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αβ χ δ CONTROLLED DOCUMENT 1
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
REVIEW
Annual Review is monitored in Q-Pulse – check there if there is any doubt whether this is
the current version of this document
1 INTRODUCTION
2 PRINCIPLE
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.
2.4 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.
3 RESPONSIBILITY
Note that only Senior BMSs and Chief BMSs are to sign staff off as competent to
work unsupervised.
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:
4.1.2 Work areas are to be kept clean with Biocleanse (available at each
workstation).
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.
Investigation of FMH by Flow Cytometry Version 1
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Blood Sciences - Immunophenotyping
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.
5 SAMPLE REQUIREMENTS
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.
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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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.
7.2.2 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 If any of the results are outside of accepted range, the experiment
should be repeated.
7.4.4 If control results can still not be corrected, contact a senior member of
staff.
8.2 Results should be reported at the same time as the results for the AE slide
technique performed in blood transfusion.
• FACSCanto II
• Falcon Tubes
• Capped Falcon Tubes
• Pastettes
• 200-1000µL Gilson Pipette
• 20-200µL Gilson Pipette
• 1-10µL Gilson Pipette
• Universals
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
11.1.1 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.
11.1.9 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)
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.
11.1.17 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).
11.3 Analysis
11.3.1 Gate the RBCs using the Log FSC/SSC plot as shown below
11.3.2 Using the SSC/CD45 plot gate the RBC population excluding any WBC
which will be CD45 positive
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.
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.
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:
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.
or simplified to:
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
Investigation of FMH by Flow Cytometry Version 1
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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.
12.7 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
14 TRANSCRIPTION
15 REFERENCE RANGE
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.
17 REFERENCES
17.3 British Committee for Standards in Haematology (BCSH 2008). Guidelines for
the Estimation of Fetomaternal Haemorrhage.
Appendix 1
Patient
________________________
Tube 1
Consultant Tube 2
Average
____________________
Destination Bleed =
Std
____________________ Deviations =
Referral Ref (if
external)
CV =
____________________
Control Range:
1%
0.25%
Results Checked
_______
Appendix 2
Fig 1. Taken from BCSH Guidelines for the Estimation of Fetomaternal Haemorrhage