Hematology Procedures

Hema Section Process Flow

1. Verification and signing of results
2. Encoding of results on the worksheet
3. Refer to policy and procedure on receiving and releasing result.
4. Sample Preparation
5. Result validation and delta checking
6. Stamp Signature of pathologist
7. Printing of results on the worksheet
8. Printing of official results Signing of results (MT who performed the test)
9. Releasing of result to reception area
10. Sample Analysis
11. Checking of specimen suitability

Ac. T 5 Diff Analyzer: Complete Blood Count (Automated) Procedure

1. Present the sample to the probe and press the aspirate switch. The LEDs flash during sample aspiration.
2. The sample results appear on the screen.
3. Gently mix the sample by inversion for about 6-8 times. Make sure the sample is not clotted.
4. Turn the instrument ON. The instrument performs the Startup routine. The Startup must be successful or passed before sample analysis shall start.
5. Remove the cap of the sample tub.
6. Press CBC/Diff to select the desired analysis mode (CBC or CBC/Diff). The mode selected appears on the screen.
7. Print results.
8. Validate the results.
9. Retrieve the results using the sample ID in the interface. Fill in the necessary data.
10. Run three levels of cell controls (low, normal and high). Results of which must be within the range specified in the product insert.
11. When the red LED remains illuminated, remove the tube from the probe. When the green LED remains illuminated, the instrument is ready for the next analysis.
12. Enter the sample ID.

HmX AL Analyzer: Complete Blood Count (Automated) Machine start up

1. Check the waste container.
2. Turn on the monitor for the Data Management System (DMS) by pressing the switch on the side of the instrument.
3. Check status of reagents.
4. Turn on the machine by pressing the switch at the back of the analyzer.
5. On the Main Menu of the DMS, select "Diluter Functions" then "Start Up" to begin machine start up. lt must be successful or passed before starting the analysis.

HmX AL Analyzer: Complete Blood Count (Automated) Running of samples - Primary Mode

1. Press the "Esc" key.
2. Press "F12" (Seq)
3. Click "Run Samples"
4. Press F2 (Start Primary)
5. Press "F12" (Seq)
6. Press F3 (Run).
7. Input appropriate patient identifiers (sample ID and patient name).
8. Press the "Enter" key
9. Put the specimen tube into the cassette making sure any bar code labels are visible through the top of the cassette.
10. Press ' Enter"
11. Press the "Enter" key twice.
12. From the Main Menu, select "Sample Analysis" Click "Worklist."
13. Input the cassette number in use.
14. Load the cassette into load elevator of the machine.
15. Click ' Sample Analysis."

HmX AL Analyzer: Complete Blood Count (Automated) Running of samples - Secondary Mode

1. Press F3 (Secondary)
2. Remove the cap of the sample tube.
3. Click "Run Samples"
4. Type the sample ID and patient name then press "Enter" key.
5. From the Main Menu, select "Sample Analysis.
6. Mix the specimen gently but thoroughly.
7. Press and release the sample bar.
8. Listen for the beep before removing the tube.
9. Press F3 (Run)
10. Immerse the aspirator tip completely in the tube.

HmX AL Analyzer: Complete Blood Count (Automated) Procedure

1. Print the results at the back of the transaction slip.
2. Validate the results.
3. Running of samples
4. Machine start up
5. Retrieve the result using the sample ID in the interface. Fill in the necessary data.

Ac. T 5 Diff Analyzer: Running of Cell Control

1. Review the control results to ensure they are within the acceptable ranges.
2. Enter the cell control level (LOW, NORMAL OR HIGH) as the sample ID.
3. Present the sample to the probe and press the aspirate switch. The LEDs flash during sample aspiration.
4. Press CBC/Diff to select the desired analysis mode (CBC or CBC/Diff). The mode seIected appears on the screen.
5. Gently mix the sample by inversion for about 6-8 times. Inspect the vial's contents to ensure that all cells are uniformly distributed.
6. Remove the cap of the sample tube.
7. Repeat steps 4 through 8 until you have run all three levels of cell control.
8. Turn the instruments ON. The instrument performs the Startup routine. The Startup must be successful or passed before sample analysis shall start.
9. When the red LED remains illuminated, remove the tube from the probe. When the green LED remains illuminated, the instrument is ready for the next analysis.
10. Log the results in the designated log sheets.
11. Print quality control result.

