The Fetal Cell Count™ kit is intended for the discrimination and quantitative detection of human fetal red blood cells in maternal blood. The Fetal Cell Count™ kit is based on a sensitive and accurate flow cytometry method, which offers a dual fluorescent detection of two intracellular antigens – fetal hemoglobin (HbF) and carbonic anhydrase (CA). The anti-HbF antibody is specific to the γ-chain of human HbF and can be used to detect fetal HbF as well as maternally expressed HbF in RBCs (F-cells). Analysis of human CA using the anti-CA antibody exclusively detects adult maternal RBCs. Simultaneous use of both anti-HbF and anti-CA antibodies in the unique 2-color flow cytometry assay discriminates between fetal HbF and maternal F-cells in human peripheral whole blood samples.
Shortly after birth, a switch normally occurs from HbF production to adult HbA expression. Some adults continue to express HbF in RBCs (F-cells), particularly in some cases of b-thalassemia. During pregnancy, fetal cells circulate in maternal blood and can be detected by a variety of methods. The Fetal Cell CountTM kit distinguishes maternal F-cells from fetal RBCs using a 2-color flow cytometry strategy. This is in contrast to the commonly used manual Kleihauer-Betke (KB) test which cannot discern maternal F-cells from fetal RBCs. The origin of circulating HbF is important to identify when doing studies of fetomaternal haemorrhage (FMH), the presence of rhesus D (RhD) antigens, β-thalassemia during pregnancy, and HbF detection.
|Reagent A||Fixative Solution (A) – Containing < 0.1% sodium azide||2.5 mL|
|Reagent B||Fixative Solution (B) – buffered Formaldehyde||2.5 mL|
|Reagent C||Permeabilization Solution (C) – containing sodium dodecyl sulfate (SDS)||2.5 mL|
|Reagent D (10x)||Washing Solution (10xD), 10x concentrated – PBS containing heparin||1×50 mL|
|Reagent E||Monoclonal antibody to human carbonic anhydrase conjugated with FITC, containing < 0.1% sodium azide||1.3 mL|
|Reagent F||Monoclonal antibody to human fetal hemoglobin conjugated with R-PE, containing < 0.1% sodium azide||1.3 mL
Each kit contains sufficient reagents to perform 25 tests.
Both HbF and CA are detected in RBCs obtained from EDTA anti-coagulated or heparin-treated human peripheral whole blood.
Antibody binding specificity – In-house study results concluded that the antibody directed against HbF recognizes only the g chain of HbF, while the other antibody is specific for the carbonic anhydrase (CA) antigen.
Accuracy – In-house study results have shown that both repeatability and reproducibility are optimal with coefficient of variance of 18.3% and 6.3% respectively for artificial mixtures with 1% fetal cells.
Linearity – Measurement of artificial mixtures for the (theoretical) concentration range 0.02 – 5.0 % (v/v) show a high correlation (r = 0.999), when 1 x 105 cells are measured. This correlation increases when more cells are evaluated.
Specificity – Tested samples from control blood donors did not show staining in the upper left (UL) quadrant area. These data demonstrate that there is no interference in the UL area leading to inaccurate counting of fetal cells.
Detection limit – The detection limit of the assay is based on the measurement of artificial mixtures and determined to be 0.014% when 1 x 105 cells are evaluated. Accuracy is improved when the number of events is increased.
The complete dual-color staining and analysis of up to 5 samples must be executed and interpreted by well-trained and authorized laboratory technicians and can be concluded within 90 minutes from blood collection.
Limitations of the procedure
- Personnel experienced in aseptic technique should perform the collection of the blood sample.
- The Fetal Cell Count™ kit is intended for detection using flow cytometry and not for use with immunofluorescent microscopy.
- Accurate results with flow cytometric procedures depend on correct alignment and calibration of laser as well as proper gate setting.
- The efficacy of the Fetal Cell Count™ kit with samples other than human RBCs has not been established.
- Lysis of erythrocytes and a decrease in HbF and CA contents cannot be excluded when cells are stored at room temperature for more than 72 hours (3 days). Therefore, preparation of the cells and incubation should always be performed within 3 days from blood collection.
The Fetal Cell Count™ kit is for research use only. It is not for use in diagnostic procedures.