Investigation of FMH by Flow Cytometry

LABORATORY INSTRUCTION Copy No:
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Blood Sciences - Immunophenotyping
Investigation of Fetomaternal
DOCUMENT DETAILS
Section: Immunophenotyping
Document type: Laboratory Procedure Q-Pulse ID: IP.SOP11
Revision date: 11.01.2011 Revision: 1.0
Issue date: 11.01.2010 Status: Active
Implementation Authorised
n/a 11.01.2010
period: date:
Haemorrhage by Flow Cytometry
AUTHORSHIP AND APPROVALS
Approval is monitored electronically in Q-Pulse
Author Claire Stephens Chief Biomedical Scientist
Approver SOP Validation
Committee
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Investigation of FMH by Flow Cytometry Version 1

Page 1 of 15
1 INTRODUCTION
This SOP covers the flow cytometric investigation of fetomaternal haemorrhage.
2 PRINCIPLE
2.1 Fetomaternal haemorrhage (FMH) may occur either during pregnancy or at

delivery, and if the mother is RhD negative and the baby RhD positive this can
lead to the immunisation to the D antigen. This can result in haemolytic
disease of the newborn (HDN) in subsequent pregnancies. Therefore it is
important to assess the volume of FMH to determine the dose of anti-D
immunoglobulin required by an RhD negative woman in order to prevent
sensitisation.
2.2 Guidelines for the use of anti-D immunoglobulin for Rh prophylaxis (BCSH
2006a) states that at least 500iu of anti-D immunoglobulin should be given to
RhD negative women within 72 hours of delivery of a RhD positive baby. This
dose will be sufficient to prevent sensitisation from a bleed of up to 4mL fetal
red cells. ~1% of women have a FMH of greater than 4mL and up to 0.3% of
women have a FMH of greater than 15mL. They may not be protected by a
500iu and 1500iu dose of anti-D immunoglobulin respectively, therefore it is
important for accurate confirmation of FMH in order to determine if a
supplementary dose of anti-D immunoglobulin is required.
2.3 BCSH Guidelines for the Estimation of Fetomaternal Haemorrhage 2008

suggest that due to the increased CVs in acid elution (AE) staining compared
to those obtained by flow cytometry, any patient with a bleed of 2mLs or
greater detected by AE needs to be sent for accurate confirmation by flow
cytometry.
2.4 The blood transfusion department will screen for any possible FMH and send
any positive or indeterminate samples for confirmation by flow cytometry.
Samples may also be referred from external hospitals for confirmation of
FMH.
2.5 Due to the importance of providing anti-D within 72 hours of delivery of a RhD
positive baby to a RhD negative mother, FMH tests will be performed 3 times
a week, Monday Morning, Wednesday and Friday Afternoon. The test will not
be performed out of hours at present.

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3 RESPONSIBILITY
Grade of Staff Can perform task Can perform Is NOT allowed to

unsupervised task under perform this task
when deemed supervision of
competent qualified,
competent BMS
MLA √
A&C √
Associate √
Practitioner
Trainee BMS/CS √
Band 5 BMS √
Band 6 BMS √
Registered CS √
Senior BMS √
Chief BMS √
Note that only Senior BMSs and Chief BMSs are to sign staff off as competent to
work unsupervised.
4 POTENTIALLY HAZARDOUS STEPS
All department Health & Safety Policies must be read and followed when working in
the laboratory. In particular:
4.1 Risk and COSHH assessments have been performed. The main points to note
are that:
4.1.1 Personal protective equipment (latex or nitrile gloves as well as

correctly-fastened laboratory coats) must be worn in the department
when handling patient or control samples to protect against accidental
splashing.
4.1.2 Work areas are to be kept clean with Biocleanse (available at each
workstation).
4.1.3 All biological spills must be cleaned according to “Leaking blood

samples and Spillages SOP”HGENSOP].
4.1.4 All contaminated gloves, tissues, etc. must be placed in an orange bag.
All pipette tips and Pasteur pipettes must be placed into a sharpsafe.
Orange bags must be disposed of in a yellow Clinical Waste Bin
designated for orange bags. Refer to Retention of Clinical Waste SOP
[HGENSOP].
4.2 Material Safety Data Sheets (MSDSs) for all reagents are available.
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4.3 In case of accident, notify supervisor, manager or Health and Safety

