Hemoglobin / Erythrocytes

Hemoglobin antibodies for flow cytometry

We offer antibodies to detect various types of hemoglobin. Each antibody can be used individually or combined in a panel. By using the combination of anti-HbS FITC, anti-panHb APC and anti-HbF Dy-410, the need for compensation can be eliminated

 

Anti-Hemoglobin S FITC

This is the first anti-hemoglobin S (HbS) antibody conjugate available for flow cytometry. For your convenience, it’s conjugated with FITC, so it can be measured on all available flow cytometers.

The anti-HbS antibody recognizes the abnormal β-chain of HbS. Substitution of the amino acid valine for glutamic acid in the sixth position of the β-chain results in the formation of a βs subunit. Due to the mutated subunit βs, hemoglobin becomes unstable when no oxygen is bound, resulting in sickle shaped erythrocytes.

 

Hemoglobin S antibody flow cytometry

Figure 1. Flow cytometry analysis of whole blood stained with anti-hemoglobin S FITC. Histogram: uninterrupted line: stained HbShigh whole blood sample. Interrupted line: stained normal whole blood sample. Dotplot: stained HbShigh whole blood sample.
Anti-pan Hemoglobin APC

The murine antibody against human pan hemoglobin (panHb) recognizes the α-chain of human hemoglobin. This antibody can be combined with other antibodies to determine the percentage of erythrocytes containing pathological forms of hemoglobin compared to the total erythrocyte population.

 

panHb antibody flow cytometry HbA

Figure 2.Flow cytometry analysis of normal whole blood stained with anti-pan hemoglobin APC. Histogram: uninterrupted line: stained normal whole blood sample. Interrupted line: blanc sample. Dotplot: stained normal whole blood sample.

Anti-Hemoglobin F Dy-410

Anti-hemoglobin F antibody recognizes the γ-chain of hemoglobin F (HbF). HbF is predominantly expressed by fetal erythrocytes. In some cases, HbF is also expressed by adult erythrocytes. These so called F-cells, can represent up to 25% of the total erythrocyte population.

 

anti hemoglobin f antibody flow cytometry fetal hemoglobin

 

Figure 3. Flow cytometry analysis of normal whole blood with a 3% spike of commercial cord blood stained with anti-hemoglobin F Dy-410. Histograms: uninterrupted line: stained whole blood sample with a 3% spike of HbFhigh whole blood, interrupted line: unstained whole blood sample with a 3% spike of HbFhigh whole blood.

 

Co-staining samples with anti-HbS and anti-HbF antibodies

In many cases, the anti-HbS and anti-HbF antibodies will be used in a combined staining to identify double positive cells. Results from such a double staining are displayed in figure 4. The double staining was performed on normal blood (AA), cord blood (CB) and HbShigh and HbFhigh blood (SF). The AA sample shows no staining with the anti-HbS antibody and a small population of HbF+ cells, the F cells. Staining of the CB sample also resulted in no signal with the anti-HbS antibody and a 96% HbFHigh population. The small population in the lower left quadrant reflects the initiated transition towards HbA in the new born. The anti-HbS antibody stained 98.4% of all red blood cells in the SF sample, with 67.8% being double positive for HbF. For more detailed information on the data, please review this presentation on the experiments here.

Hemoglobin S and hemoglobin F flow cytometry sickle cell

Figure 3. Flow cytometry analysis of whole blood samples stained with anti-hemoglobin S and anti-hemoglobin F. AA: normal adult blood, CB: cord blood and SF: HbShigh and HbFhigh blood. Data kindly provided by TransLab, Boston Children’s Hospital, USA. For more detailed information on the data, please review the full presentation here.

Complementary permeabilization reagent

IQ Perm

IQ Perm can be used as a permeabilization buffer when working with the above mentioned antibodies. Detection of these intracellular antigens or substrates often require rapid fixation, usually by formaldehyde or glutaraldehyde, followed by treatment with a permeabilizing reagent.

IQ Perm is such a permeabilizing reagent which utilizes Triton X‐100 as the major active component that allows entry of antibodies and other probes. IQ Perm is suitable for permeabilization of erythrocytes and leukocytes. Our experience has shown that IQ Perm is used at various dilutions, depending on the cells of interest.

IQ Perm is provided as a concentrate in 40 mL volume. This bottle is sufficient for 105 ‐ 265 individual tests, depending upon the application using approximately one million cells for each assay.

 

Details on the available antibodies are listed here and details on IQ Perm can be found here

Ready to measure hemoglobin with flow cytometry?

Place an order by sending an email to orders@iqproducts.nl or via the order form.

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The Netherlands

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