monitor. You do not need to power off the monitor separately. The computer does not need to be shut down on a routine basis. Power Off the computer only when circumstances are indicated.
Daily Shutdown
Beckman Coulter recommends that the SPM remain in cleaner for at least 30 minutes every 24
hours. If the SPM has power, and is in Shutdown, but the pneumatics is off, automatic cycling
occurs every 24 hours to prevent flow cell and sample line clogging.
Shutdown can be initiated manually or automatically.
Shutdown removes diluent from the Unicel DxH 800 and replaces it with cleaner.
At the end of the time with cleaner, the cleaner is replaced with diluent. Then, the compressor automatically shuts off.
Shutdown at the Mobile Examination Center (MEC)
Shutdown will be set automatically:
- Select Calendar Icon on top of DxH monitor screen.
NHANES 2013-
From the Daily Checks screen, select the Shutdown button to display the Manual Shutdown dialog box.
- Select Autoconfiguration at the bottom of the screen.
- Select Configure Shutdown
- Check Enable Automatic Shutdown: Fill in hour 07, Min 00.
- √ Daily.
- Make sure each of the 7 days is checked.
- Time in cleaner 0 hour(s) 30 Minutes (s).
- OK.
Daily Checks (formerly known as StartUp) (Menu > QA > Daily Checks) is programmed to immediately follow the StartUp:
- Select Autoconfiguration at the bottom of the screen.
- Select Configure Daily Checks.
- Check Enable Automatic Daily Checks.
- Choose radio button Perform Daily Checks after Shutdown.
- OK.
Latron CP-X control should be run immediately following daily checks.
Cancel Automatic Shutdown
At certain times Automatic Shutdown will have to be cancelled. To cancel Automatic Shutdown:
From the Daily Checks screen, select Auto Configuration.
Select Configure Shutdown.
Remove the check mark from the Enable Automatic Shutdown box.
To perform a manual shutdown when Auto Configuration is disabled, please see the information
that follows (Section 6.8.4).
NHANES 2013-
Daily Checks Screen
The results of Daily Checks display on eight tabbed views: Summary (default view), System, Background, Supplies, CBC, Diff, Retic, and NRBC.
When Daily Checks pass, the Daily Checks icon is neutral. Select the individual tabs to view those results.
If any of the Daily Checks do not pass, the following occurs:
The Daily Checks icon is red.
The problematic result is backlit in red on the Summary tab.
The tab that contains the problematic result has a red indicator.
If there are any failures, you must Review Daily Checks (Event Logs > General Tab) in order to proceed with further analysis.
The Daily Checks must pass or be reviewed in order to run specimens.
From any tab on the Daily Checks screen, select the Review button on the Local Navigation bar to indicate that the Daily Checks results have been reviewed. NOTE: If Daily Checks pass, the Review button is disabled.
You have to review only results that are questionable.
Additional Tasks From the Daily Checks Screen
Select the Histogram button at the right of the screen on the DIFF, RETIC, or NRBC tabs to display the VCSn Ramp Tests Histogram screen.
NOTE: The VCSn Ramp Tests Histogram screen is not for routine use; however, you may at some time be asked to access this screen when on the phone with a Beckman Coulter representative.
Prime the SPM.
Prime provides a method to remove cleaner in case an error occurred during a Shutdown cycle.
Select the Prime button at the bottom of the Daily Checks screen to prime the SPM.
NHANES 2013-
A DxH 800 dialog box will display the following message: “You have requested to perform a Prime Cycle.”
Select OK to continue or Cancel.
Sample Analysis
Status
The SPM must be online to run samples.
You can view the status of the SPM in the Status Mode area at the upper left corner of any screen, except the home screen.
Cassette Presentation
Ensure that the SPM is set up for the appropriate test for your workflow.
Ensure that your specimens have been collected and stored properly.
Load the specimens into the cassettes. Narrow tubes with small internal diameters will require manual premixing prior to analysis to ensure proper cell and plasma distribution and to avoid possible erroneous results. Premix these tubes before placing them in the cassette and then analyze the cassette by placing it in the Stat position of the input buffer.
Place the cassettes in the input buffer to the right of the SPM. The SPM automatically begins cycling the cassettes.
After the SPM cycles the samples, review the sample results at the System Manager. To avoid serious injury, do not place your hand through the cassette presentation opening on the SPM.
Single-Tube Presentation
Ensure that your specimens have been collected and stored properly.
Select the Single-Tube Presentation icon at the top of any screen to display the Single-Tube Presentation dialog box.