HmX AL Analyzer: Running of Cell Control

1. Preparation of 5C cell control
2. Ensure that start up is complete.
3. Validate the results.
4. Running of Latron Control
5. Remove the cassette from the unload elevator of the machine.
6. Bring Latron Primer and Control at room temperature.
7. Running of Latron Primer
8. Log the results in the designated logsheet.
9. Running of 5C Cell Control

HmX AL Analyzer: Running of Latron Primer (Primary Mode)

1. Press "F3" (Run).
2. Press the "F2" key (File).
3. Press F4 (Primer).
4. From the Main menu of the DMS so11ware, select "Controls."
5. Select "ControI Run"
6. Select the current Latron File (active lot number)
7. After the analysis, review the primer results at the DMS

HmX AL Analyzer: Running of Latron Primer (Secondary Mode)

1. Immerse the aspirator tip completely in the Primer. (Do not place the tip against the side of the bottle).
2. Press and release the sample bar.
3. Listen for the beep or verify the status line message changes from "Aspirating" to 'Diluting' before removing the bottle.

HmX AL Analyzer: Running of Latron Control

1. Run the Latron Control in the Secondary mode.
2. Press "F3" [Control(Secondary)]
3. Press "F3" (Run).
4. Gently invert the Latron Control bottle 5 to 8 times,
5. Press the "Esc" key to close the primer Run Window.

HmX AL Analyzer: Run the Latron Control in the Secondary mode

1. Listen for the beep or verify the status line message changes from "Aspirating" to "Diluting before removing the bottle.
2. Press and release the sample bar.
3. Immerse the aspirator tip completely in the Primer. (Do not place the tip against the side of the bottle.)
4. After the analysis, review the results in the Control Run Screen.

HmX AL Analyzer: Preparation of 5C cell control

1. Warm control vials at ambient temperature for 10-15 minutes before mixing.
2. After the second mixing, check the sides and bottom of the tube to verify the control is mixed completely.
3. Mix gently by hand using the 8x8x8 method twice.

HmX AL Analyzer: Running of 5C Cell Control

1. Run the SC Cell Control in Primary Mode
2. Press F3 (Run).
3. Select the current 5C Cell Control File (active lot number)
4. Select "Control Run"
5. From the Main menu of the DMS software, select "Controls."
6. Press F2 ( Start Primary).
7. Place the control tubes into the cassette making sure any bar code labels are visible through the top of the cassette.
8. Press the "F2" key (File).
9. Load the cassette in the autoloader

Running of Sample (ACL ELITE PRO)

1. Check status of reagents and consumables.
2. Fill in the necessary data.
3. Print the results at the back of the transaction slip.
4. After the analysis, validate the results.
5. Running of samples
6. Reagent preparation
7. Machine start up
8. Retrieve the result in the interface
9. Remove the sample from the sample tray.
10. Transfer the result from the DMS to the interface by clicking the "Host Communication" icon in the lower right side of the Database view screen.

Running of Sample (ACL ELITE PRO): Reagent Preparation

1. PT Fibrinogen HS Plus
2. APTT Reagent Synthasil
3. Calibration Plasma (Normal Pool)
4. Normal Control Assayed (Normal Control)

Running of Sample (ACL ELITE PRO): Machine start up

1. Input username and password in the monitor for Data Management System (DMS).
2. Turn on the analyzer.

Running of Sample (ACL ELITE PRO): Running of samples

1. Input load list number.
2. Click the "Run" icon to start.
3. Select the test/s.
4. Select the sample position.
5. Click "Multi-test session" or "Single test session".
6. Place the sample in the Sample tray
7. On the home screen of the DMS, click "Analysis".
8. Click the box beside "Loadlist No."
9. Click the "✅" icon.
10. Type the sample ID and the patient name.

