COMPLETE BLOOD COUNT (CBC)

Effective Date: 
Fri, 08/16/2013
Reviewed: 
Tue, 01/10/2017
Revised: 
Fri, 08/16/2013
Policy: 

 

Principle

The Coulter AcT2 Hematology Analyzer performs a Complete Blood Count (CBC),
Platelet Count, and a Three-Part Differential. Whole blood is aspirated, diluted, and then divided into two samples. One sample is used to analyze the red blood cells and platelets while the second sample is used to analyze the white blood cells
and hemoglobin.

Electrical impedance is used to count the white blood cells, red blood cells, and platelets as they pass through an aperture. As each cell is drawn through the aperture, a change in electrical resistance occurs generating a voltage pulse. The number of pulses during a cycle corresponds to the number of cells counted. The amplitude of each pulse is directly proportional to the cell volume.

Lyse reagent is added to the diluted sample and used to count the white blood cells. After the white blood cells have been counted and sized, the remainder of the lysed dilution is transferred to the Hgb Flow Cell to measure Hemoglobin concentration.

The AcT2 uses electronic sizing to determine a three part automated differential. The percentage and absolute counts are determined for lymphocytes, neutrophil, and mid-size population of monocytes, basophils, eosinophils, blasts, and other immature cells.

The AcT diff2 analyzer has two operating modes: Open Vial Whole Blood and Closed Vial Whole Blood.  Whole blood samples can be analyzed in either mode.  Prediluted samples can be analyzed at the Open Vial Station.

Specimen Collection and Handling

HANDLE BLOOD AS A POTENTIAL BIOHAZARD CAPABLE OF TRANSMITTING INFECTION. ALWAYS WEAR PROTECTIVE GLOVES AND LAB COAT WHEN PROCESSING SPECIMENS.

Draw specimen in into a lavender-top Vacutainer tube containing K2EDTA. Thoroughly mix blood with EDTA anticoagulant. If hemolysis or small clots are observed, discard specimen.
Mix venous blood sample at least 8 times by hand inversion. Gently turn capped sample upside down then back straight up. Alternatively, use a mechanical mixer for at least 5 minutes.  Do not test samples that are incorrectly filled or that are clotted. If hemolysis or small clots are observed, discard specimen.

Analyze venous blood samples within 24 hours of collection. Do not refrigerate samples for platelet and differential counts. If platelet or differential results are not required, store anticoagulated whole blood specimens at 2- 8 C. Warm samples to room temperature (16-35 C or 61-95 F) before testing.
 

Equipment and Materials

Equipment Performance Parameters
The AcT diff2 Analyzer operates at ambient temperature (16- 35 C or 61- 95 F) at humidity no higher than 85% without condensation.

Materials, Reagents 
COULTER diff AcT Tainer reagent pack or diff AcT Pak™, both of which contain diluent (Reagent 1) and lytic reagent (Reagent 2).  The diff AcT Tainer reagent pack also contains AcT Rinse™ Shutdown Diluent (Reagent 3).

Reagent 1 is an isotonic electrolyte solution that dilutes whole blood samples, stabilizes cell membranes for accurate counting and sizing, conducts aperture current, rinses instrument components between analyses, and prevents duplicative cell counts by using the sweep-flow process.

Reagent 2 is a lytic reagent that lyses red blood cells for white blood cell count and hemoglobin measurement.  Caution: Eye irritant.  Avoid contact with skin and eyes.  Avoid breathing gas.  Contact with acid liberates poisonous gas.

Reagent 3, AcT Rinse Shutdown Diluent, prevents protein buildup that occurs in and around the apertures.  Caution:  Avoid eye and skin contact.  Do not ingest.

Reagent Preparation
No reagent preparation is required. Appropriate safety precautions for handling reagents are contained on the respective Material Safety Data Sheets located in the laboratory’s MSDS/ HAZARDS binder.

Reagent Storage
Store reagents at ambient room temperature (2-25 C).  Keep containers closed. Discard reagents at the expiration date. Replace reagents when the screen prompt appears or when the reagent container is empty.

Reagent Tracking
When opening new reagents, log on the Reagent Log indicating date opened, lot number, and expiration date.
 

