Standard Operating Procedure for Hematology

Blood Collection for Hematology

Purpose:

This SOP describes the procedure for blood collection for hematological analysis.

Responsibility:

It is the responsibility of the back office as well as laboratory personnel to carry out this procedure to ensure that all
steps are completed competently and safely.

Equipment/ Reagent Requirements:

● Blood collection system

● Personal protective equipment
● Blood collection tube: EDTA

Procedure:

1. Draw blood directly into evacuated EDTA tube. Fill tube to the black mark or the arrow on the tube label, which
indicates that the correct amount of blood has been drawn. Underfilling or overfilling of the tube can affect
laboratory results due to incorrect blood-to-additive ratio.
2. The blood collection tube is properly labeled with a unique patient identification or is barcoded with a label that
is generated electronically.
3. Invert the tube 8-10 times to avoid the formation of micro clots.
4. The time and date of the sample is recorded into the laboratory information system (LIS).
5. The tube is inverted on a rocker ready for processing.

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 Standard Operating Procedure for Hematology

Whole Blood Analysis (Complete Blood Count: 5-part differential)

Purpose

Description of automated test method used for whole blood analysis using the Sysmex XN-430 hematology analyzer.

Responsibility

All hematology laboratory personnel are required to be knowledgeable of this procedure. New employees are
trained and assessed for competence before they can handle patient samples. Documentation of training on this
procedure can be found in the Personnel Files.

Principle of the Test

Clinical Aspects

Knowing the proportions of the different components (neutrophils, lymphocytes, monocytes, red blood cells,
basophils, hemoglobin of peripheral blood is vital in distinguishing a diseased and normal blood. Complete blood
count (CBC) is used to evaluate one’s overall health and detect a wide range of disorders including anemia and
infection.

System Overview

All XNs’ complete blood counts (CBC) include nucleated red blood cell (NRBC) counts automatically to levels as low as
1/1000 WBC. This ensures a reliable white blood cell count, even at high NRBC concentrations. WBC values are
provided automatically if NRBC are present. It also saves precious time by reliably replacing the manual NRBC
counting and white blood cell correction. It avoids interferences from non-cellular particles such as lyse-resistant RBC
or lipids. And it provides information on microcytes and macrocytes in addition to the red blood cell count.

If you screen neonatal or young children’s samples, you and the patients can both benefit. Because the analyzer
requires only 88µL per analysis, you can reduce the sample size. And thanks to auto-correction for NRBC, you know
your white blood cell values in neonate samples are going to be correct.

Measurement Principles

Analysis of WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, RDW-SD, RDW-CV, MPV, NEUT#, LYMPH#, MONO#, EO#,
BASO#, IG#, NEUT%, LYMPH%, MONO%, EO%, BASO%, IG%

WBC DIFF: Fluorescent Flow Cytometry

RBC/PLT: DC Impedance method with hydrodynamic focusing

HGB: Cyanide-free SLS method

Specimen Handling and Storage

- Some anticoagulants will alter test results due to their effects on hemolysis and blood platelet agglutination.
Therefore, use EDTA-2K, or 3K as the anticoagulant.
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 Standard Operating Procedure for Hematology

- Complete blood count is only stable for 24 hours.

- Ensure that tube is inverted 8-10 times before processing
- If stored in the refrigerator, samples should be returned to room temperature, for approximately 30 minutes
before analysis.

Causes for Rejection

- Tube is underfilled

Required sample volume (Chart 1):

- No patient identifier on tube

- Mislabeled tubes

Specimen Interferences

Some abnormal samples may interfere with automated cell counting methods. The following is a list of possible
substances that may interfere with the listed parameters

- WBC: Cold agglutinins platelet aggregation, nucleated RBC’s cryoglobulins, lyse-resistant RBCs in patients with
haemoglobinopathies, severe liver disease or neonates.
- RBC: Cold agglutinins, severe microcytosis, fragmented RBCs, large numbers of giant platelets, in vitro hemolysis.
- Hgb: Lipemia, abnormal proteins in blood plasma, severe leukocytes (above 100,000/µl). The effect of abnormal
proteins and Lipemia may be removed by plasma replacement or plasma blank procedures.
- Hct: Cold agglutinins, leukocytosis (above 100,000/µl), abnormal red cell fragility.
- PLT: Pseudo thrombocytopenia, platelet aggregation, increased microcytosis, megalocytic platelets

Potential Hazards

- Laboratory safety is a vital component of any laboratory setting. Proper PPE must be worn at all times.
- For more information on safety precautions refer to Universal Precautions Policy and Waste Disposal Policy
- If the instrument emits an abnormal odor or smoke, turn off the power immediately and disconnect the power
plug from the wall socket
- DO NO TOUCH the electrical circuits inside the cover, particularly if your hands are wet. Electrical shock may
result.

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 Standard Operating Procedure for Hematology

- When discarding waste fluid, or disassembling and assembling the related parts, always wear protective clothing,
gloves, and eyeglasses to prevent contact with contaminated fluids or parts.

Reagents

Reagents Storage Conditions

Cellpack 5 – 30o C
Sulfolyser 1 – 30o C
Cell clean 1 – 30o C
Lysercell 5 – 30o C

These reagents are supplied ready for use and are stable until the stated expiry date when stored at the recommended
storage conditions.