Running of Controls (ACL ELITE PRO)

1. Validate the results.
2. Check status of reagents.
3. Running of control.
4. Remove the sample from the sample tray.
5. Reconstitute Normal Control Assayed (Hemosil) with one mL distilled water {24 hour stability upon reconstitution).
6. Log the results in the designated quality control log sheet.
7. Click 'QC on the main screen.

Running of Controls (ACL ELITE PRO): Running of Control

1. Click "Add QC".
2. Click the Run' icon to start.
3. Place the control in the Sample tray.
4. Select the test/s.
5. On the main screen, click "Analysis".
6. Click "Multi-test session" or "Single test session".
7. Input loadlist number.
8. Click "Loadlist No."
9. Click the "✅" icon.
10. Select the sample position.

Erythrocyte Volume Fraction (Hematocrit)

1. Hold the tube against the scale so that the bottom of the column of erythrocytes (not the bottom of the tube) is aligned with the horizontal zero line.
2. Move the tube across the scale until the line marked 1.0 passes through the top of the plasma column. Check to make sure that the bottom of the column of erythrocyte is still on the zero line also check (by means of also check (by means of the heavy vertical lines) that the tube is vertical.
3. Centrifuge at 3000G (for the period of time recommended by the manufacturer of the centrifuge- usually 10 minutes).
4. The erythrocyte volume fraction reading is made exactly at the top of the column of erythrocytes
5. Allow the blood to low into the tube by capillary action. Fill about three quakers of a capillary tube with blood from a finger puncture.
6. Plug the other end of the tube, i.e., the end that has not come into contact with the blood, with soft wax or plastic modeling clay. Check that it is completely plugged to a depth of about 2mm.

Leukocyte Number Concentration Procedure

1. Leave the counting chamber on the bench for about 3 minutes to allow the cells to settle. Be careful it does not dry up
2. Immediately cover tip of pipette with a finger and remove the suction device.
3. Calculate the number of leukocytes in 1 liter of blood by multiplying the number of leukocytes counted in the four squares by 0.05. Report the result as "numbed by 10
4. Count the leukocyte in area of 4mm* of the chamber. Include in the count the leukocytes seen on the lines of two sides of each square counted, using the letter 'L" or " as your guide. This represents one of the four squares counted. Repeat the procedure in the three other.
5. Tilt pipette so that the stem is slightly above horizontal and wipe stem carefully with slightly dampened gauze, moving from pipette bulb toward tip.
6. To make a 1:20 dilution, attach a suction device to the pipette and draw a sample of whole blood to exactly 0.5 mark on pipette stem. Do not allow air bubbles to enter the pipette.
7. Place a coverslip on the hemocytometer.
8. Place a dampened gauze in the bottom of petri dish with two halves of wooden stick placed on the gauze to serve as stand for the hemocytometer.
9. Place the chamber on the stage of the microscope. Use the x 10 objective.
10. Shake pipette to mix by hand.
11. Gently rotating pipette between forefinger and thumb, draw diluent steadily into pipettes exactly to the 11 mark.
12. Dispense the mixture into the counting chamber. If the liquid overflows into the channel between the two chambers, you must start again: remove and clean the coverslip clean the counting chamber and refill with another drop.
13. Discard the first four drops.
14. Place a whole blood in a rotor for at least 1 to 2 minutes or hand invert gently for at least 8 times.