Representative and complete Datix form.
4.4 Complete safety procedures are beyond the scope of this document. Refer to
the Department Health and Safety Manual for complete safety procedures
to be followed during the performance of this as well as all other procedures
within the laboratory.
NOTE: All samples and control material should be treated as potentially

infectious samples.
5 SAMPLE REQUIREMENTS
5.1 Sample Collection
Whole blood samples should be collected in to a 6.7mL EDTA vacutainer. A

minimum of 2mLs should be collected to ensure a group and save can also be
performed on the sample.
5.2 Criteria for Sample Rejection
5.2.1 Samples >72 hours are unsuitable for analysis. A comment should be
made in WinPath and the relevant ward/hospital contacted.
5.2.2 Insufficient samples should have the comment @ins entered into
WinPath and the relevant ward/hospital contacted.
5.3 Sample Stability
Samples should be kept at room temperature and analysed within 72 hours.
6 CALIBRANTS
Not Applicable
7 INTERNAL CONTROL
RhD positive and RhD negative cells are acquired from blood transfusion. No
controls are bought in.
7.1 General
7.1.1 Acquire a RhD positive and RhD negative sample from transfusion,
ensure that no further tests are required. In the case of the RhD
negative sample ensure the sample is not from an antenatal patient.
7.1.2 Ensure the sample is well mixed and aliquot approximately 2mLs into
two labelled falcon tubes (one for RhD+, one for RhD-). Centrifuge the
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samples at 3000rpm for 10 minutes and remove plasma and buffy

coats.
7.1.3 Wash samples twice in Cellwash (centrifuge can now be returned to

2200rpm and 4 minutes).
7.1.4 Remove 500µL and place into a universal. Add 9.5mL DiaMed ID
Diluent 2 and gently mix by inversion.
7.1.5 Measure the haematocrit (HCT) on the Sysmex Full Blood Count
analyser. Adjust the concentration to give a 5% dilution (HCT should be
0.05 on analyser). It may be necessary to add more diluent or cells to
achieve this.
7.1.6 Prepare a 1% and 0.2% dilution of RhD+ cells:
1% = 2475µL RhD negative cells + 25µL RhD positive cells
0.2% = 1000µL RhD negative cells + 250µL of 1% dilution
7.1.7 Ensure the controls are well mixed prior to use.
7.2 Criteria for accepting control results
7.2.1 The 1% control is used to help differentiate between positive and

negative events. The 1% control range has been determined as 0.87-
1.12%
7.2.2 The 0.25% control is included to control the technique at the level at
which anti-D above the minimum standard dose would be required. The
0.25% control range has been determined as 0.16 – 0.27%.
7.2.3 The negative control should contain no positive events.
7.2.3 If any of the results are outside of accepted range, the experiment
should be repeated.
7.3 Factors Affecting Quality Control
• Inadequate mixing of control blood sample

• Incorrect incubation time.
• Incorrect incubation temperature.
• Poor pipetting technique
• Uncalibrated pipettes
• Contamination of reagents
• Defective flow cytometer laser

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7.4 Corrective Action
7.4.1 Out of range controls should be repeated in the first instance.
7.4.2 If the repeated control is out of range, select fresh controls.
7.4.3 If still out of range, use fresh reagents.
7.4.4 If control results can still not be corrected, contact a senior member of
staff.
7.4.2 All corrective action should be logged on the CAPA sheets.
8 EXTERNAL QUALITY ASSURANCE
8.1 Samples from UK NEQAS(FMH) will arrive via blood transfusion
8.2 Results should be reported at the same time as the results for the AE slide
technique performed in blood transfusion.
9 EQUIPMENT and SUPPLIES REQUIRED
• FACSCanto II
• Falcon Tubes
• Capped Falcon Tubes
• Pastettes
• 200-1000µL Gilson Pipette
• Universals
10 REAGENTS AND PREPARATION
NOTE: All reagents should be dated and initialled by preparer. Discard unlabelled
material.
• BD Cellwash
• BD FACSFlow
• Brad-3 FITC
• AEVZ FITC
• BD CD45 V500
• DiaMed ID Diluent 2
11 PROCEDURE
11.1 Screen Set-up