Quality Control Procedures

QC Frequency
Test 3 levels of QC samples once per day of testing using Coulter 4C PLUS cell controls.

QC Procedure Using COULTER 4C PLUS Cell Control

  1. Be sure the 4C PLUS cell control information and values have been correctly entered from the TABLE OF EXPECTED RESULTS listed on the package insert.  For information on how to enter the values, see "Entering 4C PLUS Cell Control Information" in chapter 2 of the AcT diff2 Operator’s Guide.
  2. Ensure that 4C PLUS cell control is not past its expiration date and that it is at the correct storage temperature.
  3. Allow refrigerated controls/calibrators to sit 10-15 minutes at room temperature before use . Mix by rolling slowly between the palms of the hands 8 times then invert and roll 8 times. Gently invert the tube 8 times. Inspect the tube contents to determine if all cells have been uniformly distributed. Repeat above steps if tube contents have not been uniformly distributed Inspect the tube contents to ensure that all cells are uniformly distributed; if not, repeat this step.
  4. At the Main screen, touch the QA icon.
  5. At the QA screen, touch the 4C PLUS Run icon.
  6. Select the correct control level:

   L   Low

       N   Normal

                                                            H   High                                                   

  1. The square darkens next to your selection. Make sure that the level of control you are testing matches the one selected (L, N or H).
  2. Invert the tube once or twice prior to cycling.

IMPORTANT:  Risk of misleading results if Cap Pierce Station door is opened before the sample analysis is completed.  Do not open the door.  The door will open automatically.

  1. Place the well-mixed sample in the tube holder at the Cap Pierce Station and close the door.
  2. When the tube holder door opens, remove the vial and return it to the refrigerator.
  3. Results appear on the screen.  Unless non-numeric results occur for one or more parameters, the control results are automatically stored:
  4. If Autoprint is off you can manually print results by touching the Print icon.
  5. To manually reject these results, touch the Trash icon.
  6. If results are not within the expected range, rerun the control starting at step 6. If results are still out of range, see the Special Procedures and Troubleshooting Section of the AcT diff2 Operator's Guide.
  7. Repeat steps 6 through 10 for each required control level.
  8. If the results are within the expected range, you are finished running controls.  If you do all of the above steps and the results still do not meet your performance expectations, call your Beckman Coulter Representative.
  9. First try re-running the control with the same vial of control.
  10. If QC is still not in range, open a new vial of QC material and re-run.
  11. Refer to troubleshooting procedures in the Coulter Operator’s Guide section 6.8.

       Do not report patient test results until control values are acceptable.
 

Critical Values

Critical/Alert Values are those results demonstrating such variance from normal as torepresent a pathophysiological state with potential of being life threatening unless action is taken quickly. These results must be immediately reported to the care provider and be documented in the test record as to who was contacted, the time of contact, the person making contact, and that the results were read back.

WBCs K/mm3 <3.0 or >15.0
HGB g/dl <8.0 or > 20.0
HCT % <25 or >60
Platelets  K/mm3 <100 or >600

 

Procedure: 

Sample Analysis  - Closed Vial Whole Blood Mode

  1. At the Main screen, select Closed Vial Whole Blood mode.
  2. At the Main screen, touch the Sample Results Screen icon.

NOTE:  If the door is inadvertently closed after it has opened automatically, or if it is closed at a screen where samples are not run, you can open the door by touching the Main Menu icon and then the Sample Results icon.

  1. Touch the Patient Range icon until the desired range (1, 2 or 3) appears.
  2. Verify that the sample ID is correct.  If autosequencing is on, the 9-digit sample ID number automatically increments by 1.  If autosequencing is off, manually enter the sample ID and touch the Save icon.  Be careful not to duplicate an existing sample ID number that may have been used previously. Unique sample accession numbers will be automatically assigned to specimens by the Orchard Harvest LIS.
  3. Mix the sample on the mechanical rocker prior to sampling.
  4. Be sure you are in the Closed Vial Whole Blood mode.
  5. Check the sample for clots by tilting the tube back forth and observing for clots.
  6. Place the well-mixed sample in the tube holder at the Cap Pierce Station and close the door.
  7. When the tube holder door opens, remove the tube.
  8. Sample results are automatically saved by the instrument, and the results appear on the screen.
  9. Print the results:
  • If Autoprint is on, the results print automatically.
  • If Autoprint is off, touch the Print icon.
  • If Autosequence is on, the instrument is ready to run the next sample.
  • If Autosequence is off, you must manually enter an ID number before the probe  descends for the next sample.
  • If flags appear, see the Special Procedures and Troubleshooting   Section of the AcT diff 2 Operator's Manual.