Preparation and Staining of Blood Smear

1. Collect a drop of blood of about 4mm in diameter on one end of the slides.
2. Hold the slides with one hand. Using the other hand, place the edge of the spreader just in front of the drop of blood.
3. Fix the thin smear with methanol for 2-3 minutes.
4. Wash the stain off in a stream of buffered water. Do not tip the stain off as thin will leave a deposit of stain on the smear.
5. Draw the spreader back until it touches the drop of blood.
6. Tip off the water and place the slide in a draining rack to dry.
7. Leave clean water on the slide for 2-3 minutes to differentiate the smear.
8. Write with a lead pencil on the thick part of the smear not used for examination patient's name and date of examination.
9. Examine the color of the smear. It should appear mauve-neither too blue nor too pink
10. Push the spreader to the end of the slides with a smooth movement (all the blood should be used).
11. Stain according to the manufacturer's instructions.
12. Let the blood run along the edge of the spreader.
13. Air dry smear. Adequate drying is essential to preserve the quality of the smear.
14. Check if the smear is satisfactory.

Leukocyte Differential Count

1. Note down the total number for each type.
2. Perform low power scan to review blood smear adequacy.
3. Classify 100 leukocytes using battlement track method.
4. Check slide identification.
5. Perform oil-immersion examination of the blood smear. Use oil sparingly and never substitute other types of lubricants for immersion oil, since substitutes may damage optical lenses.

Platelet Estimation procedure

1. Divide the total number of platelets counted by 10 to obtain the average number per field.
2. Count the number of identifiable platelets in 10 oil immersion fields by using only the area where 50D/o of the red cells are overlapping in doublets or triplets.
3. The number obtained is reported as Platelet Estimate: __________ x 10^9
4. Multiply the average number by the platelet estimation factor.

Platelet Estimation Factor procedure

1. Prepare smears from each sample tested.
2. Using at least 30 specimens from healthy subjects, perform machine platelet count in a hematology analyzer. Record results.
3. Divide the total number of platelets found by 10 to obtain the average number per single oil-immersion field.
4. For each of the 30 smears, count and record the number of platelets in 10 consecutive fields. Use only the area where 50% of the red cells are overlapping in doublets or triplets.
5. Add all individual PEF from 30 specimens and divide by 30 to obtain the average PEF. Round-off the number to the nearest digit and use this number as the PEF.
6. For each specimen, divide the corresponding machine count by the average number of platelets per oil-immersion field to obtain individual platelet estimation factors (PEF)

Reticulocyte Count procedure

1. Place three drops each of reagent and blood sample in a test tube.
2. Using oil immersion lens, count the number of reticulocytes per 1000 erythrocytes counted and record.
3. Allow the blood and reagent mixture to stand for 15-20 minutes.
4. With the use of a pasteur pipette (or any of equivalent), place 1 drop of the mixture on one side of clean slide.
5. Mix blood and reagent by swirling the tube.
6. Calculate the percent erythrocytes using the following formula: Number of reticulocytes counted/1000 RBC x 1,000 = Reticulocyte ct. in %
7. Prepare a thin blood dye film in usual manner and allow to dry.

Erythrocyte Sedimentation Rate (ESR) procedure

1. Allow the tube to stand in a vertical position in the Westergen rack exactly one hour at room temperature without vibration or exposure to direct sunlight.
2. Collect venous blood into an EDTA (Ethylene Diamine Tetraacetic Acid) tube or oxalated vacuum tube. Mix well but gently.
3. Check that the Westergren tube is filled with blood up to the zero mark.
4. Uncap the tube and insert gently the commercially available Westergren tube unto the tube with venous tube.
5. After exactly 60 minutes or one hour, the distance Pom the 0 mark to the top of the column of red cells is recorded in milliliters as the ESR value. If the demarcation between plasma and the red cell column is hazy, the level is taken where the full density is first apparent

Bleeding Time procedure

1. At interval of 30 seconds, blot the blood which has oozed out with absorbent or filter paper.
2. Let it dry.
3. Clean the ring finger of the patient with cotton and alcohol.
4. Start timer.
5. Make a deep puncture on the tip of the ring finger using sterile lancet.
6. Discard the first drop.
7. Record the time until the bleeding stop.

