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Anti-Com-DF (Rh-D PE/HbF-FITC) Reagent, Ready to Use
| Species Reactivity | Key Applications | Host | Format | Antibody Type |
|---|---|---|---|---|
| H | FC | Mouse | FITC | Monoclonal Antibody |
Description:
Anti-Com-DF (Rh-D PE/HbF-FITC) Reagent, Ready to Use
Trade Name:
Chemicon (Millipore)
Specificity:
This reagent recognizes both the Rh D antigen found on the red blood cells (RBC) of Rh D positive individuals and Fetal Hemoglobin (HbF) of human red blood cells.
This test is confined to use on maternal blood samples from Rh D negative women while pregnant or following delivery of Rh D positive babies. FMH during pregnancy may be caused by threatened miscarriage, chorionic villus sampling, placenta abruption, abdominal trauma or trauma induced by external cephalic version. FMH also may occur as a result of ectopic pregnancy, spontaneous abortion or termination of pregnancy. The test should be performed on maternal blood after any such event(NHMRC Guidelines, 1996). In addition, the magnitude of FMH should be estimated following delivery of Rh D positive infants(NHMRC Guidelines, 1996). The test should be carried out soon after the precipitating event or delivery in order to permit the timely administration of the appropriate amount of Rh D immunoglobulin to prevent alloimmunization occurring in the mother.
This test is confined to use on maternal blood samples from Rh D negative women while pregnant or following delivery of Rh D positive babies. FMH during pregnancy may be caused by threatened miscarriage, chorionic villus sampling, placenta abruption, abdominal trauma or trauma induced by external cephalic version. FMH also may occur as a result of ectopic pregnancy, spontaneous abortion or termination of pregnancy. The test should be performed on maternal blood after any such event(NHMRC Guidelines, 1996). In addition, the magnitude of FMH should be estimated following delivery of Rh D positive infants(NHMRC Guidelines, 1996). The test should be carried out soon after the precipitating event or delivery in order to permit the timely administration of the appropriate amount of Rh D immunoglobulin to prevent alloimmunization occurring in the mother.
Background Information:
Currently, there is a world shortage of Rh D immunoglobulin for immunoprophylaxis. Administration of the appropriate dose is encouraged (NHMRC Guidelines, 1996) to ensure adequate protection against alloimmunization while conserving a valuable resource. To determine the appropriate dose, it is necessary to quantitate the magnitude of a suspected FMH and administer Rh D immunoglobulin according to the extent of the fetal bleed. The required dose is estimated on the basis that 20mg of Rh D immunoglobulin will suppress immunization by 1ml D-positive red cells (2ml fetal blood). Pack sizes for Rh D immunoglobulin vary. In Australia, 125μg is considered to be sufficient to suppress immunization by up to 12ml of fetal blood. (NHMRC Guidelines, 1996). Assuming a maternal blood volume of 5000ml, a hemorrhage of this magnitude would be detectable in the maternal circulation as approximately 0.25% of the red cell population or 1/400 cells.
The use of flow cytometry to quantitate minor populations of Rh D and HbF positive cells has been explored by a number of workers(Nance et al, 1989-Lloyd-Evans, et al 1996). The capacity of the flow cytometer to analyze a large number of cells in a short period of time enables accurate quantitation of FMH at clinically relevant levels. Com-DF is designed for use with a flow cytometer. The test is simple to perform, rapid, reliable and objective.
The use of flow cytometry to quantitate minor populations of Rh D and HbF positive cells has been explored by a number of workers(Nance et al, 1989-Lloyd-Evans, et al 1996). The capacity of the flow cytometer to analyze a large number of cells in a short period of time enables accurate quantitation of FMH at clinically relevant levels. Com-DF is designed for use with a flow cytometer. The test is simple to perform, rapid, reliable and objective.
Species Reactivity:
Human
Application Notes:
This reagent is designed for the detection and quantitation of Rh D positive and HbF positive RBC arising in the circulation of mothers as a consequence of fetomaternal hemorrhage (FMH). The procedure requires prefixation and permeabilization of the cells followed by a single staining step and analysis using a flow cytometer.
STAINING PROCEDURE:
1. Collect maternal blood into EDTA anticoagulant. (Sample must be drawn prior to administration of Rh D immunoglobulin.)
2. Mix thoroughly and dilute the blood 1/10 in wash buffer (see Reagent Preparation), vortex gently and dispense 20 μl of the diluted blood into the bottom of a flow cytometer tube.
3. Add 1 ml of cold freshly prepared Prefixation solution (see Reagent Preparation). Vortex and incubate for 10 minutes at room temperature.