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11.1.1 Ensure all patient and control samples are mixed by gentle inversion
for a minimum of 10 times prior to use.
11.1.2 Patient samples must be performed in duplicate in conjunction with an
isotype control which will allow the detection of any non-specific
binding of the Brad-3 FITC antibody. The isotype control used is AEVZ
FITC which has been recommended by International Blood Group
Reference Laboratory (IBGRL)
11.1.3 Print FMH tube labels by double left clicking the panel label template
icon on the desktop then double left click FMH. Replace Name with
patient(s) surname.
11.1.4 Label 2 Falcon tubes 1% Control Dilution and 0.25% Control Dilution
and prepare the dilutions as stated above in 7.1
11.1.5 Label capped Falcon tubes Patient(s) Control Wash. Pipette 100µL of
each patient sample into the relevant tube.
11.1.6 Fill each wash tube with BD CellWash and gently mix. Centrifuge for 4
mins at 2200rpm.
11.1.7 Aspirate supernatant using a pastette taking care not to disturb the red
cell pellet. Gently resuspend red cells and wash once more.
11.1.8 Again aspirate supernatant using a pastette, and resuspend cells in

1mL of Cell Wash.
11.1.9 Label Capped Falcon tubes with control labels and patient labels.
11.1.10 Pipette the relevant amount of antibodies into each tube. (see the
table below)
Tube Label FITC V500

1% Control Brad-3 CD45
5µL 5µL
0.25% Control Brad-3 CD45
5µL 5µL
Negative Control Brad-3 CD45
5µL 5µL
Name Isotype Control AEVZ CD45
5µL 5µL
Name FMH 1 Brad-3 CD45
5µL 5µL
Name FMH 2 Brad-3 CD45
5µL 5µL
11.1.11 Add 50µL of washed cells to the relevant tube. 50µL of Patient D
positive 1 can be added to the Patient Isotype control tube.
11.1.12 Place caps on tubes securely and then gently vortex samples.
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11.1.13 Place in 37ºC water bath for 30 minutes with agitation at 10 and 20
minutes.
11.1.14 After incubation, remove tubes from water bath and fill with BD
CellWash.
11.1.15 Centrifuge at 2200rpm for 4 minutes.
11.1.16 Decant supernatant and add 2mL of BD CellWash to each tube.
11.1.17 Tubes are now ready for analysis. Ideally analyse as soon as
possible, if this is not possible, place tubes in the dark and analyse
within 1 hour.
11.2 Acquisition.
11.2.1 Login to FACSDiva and select FMH experiment and label appropriately
(see SOP Sample analysis using FACSCanto).
11.2.2 Ensure 500,000 RBC-WBC events are analysed.
11.3 Analysis
11.3.1 Gate the RBCs using the Log FSC/SSC plot as shown below
Figure 1. RBCs as seen on a log FSC/SSC plot
11.3.2 Using the SSC/CD45 plot gate the RBC population excluding any WBC
which will be CD45 positive

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Figure 2. SSC/CD45 plot showing RBC population without WBCs.
11.3.3 Using the patient isotype control tube, set the D Pos gate so that it is
placed as close to the negative AEVZ population as possible without
including any positive events.
Figure 3. Isotype control
11.3.4 Moving to the patient D positive tube, if there are D positive cells they
should be easily visualised within the D positive gate. The gate should
not need to be moved, from where it was set using the isotype control.