Clotting Time procedure

1. Obtain four drops of blood by prick method and place them separately on a clean slide Time simultaneously.
2. Record the results
3. At intervals of one minute, draw the tip of the needle to each of the drops successively. The appearance of fibrin at the point of the needle is the coagulation time

NEQAS Sample Processing and Testing

1. Roll the vial gently between the palms of the hands for 20 seconds in the upright position, invert vial and roll 20 seconds more.
2. Allow previously refrigerated vials to stand at room temperature (20- 24°C) for at least 15 minutes before mixing.
3. Process the samples using the routine blood count analysis method of your Laboratory.
4. Verify that the vial cap is securely in place. Mix the vial by gently end-to- end inversion until the cell is completely re-suspended.

NEQAS Data Reporting

1. Report results according to the units and decimal places specified in the Survey Result Form.
2. Fill up the Survey Result Form/s completely and send back the form to the NRL either via email or personal delivery.

Analyzing Commercial Control on DxH 560

1. • On the Daily Checks screen, select Daily Checks.
2. • The status LED turn red throughout tge Daily Checks process.
3. • Run daily checks. Select the Daily Checks icon on the Maine Menu.
4. • When the process is complete, the status LED turns green and results are displayed.

Cassette Mode Procedure

1. • Select the QC sample lot number, Click display.
2. • To view the QC result, click the QC/QA icon on the screen.
3. • On the main menu, click the Display/Run icon.
4. • Review and validate the results.
5. • Place the control tubes into the cassette making sure that the bar code labels are visible throughout the top of the cassette
6. • Load the cassette in the autoloader.
7. • Document quality control results in the designated logsheet.
8. • Select the start icon on the screen
9. • On the QC (Data View) Screen, out-of-range value are highlighted in red and flagged as L or H.

Analyzing Commercial Controls om DxH 520

1. • Store the control according to the instructions for use.
2. • Review and validate the quality control results.
3. • Mix the control according to the control's instructions for use.
4. • Select ✅ when prompted to confirm the information entered.
5. • Remove the control when the door opens and the control is finished processing.
6. • Document the quality control results in the designated logsheet.
7. • Use the barcode scanner to scan the control bar code.
8. • Fully insert the control into the tube holder and select RUN. The door automatically closes to initiate the analysis.
9. • Select the Display/Run icon to display the Sample Analysis-Patient Result screen. The door automatically opens.

Sample Analysis on DxH 560 (Closed-Vial/ Cassette Mode)

1. • Input Speciment ID.
2. • Select Start.
3. • Review the request. Put the sample into the cassette.
4. • Once done with the worklist, click Display/Run.
5. •Select New Order.
6. • Place the cassette into the autoloader.

Sample Analysis on DxH 560 (Open-vial Mode)
1. • Select Next
2. • Fully immerse the probe into the tube and select the tube icon to analyze the sample
3. • Select Open tube then Click ✅ when prompted to confirm selection.
4. • The status LED flashes red during aspiration indicating that the sample aspiration is in progress.
5. • After the analysis, the result are displayed on the screen and transmitted to the interface.
6. • Validate the result before printing.
7. • Click ✅
8. • Input the specimen ID.
9. • Mix the whole blood sample. Carefully remove the cap.
10. • On the Sample Analysis screen, select Single Tube

Sample Analysis on DxH 529 (Closed-Vial Mode)

1. • Input the Specimen ID using the on-screen keyboard
2. • Select Run and the door automatically closes.
3. • Click ✅ to save
4. • The door automatically opens.
5. • Mix the whole blood sample.
6. • Validate the results before printing.
7. • Select Next on the bottom right Sample Analysis Screen.
8. • Fully insert the sample into the tube holders.