4. Add 2ml of wash buffer, mix and centrifuge to achieve gentle sedimentation of RBC. Repeat for a total of 3 washes.
5. Carefully decant or aspirate by vacuum suction discarding the supernatant.
6. Resuspend the RBC pellet in 0.5 ml of Permeabilization solution (see Reagent Preparation) by gentle vortexing. Incubate for 3-5 minutes at room temperature.
7. Wash once with 2 ml of wash buffer.
8. Carefully discard or aspirate the supernatant.
9. Add 50μl of Com-DF™ to the RBC pellet.
10. Vortex gently and incubate at room temperature in the dark for 15 minutes.
11. Add 2 ml of wash buffer, mix and centrifuge at 600 x g.
12. Decant or aspirate, discarding the supernatant.
13. Resuspend the RBC pellet in 0.6ml of fixing solution (see Reagent Preparation) and proceed to data acquisition by flow cytometry. Store cells at 4ºC in the dark prior to analysis if preferred.
ANALYSIS: Standards: To optimize your flow cytometer settings, use of one of the following standards is recommended.
Chemicon Fetal Control Kit (FT100), which consists of 3 stabilized, prepared controls of human RhD negative adult erythrocytes, supplemented with human RhD positive cord blood erythrocytes. The three controls represent no fetal cells, low and high levels of fetal RhD positive RBC.
Blood samples from ABO compatible, D-negative and D-positive donors arranged to give:
· 100% D-negative cells
· a mixture comprising 99.75% D-negative and 0.25% D-positive cells
These standards should be prepared fresh and 20 ml of a 1:10 dilution in wash buffer run in parallel with each batch of samples tested.
Data acquisition: For each test, collect 50,000 events gating on the singlet RBC population.
Data analysis: The vertical scale of fluorescence intensity histograms should be adjusted so that small HbF and D-positive populations can be visualized. Results should be expressed to 2 decimal places.
LIMITATIONS
OF THE TEST: 1. This test has the capacity to quantify FMH.
2. In cases of ABO incompatibility between mother and child, the natural ABO antibodies of the mother may destroy fetal cells in the maternal circulation before testing is performed.
STAINING PROCEDURE:
1. Collect maternal blood into EDTA anticoagulant. (Sample must be drawn prior to administration of Rh D immunoglobulin.)
2. Mix thoroughly and dilute the blood 1/10 in wash buffer (see Reagent Preparation), vortex gently and dispense 20 μl of the diluted blood into the bottom of a flow cytometer tube.
3. Add 1 ml of cold freshly prepared Prefixation solution (see Reagent Preparation). Vortex and incubate for 10 minutes at room temperature.
4. Add 2ml of wash buffer, mix and centrifuge to achieve gentle sedimentation of RBC. Repeat for a total of 3 washes.
5. Carefully decant or aspirate by vacuum suction discarding the supernatant.
6. Resuspend the RBC pellet in 0.5 ml of Permeabilization solution (see Reagent Preparation) by gentle vortexing. Incubate for 3-5 minutes at room temperature.
7. Wash once with 2 ml of wash buffer.
8. Carefully discard or aspirate the supernatant.
9. Add 50μl of Com-DF™ to the RBC pellet.
10. Vortex gently and incubate at room temperature in the dark for 15 minutes.
11. Add 2 ml of wash buffer, mix and centrifuge at 600 x g.
12. Decant or aspirate, discarding the supernatant.
13. Resuspend the RBC pellet in 0.6ml of fixing solution (see Reagent Preparation) and proceed to data acquisition by flow cytometry. Store cells at 4ºC in the dark prior to analysis if preferred.
ANALYSIS: Standards: To optimize your flow cytometer settings, use of one of the following standards is recommended.
Chemicon Fetal Control Kit (FT100), which consists of 3 stabilized, prepared controls of human RhD negative adult erythrocytes, supplemented with human RhD positive cord blood erythrocytes. The three controls represent no fetal cells, low and high levels of fetal RhD positive RBC.
Blood samples from ABO compatible, D-negative and D-positive donors arranged to give:
· 100% D-negative cells
· a mixture comprising 99.75% D-negative and 0.25% D-positive cells
These standards should be prepared fresh and 20 ml of a 1:10 dilution in wash buffer run in parallel with each batch of samples tested.
Data acquisition: For each test, collect 50,000 events gating on the singlet RBC population.
Data analysis: The vertical scale of fluorescence intensity histograms should be adjusted so that small HbF and D-positive populations can be visualized. Results should be expressed to 2 decimal places.
LIMITATIONS
OF THE TEST: 1. This test has the capacity to quantify FMH.
2. In cases of ABO incompatibility between mother and child, the natural ABO antibodies of the mother may destroy fetal cells in the maternal circulation before testing is performed.
Presentation:
Supplied in phosphate buffered saline, pH 7.4, containing 0.1% sodium azide and 0.2% bovine serum albumin.
Blood collection tubes containing EDTA as anti-coagulant.
Test tubes compatible with the sampling system of the flow cytometer to be used.
Micropipettors.
Vortex mixer.
Benchtop centrifuge suitable for sedimentation of RBC.
Wash buffer: Phosphate buffered saline, pH 7.4, containing 0.1% sodium azide and 0.2% bovine serum albumin.
Prefixation Solution: 0.05% Glutaraldehyde in PBS
Permeabilization Solution: 0.1% Triton X-100 in PBS containing 0.1% bovine serum albumin
Fixing solution: 1% paraformaldehyde in phosphate buffered saline.
Flow cytometer equipped with a laser capable of exciting FITC and R-Phycoerythrin (488nm wavelength) and a filter configuration suitable for detection of the emitted signals.
Blood collection tubes containing EDTA as anti-coagulant.
Test tubes compatible with the sampling system of the flow cytometer to be used.
Micropipettors.
Vortex mixer.
Benchtop centrifuge suitable for sedimentation of RBC.
Wash buffer: Phosphate buffered saline, pH 7.4, containing 0.1% sodium azide and 0.2% bovine serum albumin.
Prefixation Solution: 0.05% Glutaraldehyde in PBS
Permeabilization Solution: 0.1% Triton X-100 in PBS containing 0.1% bovine serum albumin
Fixing solution: 1% paraformaldehyde in phosphate buffered saline.
Flow cytometer equipped with a laser capable of exciting FITC and R-Phycoerythrin (488nm wavelength) and a filter configuration suitable for detection of the emitted signals.
Storage Conditions:
Store at 2-8°C, for up to 6 months. DO NOT FREEZE. Protect from light.
WARNINGS AND PRECAUTIONS
Test specimens are potentially infectious and should be handled with appropriate precautions.
The reagent contains sodium azide as a preservative. Unwanted reagent should be disposed of with liberal volumes of water to prevent potential hazards arising from the build-up of this material in metal plumbing.
WARNINGS AND PRECAUTIONS
Test specimens are potentially infectious and should be handled with appropriate precautions.
The reagent contains sodium azide as a preservative. Unwanted reagent should be disposed of with liberal volumes of water to prevent potential hazards arising from the build-up of this material in metal plumbing.
UniProt Number:
Entrez Gene Number:
Gene Symbol:
- RHD
- RhII
- RhK562-II
- Rh4
- Dpolypeptide
- RHDel
- RH30
- MGC165007
- RhDCw
- RHCED
- RH
- RHXIII
- RhPII
- DIIIc
- Rh30a
- RHDVA(TT)
- RHPII
- RhPI
- CD240D
Components:
Com-DF: This is ready to use at 50μL/test and no further preparation is required.
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
The Rh blood group system is the second most clinically significant of the blood groups, second only to ABO. It is also the most polymorphic of the blood groups, with variations due to deletions, gene conversions, and missense mutations. The Rh blood group includes this gene, which encodes the RhD protein, and a second gene that encodes both the RhC and RhE antigens on a single polypeptide. The two genes, and a third unrelated gene, are found in a cluster on chromosome 1. The classification of Rh-positive and Rh-negative individuals is determined by the presence or absence of the highly immunogenic RhD protein on the surface of erythrocytes.
Key Applications:
Flow Cytometry
UniProt Summary:
FUNCTION: SwissProt: Q02161 # May be part of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane.
SIZE: 417 amino acids; 45180 Da
SUBCELLULAR LOCATION: Membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Restricted to tissues or cell lines expressing erythroid characters.
SIMILARITY: SwissProt: Q02161 ## Belongs to the ammonium transporter (TC 2.A.49) family. Rh subfamily.
SIZE: 417 amino acids; 45180 Da
SUBCELLULAR LOCATION: Membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Restricted to tissues or cell lines expressing erythroid characters.
SIMILARITY: SwissProt: Q02161 ## Belongs to the ammonium transporter (TC 2.A.49) family. Rh subfamily.
Qty/Pk:
100 assays
Brand Family:
Chemicon
Format:
FITC
Host:
Mouse
Product Name:
Anti-Com-DF (Rh-D PE/HbF-FITC) Reagent, Ready to Use
Antibody Type:
Monoclonal Antibody