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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects' erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

Abstract

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100–1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer's disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

Introduction

CR1 (complement receptor type 1, CD35) is a 200 kDa transmembrane glycoprotein present on the surface of many cell types, such as erythrocytes1, B lymphocytes2, monocytic cells, some T cells, follicular dendritic cells3, fetal astrocytes4, and glomerular podocytes5. CR1 interfering with its ligands C3b, C4b, C3bi6,7,8,9, a subunit of the first complement component, C1q10 and MBL (mannan-binding lectin)11 inhibits the activation of complement and is involved in humoral and cellular immune response.

In primates, including humans, erythrocyte CR1 is involved in the transport of immune complexes to the liver and spleen, to purify the blood and prevent their accumulation in vulnerable tissues such as the skin or kidneys12,13,14. This phenomenon of immune adhesion between immune complexes and erythrocytes depends on the number of CR1 molecules15. In humans, the mean density of CR1/E is only 500 (i.e., 500 molecules of CR1 per erythrocyte). This density varies from one individual to another (100–1,200 CR1/E) and from one erythrocyte to another in the same individual. Some individuals of "null" phenotype express fewer than 20 CR1/E16.

The density of CR1/E is regulated by two co-dominant autosomal alleles linked to a point mutation in intron 27 of the gene coding for CR1*117,18. This mutation produces an additional restriction site for the HindIII enzyme. The restriction fragments obtained after digestion with HindIII in this case are 7.4 kb for the allele linked to a strong expression of CR1 (H: high allele) and 6.9 kb for the allele linked to low CR1 expression (L: low allele). This link is found in Caucasians and Asians but not in people of African descent19.

The level of expression of erythrocyte CR1 is also correlated with the presence of point nucleotide mutations in exon 13 encoding SCR 10 (I643T) and in exon 19 encoding SCR16 (Q981H). It is high in homozygous 643I/981Q and low in homozygous 643T/981H individuals20. Thus, "low" individuals express around 150 CR1/E, "medium" individuals express around 500 CR1/E, and "high" individuals express around 1,000 CR1/E.

In addition to this erythrocyte density polymorphism, CR1 is characterized by a length polymorphism corresponding to four allotypes of different sizes: CR1*1 (190 kDa), CR1*2 (220 kDa), CR1*3 (160 kDa), and CR1*4 (250 kDa)21 and an antigenic polymorphism corresponding to the blood group KN22.

We present our method based on flow cytometry to determine the density of CR1/E. Using three subjects whose CR1/E density is known, expressing a low density level (180 CR1/E), a medium density level (646 CR1/E), and a high density level (966 CR1/E), it is easy to measure the mean fluorescence intensity (MFI) of their erythrocytes or red blood cells (RBC), or RBC MFI, after anti-CR1 immunostaining using a flow cytometer. One can then plot a standard line representing the MFI as a function of CR1/E density. Measuring the MFI of subjects whose CR1/E density is not known and comparing it to this standard line, it is possible to determine the individuals' CR1/E density. This technique has been used for many years in the laboratory, and has enabled us to detect a reduction in the expression of erythrocyte CR1 in many pathologies such as systemic lupus erythematosus (SLE)23, Acquired immunodeficiency syndrome (AIDS)24, malaria25, and recently Alzheimer's disease (AD)26,27. The development of drugs targeting CR1 to couple with erythrocytes, as in the case of anti-thrombotic drugs28 requires the evaluation of CR1/E density, and the availability of a robust technique to quantify CR1.

The protocol presented runs in singlicate. It is adaptable to determine the density of CR1/E on many individuals using specific commercially available 96 well plates (see Table of Materials). To this end, it is easy to adapt our method to any 96 well plate. For each sample, a cell suspension of erythrocytes (0.5 x 106–1 x 106 erythrocytes) is distributed per well. For each well, first the primary anti-CR1 antibody is added, then streptavidin PE, the secondary anti-streptavidin antibody, and again streptavidin PE, using the same dilutions as those of our method, but by adapting volumes and respecting proportionality.

The blood samples from subjects of the range and from subjects to be quantified for CR1 should be drawn at the same time, stored in the refrigerator at 4 °C, and handled at 4 °C (on ice and/or in the refrigerator).

Protocol

The protocol for human blood collection and handling was reviewed and approved by the regional ethics committee (CPP Est II), and the protocol number is 2011-A00594-37. Because the following protocol describes the handling of human blood, institutional guidelines for disposing of biohazardous material should be followed. Laboratory safety equipment, such as lab coats and gloves, should be worn.