Sample Analysis  - Open Vial Whole Blood Mode

  1. At the Main screen, select Open Vial Whole Blood mode.
  2. At the Main screen, touch the Sample Results Screen icon.
  3. Touch the Patient Range icon until the desired range (1, 2 or 3) appears.
  4. Verify that the sample ID is correct.  If autosequencing is on, the 9-digit sample ID number automatically increments by 1.  If autosequencing is off, manually enter the sample ID and touch the Save icon.  Be careful not to duplicate an existing sample ID number that may have been used previously.
  5. Mix the sample thoroughly on the mechanical rocker.
  6. Be sure you are in the Open Vial Whole Blood mode.
  7. Present the well-mixed sample to the probe so that the tip is well into the tube, and press the aspirate switch.
  8. When you hear the beep, remove the sample, and put the cap back on the tube.
  9. The analyzer displays the sample results on the screen and automatically saves them.
  10. Print the results:
  • If Autoprint is on, the results print automatically.
  • If Autoprint is off, touch the Print icon.
  • If Autosequence is on, the instrument is ready to run the next sample.
  • If Autosequence is off, you must manually enter an ID number before the probe descends for the next sample.
  • If flags appear, see the Special Procedures and Troubleshooting Section of the AcT diff2 Operator’s Manual.

Test Results

WBC - White Blood Cell or leukocyte count
              LY#          Lymphocyte number
              LY%         Lymphocyte percent (or ratio)
              MO#         Mononuclear cell number
              MO%        Mononuclear cell percent (or ratio)
              GR#          Granulocyte number
              GR%         Granulocyte percent (or ratio)
RBC - Red Blood Cell or erythrocyte count
Hgb - Hemoglobin concentration
Hct - Hematocrit (relative volume of erythrocytes)
MCV - Mean Corpuscular (erythrocyte) Volume
MCH - Mean Corpuscular (erythrocyte) Hemoglobin
MCHC - Mean Corpuscular (erythrocyte) Hemoglobin Concentration
Plt - Platelet or thrombocyte count
RDW - Red Cell (erythrocyte volume) Distribution Width
MPV - Mean Platelet (thrombocyte) Volume

Flagged Results

Parameter
Range
Action

WBC, RBC, HGB, PLT

Exceeds linearity

Dilute and re-run

WBC

<4.0 or  >15,000

Slide review

PLT

<100,000  or >500,000

Slide review

MCV

<75 or >105 fl

Slide review

MCHC

> 36

Check for cold agglutinin by placing in 37C incubator for intervals of 15 min; check for lipemia, hemolysis.

RDW

>22%

Slide review

No diff or incomplete diff

 

Manual differential

Neut #

<1.0 or >8.0

Slide review

                   Neut %

>85%

Slide rev. for bands

Bands,Metas,Myelos,Blasts,nRBCs

>5%  on slide rev.

Manual differential

Eosinophils >10% on slide rev. Manual differential
Basophils >5% on slide rev. Manual differential

Lymph%

> 50.0

Slide review

Mono#

>1.5

Slide review

-----

Total voteout

Thoroughly mix and repeat specimen, zap apertures if persists

+++++

Results over range for Plt, WBC, RBC, HGB, HCT GRAN, LYM

Check bath shield; make a dilution with normal saline

MCV +++++

<50 or >130

Use spun crit or slide rev to verify

XXXXX

Aperture alert

Check sample for clots; if persists, repeat with known sample; if persists, zap apertures.

…..