When using reagents be cautious of the following:

- After unpacking, be sure not to allow dust, dirt, or bacteria to come in touch with the reagent
- Do not us the reagents which are out of expiration date
- Handle a reagent gently to prevent the formation of bubbles
- Take care not to spill a reagent. If it spills, wipe off immediately
- Refer to the MSDS provided for each reagent

Reagent Replacement: (Diluent and Hemolytic Agent)

1. The “Help” dialog box will appear when the system detects and empty/ expired reagent container
2. Read the error message to determine which reagent is empty, then touch [Execute]
3. Touch the name of the reagent to be replaced
4. Place a check mark next to “Replace the reagent” then place the cursor in the reagent code text box
5. Using the handheld reader scan the reagent code on the new reagent container
a. NOTE: Scan Reagent Code 2 when available on the reagent container
6. Open the cap on the new container
7. Remove cap from expired/empty container and carefully remove spout
8. Insert spout straight into new container
9. Tighten cap on the new container and move container into position
10. Touch [Execute] from the Reagent Replacement dialog box

Reagent Replacement: (Replacing the Dye)

1. The “Help” dialog box will appear when the system detects and empty/ expired reagent container
2. Read the error message to determine which reagent is empty, then touch [Execute]
3. Pull out the dye holder drawer
4. Remove the entire dye holder (cover and cartridge)
5. Slowly remove the dye cover, making sure the dye does not drip
6. Remove the old reagent cartridge from the holder
7. Install the new dye cartridge into the holder
8. Place the dye cover over the cartridge
9. Replace the dye holder and close the drawer
a. NOTE: The analyzer will beep as the information is automatically registered
b. Reagent replacement will begin automatically; when complete the dialog box will close

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 Standard Operating Procedure for Hematology

PROCEDURE
Instrument Start Up

- Refer to Chapter 1: Basic Operation 1.2 Startup

- Before turning ON the instrument power, check the following tube and cable connections, bent tubes, and any
objects on the instrument

Processing Quality Control

1. Bring QC material to room temperature for 15 minutes

2. Make sure the analyzer is in “Ready” state
3. Verify the analyzer is in WB (whole blood) mode
4. To change modes, touch [Mode] on the control menu
5. Touch the analysis mode, then touch [OK]
6. Touch the [Manual] icon and barcode the tube
7. Mix the QC material according to the package insert, until the cell button is suspended
8. Put sample tube in tube holder with cap closed
9. Once aspiration is complete, remove the sample tube from sampler tuber tube holder
10. Touch [Accept] to plot the QC data to the Levy-Jennings chart or press [Cancel] and rerun the QC material

QC Result review: Levy-Jennings Charts

1. On the toolbar, touch [QC File]

2. Select a QC File
3. Touch the [QC Chart] button on the toolbar
a. The L-J Charts will display for selected QC file

Processing Whole Blood Samples

1. Make sure the analyzer is in the “Ready” state

2. Touch [Mode] in the control menu and make sure it is set to Whole Blood
3. Touch [Manual] on the right edge of the control menu.
4. Touch a field to:
a. Scan the barcode or manually enter the sample ID
b. If necessary, place a check next to [Cap Open]
5. Touch [OK]
6. Mix the sample according to your laboratory protocol for processing patient whole blood specimens
7. Place the sample tube in the sampler tube holder
8. Press the start switch
9. After analysis is complete, remove the sample tube

Manual Mode: Low WBC

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 Standard Operating Procedure for Hematology

1. Make sure the button on the right edge of the control menu is [Manual]. When the mode is set to [Sampler]
press the mode switch button on the main unit
2. Make sure the instrument is in the “Ready” state
3. Touch [Mode] and touch the analysis mode you want to set: [Low WBC]
4. Touch [OK]
5. To Return to whole blood mode
6. Touch the [Analysis Mode] button
7. Select Whole Blood
8. Touch [OK]

Process Whole Blood Samples: Micro Collection Tubes

1. Make sure the instrument is in the ready state

2. Touch [Mode] in the control menu
3. Touch a field to:
a. Scan the barcode or manually enter the sample ID
b. Place a check mark next to [Raised Bottom Tube]
4. Touch [OK]
5. Mix the sample according to your laboratory protocol for processing Raised Bottom Tubes
6. Place the tube in the regular sample tube holder
7. Press the start switch
8. After analysis is complete, remove the sample tube

QC material used: XN-L

Logging Off:

- Touch the [Menu] button on the toolbar

- Touch the [LOGOFF] icon
- Touch [YES]

Daily Maintenance: Shutdown

1. Make sure the analyzer is in the “Ready” state

2. Touch the [Menu] button on the toolbar

3. Touch the [Shutdown] icon

4. Touch [OK]

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*NOTE: The analyzer will automatically power off once the shutdown sequence is complete in approximately 2
minutes. During shutdown, no sample tubes will be processed.

Weekly Maintenance: Routine Cleaning (Cell Clean Auto)

1. Make sure analyzer is in the “Ready” state

2. Touch the [Maintenance] icon in the [Menu] screen
3. Touch the [Rinse Instrument] icon
4. Touch [Routine Cleaning]
5. If the sample tube holder is not ejected, press the sample tube holder open/close switch
6. Press CELLCLEAN AUTO in the sample holder
7. Press the start switch
8. When aspiration completes, remove CELLCLEAN AUTO from the tube holder
9. When the process finishes, approximately 15 minutes, the dialog box closes, and the instrument power
automatically turns OFF.

Name Title Signature Date

Approved By: Sharlene Lansdale Lab Manager

Approved By: Mondith Blanche Lab Consultant

Reviewed By: Lawrence D. Sher, M.D. Lab Director

Revised 10/5/22