1. Erythrocyte washing

NOTE: The day before handling, prepare a PBS-BSA buffer with phosphate buffered saline (PBS) containing 0.15% of bovine serum albumin (BSA) and place it in the refrigerator at 4 °C. This buffer will be used as a washing buffer and as a dilution buffer.

  1. Pipette 20 mL of PBS-BSA into a 50 mL tube.
  2. Aspirate 250 µL of sodium ethylenediamine tetraacetic acid (EDTA) anticoagulated whole blood from blood storage tubes and add to the tube containing the 20 mL of PBS-BSA. Close the tube by screwing the cap. Mix gently by inverting the tube 2x.
  3. Centrifuge the tube for 10 min at 4 °C at 430 x g. Remove and discard the supernatant using a 10 mL pipette. Resuspend the pellet in the residual volume of supernatant by gentle and careful pipetting.
  4. Add 20 mL of cold PBS-BSA (4 °C) into the tube containing the pellet. Centrifuge the tube for 10 min at 4 °C at 430 x g. Remove and discard the supernatant using a 10 mL pipette.
  5. Add 20 mL of cold PBS-BSA (4 °C) into the tube containing the pellet. Centrifuge the tube for 10 min at 4 °C at 430 x g. Leave the tube in the centrifuge at 4 °C and go to section 2.

2. Erythrocyte dilution

  1. Pipette 3 mL of cold PBS-BSA into a 50 mL tube and store it at 4 °C on a rack in ice.
  2. Put the centrifuged tube containing the erythrocytes (step 1.4) on a rack placed in ice.
  3. Pipette 8 µL of pelleted erythrocytes using the pipette and add to the 50 mL tube containing the 3 mL of PBS-BSA to obtain the erythrocyte dilution. Mix the tube gently by hand to obtain a homogeneous cell suspension of erythrocytes.

3. Erythrocyte immunostaining

  1. Pipette 100 µL of erythrocyte dilution (obtained in section 4) and add to 1.4 mL tubes.
  2. Centrifuge the tubes for 5 min at 4 °C at 430 x g. During centrifugation, prepare a dilution of biotinylated anti-CR1 J3D3 antibody at a concentration of 0.05 μg/μL in PBS-BSA buffer.
  3. Once the centrifugation is done, remove and discard the supernatant.
  4. Add 20 µL of biotinylated anti-CR1 J3D3 directly to the pellet. To prepare the negative control, add 20 µL of PBS-BSA buffer instead. Mix the tubes gently and incubate for 45 min at 4 °C.
  5. After 45 min of incubation, add 750 µL of PBS-BSA to the tubes. Centrifuge the tubes for 5 min at 4 °C at 430 x g. Remove and discard the supernatant. Repeat.
  6. In the meantime, prepare a 1:10 dilution of streptavidin-phycoerythrin diluted in PBS-BSA buffer. Pipette 20 µL of the 1:10 dilution of streptavidin-phycoerythrin and add to the tubes. Mix the tubes gently and incubate for 45 min at 4 °C.
  7. Add 750 µL of PBS-BSA buffer into the tubes. Mix well and centrifuge for 5 min at 4 °C at 430 x g. Remove and discard the supernatant. Repeat.
  8. During centrifugation, prepare a 1:100 dilution of biotinylated anti-streptavidin antibody diluted in PBS-BSA buffer.
  9. Once the centrifugation is done, remove and discard the supernatant.
  10. Pipette 20 µL of the 1:100 dilution of biotinylated anti-streptavidin into the tubes. Mix the tubes gently. Incubate the tubes for 45 min at 4 °C.
  11. After 45 min of incubation, pipette 750 µL of PBS-BSA buffer and add into the tubes. Centrifuge the tubes for 5 min at 4 °C at 430 x g. Remove and discard the supernatant. Repeat.
  12. Pipette 20 µL of the 1:10 dilution of streptavidin-phycoerythrin and add into the tubes. Mix the tubes gently. Incubate the tubes for 45 min at 4 °C.
  13. Pipette 750 µL of PBS-BSA buffer and add into the tubes. Mix well and centrifuge the tubes for 5 min at 4 °C at 430 x g. Remove and discard the supernatant. Repeat this step 2x.

4. Immunostained erythrocyte fixation

  1. During the last centrifugation, prepare the fixation buffer, a 1:100 dilution of 37% formaldehyde using the washing buffer PBS-BSA.
  2. Pipette 450 μL of fixation buffer and add into immunostained erythrocyte tubes (from step 3.13) while vortexing for 5 s.
  3. Pipette all fixed cells into 5 mL round bottom tubes and store in the refrigerator.
    NOTE: The protocol can be paused here for up to 48 h.