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Figure 4. RhD positive
11.3.5 Print the patient D positive result.
12 RECORDING AND CALULATION OF RESULTS
12.1 Attach the printed sheet to the Fetomaternal Haemorrhage Report Sheet. All
patient details should be filled out correctly, including the Batch No of Brad-3
and AEVZ used. (See appendix 1)
12.2 The % D positive fetal cells in all RBCs needs to be calculated. This is done
by:
Number of D Positive Events x 100 = % D Positive Fetal Cells

Number of RBC-WBC Events
Fill this result in the relevant area on the report sheet for controls and patients.
12.3 FMH is calculated using the formula (Mollinson 1972), which assumes that:
• The maternal red cell volume is 1800mL
• Fetal cells are 22% larger than maternal cells.
% D Positive Fetal Cells x 1800 x 1.22 = Bleed (mL)

100
or simplified to:
% D Positive Fetal Cells x 18 x 1.22 = Bleed (mL)
Fill this result in the relevant area on the report sheet for controls and patients.
12.4 Using the FMH Calculation Excel worksheet located on the desktop of both
Immunophenotyping computers, calculate the average bleed, standard
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deviation and coefficient of variation for patient bleeds. The patient results
should be less than 2 standard deviations and the CV should be <10%. If they
are greater then the experiment should be repeated.
12.5 Check the control results are within range, if out of range see section 7.4.
12.6 The amount of anti-D to be administered should be calculated. (see 15)
12.7 For bleeds >4mL a repeat sample should be obtained at 48-72hours post anti-
D administration. (See appendix 2)
12.8 Results should then be reported onto WinPath, and phoned to any external
hospital. If the results is from an internal source, contact the transfusion
laboratory on ext 60344 to inform them of the result.
13 UNITS
Results are reported in mLs
14 TRANSCRIPTION
Results are manually transcribed on the WinPath computer system.
15 REFERENCE RANGE
The amount of anti-D to be administered can be calculated as:

125iu for every 1mL bleed
Therefore it is recommended for bleeds:

<4mL 500iu of anti-D should be administered
4-10mL 1250iu anti-D should be administered
10-12mL 1500iu anti-D should be administered
16 LIMITATIONS OF ANALYSIS
16.1 This assay is only suitable for RhD Negative woman. For RhD positive women
who have had an intrauterine death or stillbirth and suspect FMH, estimation
should be performed using AE.
16.2 The possibility that the anti-D reagent may not have detected D variant cord
cells, although rare, should be taken into account when discrepancies arise
between the results of AE and flow cytometry on a sample.
16.3 The Brad-3 monoclonal anti-D has been shown to react with all RhD positive
cells except those of the rare DVI or Rohar types.
16.4 HbF is not quantitated by this assay at present.

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17 REFERENCES
17.1 IBGRL BRAD-3 Product insert
17.2 IBGRL AEVZ Product insert
17.3 British Committee for Standards in Haematology (BCSH 2008). Guidelines for
the Estimation of Fetomaternal Haemorrhage.

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Appendix 1
Fetomaternal Haemorrhage Report Sheet

Brad-3 Batch No: AEVZ Batch No:
In Lab (Date and Time)
Estimate of Maternal Red Cell Volume: 1800mL

Sample date: ___________
Fetal Red Cell Volume Correction Factor: 1.22
Analysis date: ____________
LAB NUMBER BARCODE % Fetal D Pos Cells x 18 x 1.22 = Bleed (mL)
Please stick form label here Controls % D Pos Bleed Accepted

Cells (Y/N)
1% Control
D.O.B. 0.25% Control
_________________ M / F
Neg Control
Hospital Number / MRN
Patient
________________________
Tube 1
Consultant Tube 2
Average
____________________
Destination Bleed =
Std
____________________ Deviations =
Referral Ref (if
external)
CV =
____________________
Patient Results should NOT be accepted if there is more than

2 Std Deviation difference and CV is >10%
Control Range:
1%
0.25%

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Audit Trail (Please initial)
Booked-in (Winpath) Interim Faxed (if external) _______