Incomplete calculation

Address voteout (above)

Reportable Ranges
The operating range listed below is the range of results over which the AcT diff2 Series AnalyzerSeries instruments display, print and transmit results.  The linear (reportable) range is also listed below. Linearity limits apply only to directly measured parameters. The AcT diff2 Series Analyzer flags values between the linear range and the operating range.

Parameter

Operating Range

Linearity Limit/

Reportable Range

Units

WBC

0.0 ‑ 150

0-99.9

x 103 cells/ mL

RBC

0.00 ‑ 8.00

0-7.0

x 106 cells/m L

        Hgb

00.0 ‑ 30.0

0-25.0

g/dL

MCV

50.0 ‑ 130.0

 

fL

        Plt

000 ‑ 3000

0-999.0

x 103 cells/ mL

LY%

0 ‑ 100

 

%

MO%

0 - 100

 

%

GR%

0 - 100

 

%

        LY#

0 - 99.9

 

x 103 cells/ mL

MO#

0 - 99.9

 

x 103 cells/ mL

        GR#

0 - 99.9

 

x 103 cells/ mL

 Reference Ranges

 

Parameter

Units

Our Reference Ranges

WBC

x103 cells/ uL

<21 yrs 4.5-13.2

>21 yrs 3.4-10.0

RBC

x106 cells/ uL

Males 4.4-5.9

Females 4.0-5.2

HGB

g/dL

Males 13.5-17.5

Females 12.0-15.5

HCT

ratio

Males 41-53.0

Females 36-46.0

MCV

fL

80.0-100.0

MCH

pg

26.0-34.0

MCHC

g/dL

31.0-36.0

PLT

x103 cells/ uL

140-450

LY

#

<21 yrs 1.0-6.1

>21 yrs 1.0-3.4

MO

#

<21 yrs 0-1.4

>21 yrs 0-0.8

GR

#

<21 yrs 1.8-8.0

>21 yrs 1.8-6.8

Limitations of the Procedure

 K2EDTA is the recommended anticoagulant. K3EDTA and Na2EDTA are also acceptable. Use of other anticoagulants can yield misleading results.

 

Interfering Substances

These can also yield misleading results for the parameters listed below:

  • WBC: Certain unusual RBC abnormalities that resist lysing, nucleated RBCs, fragmented WBCs, any unlysed particles greater than 35 fL, very large or aggregated platelets as when anticoagulated with oxalate or heparin.
  • RBC: Very high WBC count, high concentration of very large platelets, agglutinated RBCs and RBCs smaller than 36 fL.
  • Hgb: Very high WBC count, severe lipemia, certain unusual RBC abnormalities that resist lysing, anything that increases the turbidity of the sample such as elevated levels of triglycerides.
  • MCV: Very high WBC count, high concentration of very large platelets, agglutinated RBCs, RBC fragments that fall below the 36‑fL threshold, rigid RBCs.
  • Plt: Very small red blood cells near the upper threshold, cell fragments, clumped platelets as with oxalate or heparin, platelet fragments or cellular debris near the lower platelet threshold.
  • Hct: Known factors that interfere with the parameters used for its computation, RBC and MCV.
  • MCH: Known factors that interfere with the parameters used for its computation, Hgb and RBC.
  • MCHC: Known factors that interfere with the parameters used for its computation, Hgb, RBC and MCV.
  • LY,MO,GR: Known factors that affect the WBC count as listed above, such as high triglycerides, that can affect lysing.

 

Key Points: 

 

Use the Operator's Guide for:

                               Getting started and running the instrument day‑to‑day

Reviewing unusual results (how to read a result report and what flags mean)

Performing special procedures such as cleaning, replacing, or adjusting a component of the instrument

Troubleshooting problems with your instrument.

 

Use the Reference Manual for:

What the instrument does and methods it uses

Instrument specifications and requirements

How to interface your analyzer to your laboratory's host computer

How to safely use the instrument.

 

Use the Installation and Training Guide for:

Initially setting up the instrument and printer

Powering up the instrument

Customizing the software.

 

Use the Operating Summary for:

Running your instrument using a quick reference set of procedures

Verifying screen icon

 

References

Coulter AcT diff2 Analyzer Operator's Guide PN 4237495B (June 2003) Copyright Beckman Coulter 1999,2003