5. Flow cytometry analysis of stained erythrocytes

NOTE: It is advisable to refer to the operator's manual for the cytometer (see Table of Materials) to know how to perform the cytometric readings. The suggested parameters below apply to the instrument used and must be optimized for each cytometer.

  1. Turn on the flow cytometer, then turn on the computer. Let the optical system temperature stabilize by leaving it on for 30 min. Check the cytometer window in the software to ensure that the cytometer is connected to the workstation (the message Cytometer Connected is displayed).
  2. Check that the buffer container is full and that the waste container is empty. Remove air bubbles in the buffer filter and the buffer line using the purge system. Prime the fluidics system by pressing the Prime button on the console of the cytometer. Wait until the indicator light changes from red to green.
  3. To clean the fluidics, install a tube containing 3 mL of a cleaning solution on the sample injection port and allow the cleaning solution to run for 5 min with a high sample flow rate. Repeat this with the rinse solution with distilled water. Leave the tube containing water on the sample injection port.
  4. To prepare the calibrating beads, pipette 400 µL of PBS into the bottom of a round bottom tube. Mix the bead stocks strongly by vortexing for 30 s. Add a drop to the round bottom tube containing PBS. Mix carefully by vortexing for 30 s.
  5. Run the performance check. Open the cytometer Setup and Tracking module in the software (Figure 1A). Verify that the cytometer configuration is correct for the experiment using PE immunostaining. Verify that the calibrating bead batch is correct with the configuration.
  6. Install the bead tube on the sample injection port and let it run with a low sample flow rate. Run the performance check, which takes approximately 5 min to complete). Once the performance check is complete, verify that the cytometer performance is satisfactory (Figure 1B). Close the cytometer Setup and Tracking module in the software.
  7. To set up an experiment and create application settings, click the New Experiment button on the browser toolbar and open the new experiment. Specify the parameters by selecting appropriate cytometer settings: Forward Scatter (FSC), Side Scatter (SSC), and PE from the drop-down menu of the experiment (Figure 1C). Select Linear Mode for FSC parameter and Logarithm Mode for SSC and PE parameters.
  8. In the open experiment, select Cytometer Settings (Figure 1D), then select Application Settings, and create a global worksheet (Figure 1E). Use the gray boxes and crosshairs to guide the optimization.
  9. Load the unstained control tube onto the cytometer and run Acquisition. Ensure that the population of interest (i.e., RBCs) is on scale by optimizing the FSC and SSC voltages. Optimize the FSC threshold value to eliminate debris without interfering with the population of interest.
  10. Draw a gate around the RBCs on the FSC vs. SSC plot. Display the RBC population in the dot plot of PE fluorescence. If needed, increase the fluorescence of the photomultiplier tube (PMT) voltages to place the negative population within the gray boxes. Unload the unstained control tube from the cytometer.
  11. Verify that the positive populations are on scale. Load the stained control tube onto the cytometer and run Acquisition. Lower the PMT voltage for the positive population if it is off scale until the positive population can be seen entirely on scale. Then unload the stained sample.
  12. To record and analyze samples, on a new global worksheet, create the following plots for previewing the data: 1) FSC vs. SSC, and 2) PE fluorescence histogram. Load the first sample onto the cytometer and run Acquisition.
  13. Draw an RBC gate around the erythrocytes on the FSC vs. SSC plot. Display the RBC population in the PE fluorescence histogram. In the Statistics view, select the mean for PE fluorescence parameters on GR populations (Figure 1F).
  14. In the Acquisition dashboard, select all events in the stopping gate and 10,000 events to record (Figure 1G). Click Record Data. When the event recording has completed, remove the first tube from the cytometer. The global worksheet plots should look like those in Figure 2.
  15. Load the following samples and record them.

6. Determination of the density of erythrocyte CR1

  1. Take the values of the mean fluorescence intensities of the samples corresponding to the "low" subject (Figure 3, Table I, RBC MFI), "medium" subject (Figure 4, Table D, RBC MFI), "high" subject (Figure 4, Table I, RBC MFI), and to the negative control sample (Figure 3, Table I, RBC MFI).
  2. On a graph representing the mean fluorescence intensity as a function of the density of CR1, place the four points corresponding to the negative control, "low" subject, "medium" subject, and "high" subject (blue points, Figure 6).
  3. Draw the regression line to get the calibration line and its equation.
  4. Take the values of the mean fluorescence intensity of the samples corresponding to the subjects whose density is to be determined. (Figure 5, Tables D and I, RBC MFI).
  5. Obtain the equation by replacing "Y" using the values of the mean fluorescence intensities, and calculate the density of CR1/E (Figure 6).
  6. Check on the graph that the mean fluorescence intensity values and the determined CR1/E density correspond to a point on the calibration line (Figure 6).

Results

The erythrocytes of three subjects whose density of CR1 is known ("low" subject [180 CR1/E], "medium" subject [646 CR1/E], and "high" subject [966 CR1/E]), and of two subjects whose CR1 density needed to be determined were immunostained by an anti-CR1 antibody coupled to an amplification system using the phycoerythrin fluorochrome. At the beginning, the CR1 density of the subjects from the low-high range was determined by the Scatchard method29 using radiolabeled antibodies...

Discussion

Several techniques are available to determine the density of erythrocyte CR1 (CR1/E). The first techniques used were the agglutination of red blood cells by anti-CR1 antibodies31 and the formation of rosettes in the presence of erythrocytes coated with C3b32. These rudimentary techniques were rapidly replaced by immunostaining methods using radiolabeled anti-CR1 antibodies1,33. It is also possible to measure the con...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank all the members of the URCACyt, flow cytometry technical platform, the staff of the Department of Immunology, and the staff of the Department of Internal Medicine and Geriatrics, who contributed to optimizing and validating the protocol. This work was funded by Reims University Hospitals (grant number AOL11UF9156).

Materials

NameCompanyCatalog NumberComments
1000E Barrier TipThermo Fischer Scientific , F-67403 Illkirch, France2079Esample pipetting
1-100 µL Bevelled, filter tipStarlab GmbH, D-22926 Ahrenburg, GermanyS1120-1840sample pipetting
Biotinylated anti-CR1 monoclonal antibody (J3D3)Home production of non-commercial monoclonal antibody, courtesy of Dr J. Cookimmunostaining
Blouseprotection
Bovin serum albumin (7,5%)Thermo Fischer Scientific , F-67403 Illkirch, France15260037cytometry
CentrifugeThermo Fischer Scientific , F-67403 Illkirch, France11176917centrifugation
Clean SolutionBD, F-38801 Le Pont de Claix, France340345cytometry
Comorack-96Dominique DUTSCHER SAS, F-67172 Brumath944060Prack
Cytometer Setup & Tracking Beads KitBD, F-38801 Le Pont de Claix, France655051cytometry
Formaldehyde solution 36.5 %Sigma Aldrich, F-38070 Saint Quentin Fallavier, FranceF8775-25MLFixation
10 µL Graduated, filter tipStarlab GmbH, D-22926 Ahrenburg, GermanyS1121-3810sample pipetting
LSRFORTESSA Flow CytometerBD, F-38801 Le Pont de Claix, France647788cytometry
Microman Capillary PistonsDominique DUTSCHER SAS, F-67172 Brumath067494sample pipetting
Micronic 1.40 mL round bottom tubesDominique DUTSCHER SAS, F-67172 BrumathMP32051mix
Micropipette Microman - type M25 -Dominique DUTSCHER SAS, F-67172 Brumath066379sample pipetting
Phosphate buffered Saline (PBS)Thermo Fischer Scientific , F-67403 Illkirch, France10010031cytometry
Pipette PS 325 mm, 10 mLDominique DUTSCHER SAS, F-67172 Brumath391952sample pipetting
powder-free Nitrile Exam glovesMedline Industries, Inc, Mundelein, IL 60060, USA486802sample protection
Reference 2 pipette, 0,5-10 µLEppendorf France SAS, F-78360 Montesson, France4920000024sample pipetting
Reference 2 pipette, 20-100 µLEppendorf France SAS, F-78360 Montesson, France4920000059sample pipetting
Reference 2 pipette, 100-1000 µLEppendorf France SAS, F-78360 Montesson, France4920000083sample pipetting
Rinse SolutionBD, F-38801 Le Pont de Claix, France340346cytometry
Round bottom tubeSarstedt, F-70150 Marnay, France55.1579cytometry
Safe-Lock Tubes, 1.5 mLEppendorf France SAS, F-78360 Montesson, France0030120086mix
streptavidin R-PETebu Bio, F-78612 Le Perray-en-Yvelines, FranceAS-60669immunostaining
Tapered Centrifuge Tubes 50 mLThermo Fischer Scientific , F-67403 Illkirch, France10203001mix
Vector anti streptavidin biotinEurobio Ingen, F-91953 Les Ulis, FranceBA-0500immunostaining
Vortex-Genie 2Scientific Industries, Inc, Bohemia, NY 111716, USASI-0236mix

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