_______
Results Phoned (if external) _______
Results Entered
_______ Panel set up by _______
Results Checked
_______
Appendix 2

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Fig 1. Taken from BCSH Guidelines for the Estimation of Fetomaternal Haemorrhage

Page 15 of 15

Investigation of FMH by Flow Cytometry

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LABORATORY INSTRUCTION Copy No:

DISTRIBUTION LIST - LOCATION OF CONTROLLED COPIES OF THIS VERSION

CHANGES INCLUDED IN THIS REVISION

Investigation of FMH by Flow Cytometry Version 1

This SOP covers the flow cytometric investigation of fetomaternal haemorrhage.

2.1 Fetomaternal haemorrhage (FMH) may occur either during pregnancy or at

2.3 BCSH Guidelines for the Estimation of Fetomaternal Haemorrhage 2008

Investigation of FMH by Flow Cytometry Version 1

Grade of Staff Can perform task Can perform Is NOT allowed to

4 POTENTIALLY HAZARDOUS STEPS

4.1.1 Personal protective equipment (latex or nitrile gloves as well as

4.1.3 All biological spills must be cleaned according to “Leaking blood

4.3 In case of accident, notify supervisor, manager or Health and Safety

NOTE: All samples and control material should be treated as potentially

5.1 Sample Collection

Whole blood samples should be collected in to a 6.7mL EDTA vacutainer. A

5.2 Criteria for Sample Rejection

5.3 Sample Stability

Samples should be kept at room temperature and analysed within 72 hours.

samples at 3000rpm for 10 minutes and remove plasma and buffy

7.1.3 Wash samples twice in Cellwash (centrifuge can now be returned to

7.1.6 Prepare a 1% and 0.2% dilution of RhD+ cells:

1% = 2475µL RhD negative cells + 25µL RhD positive cells

0.2% = 1000µL RhD negative cells + 250µL of 1% dilution

7.1.7 Ensure the controls are well mixed prior to use.

7.2 Criteria for accepting control results

7.2.1 The 1% control is used to help differentiate between positive and

7.2.3 The negative control should contain no positive events.

7.3 Factors Affecting Quality Control

• Inadequate mixing of control blood sample

Investigation of FMH by Flow Cytometry Version 1

7.4 Corrective Action

7.4.1 Out of range controls should be repeated in the first instance.

7.4.2 If the repeated control is out of range, select fresh controls.

7.4.3 If still out of range, use fresh reagents.

7.4.2 All corrective action should be logged on the CAPA sheets.

8 EXTERNAL QUALITY ASSURANCE

8.1 Samples from UK NEQAS(FMH) will arrive via blood transfusion

9 EQUIPMENT and SUPPLIES REQUIRED

10 REAGENTS AND PREPARATION

11.1 Screen Set-up

Investigation of FMH by Flow Cytometry Version 1

11.1.8 Again aspirate supernatant using a pastette, and resuspend cells in

Tube Label FITC V500

11.1.15 Centrifuge at 2200rpm for 4 minutes.

11.1.16 Decant supernatant and add 2mL of BD CellWash to each tube.

11.2.2 Ensure 500,000 RBC-WBC events are analysed.

Figure 1. RBCs as seen on a log FSC/SSC plot

Investigation of FMH by Flow Cytometry Version 1

Figure 2. SSC/CD45 plot showing RBC population without WBCs.

Figure 3. Isotype control

Investigation of FMH by Flow Cytometry Version 1

Figure 4. RhD positive

11.3.5 Print the patient D positive result.

12 RECORDING AND CALULATION OF RESULTS

Number of D Positive Events x 100 = % D Positive Fetal Cells

% D Positive Fetal Cells x 1800 x 1.22 = Bleed (mL)

% D Positive Fetal Cells x 18 x 1.22 = Bleed (mL)

12.6 The amount of anti-D to be administered should be calculated. (see 15)

Results are reported in mLs

Results are manually transcribed on the WinPath computer system.

The amount of anti-D to be administered can be calculated as:

Therefore it is recommended for bleeds: