MONOCLONAL ANTIBODIES DIRECTED TO HUMAN FETAL …tools.thermofisher.com/content/sfs/manuals/L13012_ms_x_hu_HbF_mab.pdfof fetal red blood cells. Fetal cells are identified by the presence

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PI: L13012 MAN0006693 Rev 2.00 Effective Date: 11 JUL 2012 © 2007 Invitrogen Corporation. All right reserved. These products may be covered by one or more Limited Use Label Licenses (see the Life Technologies Catalog or www.lifetechnologies.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.

MONOCLONAL ANTIBODIES DIRECTED TO HUMAN FETAL HEMOGLOBIN (HbF)

Invitrogen FITC-conjugated Monoclonal Antibody Directed to Fetal Hemoglobin CATALOG No. MHFH01 100 tests CATALOG No. MHFH01-4 400 tests Invitrogen R-PE-conjugated Monoclonal Antibody Directed to Fetal Hemoglobin CATALOG No. MHFH04 100 tests CATALOG No. MHFH04-4 400 tests Invitrogen TRI-COLOR®-conjugated Monoclonal Antibody Directed to Fetal Hemoglobin CATALOG No. MHFH06 100 tests

FOR IN VITRO DIAGNOSTIC USE

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PI: L13012 MAN0006693 Rev 1.00 Effective Date: 15 MAR 2012 © 2007 Invitrogen Corporation. All right reserved. These products may be covered by one or more Limited Use Label Licenses (see the Life Technologies Catalog or www.lifetechnologies.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.

I. INTENDED USE

Invitrogen’s Fetal Hemoglobin (HbF) monoclonal antibody is conjugated with either fluorescein isothiocyanate (FITC), R-phycoerythrin (R-PE) or R-PE conjugated to the indodicarbocyanine dye, Cy5 (TRI-COLOR, TC). These reagents are intended for the identification followed by enumeration of fetal red blood cells. Fetal cells are identified by the presence of fetal hemoglobin by a flow cytometric method. The presence of fetal cells in the maternal circulation, resulting from fetal-maternal hemorrhage, may be attributed to a variety of causes including fetal trauma, various obstetrical emergencies and placental trauma. Hemorrhage of Rh+ fetal blood into Rh- maternal blood may result in the formation of Rh-reactive antibodies in the mother. This Rh immunization may be prevented by the administration to the mother of Rh immune globulin (RhIg, RhoGAM®, Ortho Diagnostic Systems) soon after delivery. Invitrogen’s Fetal Hemoglobin antibody may be used as an aid in identifying fetal-maternal hemorrhage and determining the need for immunoprophylaxis with immune globulin.

II. SUMMARY AND EXPLANATION

The passage of erythrocytes from an Rh+ fetus into the circulation of an Rh- mother results in the formation of Rh antibodies in the mother. The formed Rh antibodies remain in the mother and are easily transmitted through the placenta to the fetus in a subsequent pregnancy. These transmitted Rh antibodies may react with Rh+ cells in the fetus, causing a disease process referred to as Rh hemolytic disease or erythroblastosis (1). This process of immunization of the mother by Rh incompatible fetal blood has been associated with an increased risk of pregnancy complications, and potentially fetal death, unless Rh immune globulin (RhoGAM) is administered to the mother soon after delivery of the first Rh-incompatible fetus (2). The escape of fetal cells into the maternal circulation has been observed as early as the first trimester of pregnancy, and may increase as gestational age increases. However, the volume of fetal blood in the maternal circulation is reported to be small in most instances, with 98% of women having less than 2.0 ml of whole fetal blood in their peripheral blood. Substantial fetal bleeds of 50 ml are infrequent, and fetal blood volumes of 250-300 ml, or 6% fetal cells in the maternal circulation, are considered to be exceptional (3). Because the clinical significance of small volumes of fetal-maternal hemorrhage remains controversial, standard medical practice has been to administer RhIg (RhoGAM) to all Rh- women following delivery of an Rh+ infant (2). Fetal hemoglobin tests have been used as a guide in determining the amount of RhIg to administer clinically. This is done by using the measured percentage of fetal hemoglobin-containing red cells to calculate the partial volume of fetal blood in the maternal circulation. The Kleihauer-Betke (KB) test has been used successfully since 1957 to detect fetal-maternal hemorrhage and to identify the approximate volume of fetal red blood cells in the maternal circulation. This test is the accepted standard and is currently performed in essentially the same manner as originally described (4). The KB test is based on acid elution of maternal hemoglobin from maternal red cells, resulting in pale "ghost" cells in the maternal blood smear. Acid-stable fetal hemoglobin in fetal erythrocytes stains pink with erythrocin. Both maternal and fetal erythrocytes are counted under microscopic examination and the quantity of fetal blood in the maternal circulation is calculated from the estimated total maternal blood volume. The test has been shown to be useful for determining fetal-maternal hemorrhage resulting from maternal trauma, and placental defects, as well as Rh immunization (5); although, it has been claimed that the test is not always accurate in the context of acute trauma (6). Despite widespread use, the KB acid elution method is limited to a detection sensitivity of about 0.5% of fetal cells, and errors frequently arise due to inter-observer subjectivity, as well as the need to manually count substantial numbers of





maternal and fetal erythrocytes to determine the percentage of fetal cells present (7).

More recently, flow cytometric methods have been employed to enumerate fetal cells in the maternal circulation following an Rh-incompatible delivery. This method is claimed to circumvent the subjectivity and limited sensitivity attributed to the KB method. Flow cytometric methods are reported to be less labor-intensive and are compatible with multiple sample and high volume laboratory testing, but do require more elaborate instrumentation (8). Indirect immunofluorescence flow cytometry using FITC-labeled anti-IgG reagents has been reported to be more accurate than the acid elution method in quantitating fetal cells, and to demonstrate good precision at fetal cell frequencies of approximately 0.1% (8, 9). Flow cytometric methods have also demonstrated an ability to detect very small fetal bleeds in the range of 1 fetal cell per 100,000 maternal red cells; whereas, the sensitivity needed for routine clinical use is much less, or approximately 1 fetal cell per 200 maternal red cells (8). The ability to detect fetal cells has been substantially improved with the use of fixation and permeabilization procedures. These procedures permit the immunostaining of intracellular hemoglobin in intact red cells, followed by fluorescence-activated cell sorting to detect rare events (10), or routine flow cytometry. Fetal red cells have also been analyzed for beta chain/hemoglobin A (HbA) content in the study of thalassemia (11), and for their identification by monoclonal antibodies specific for fetal hemoglobin (11, 12, 13).

III. CLINICAL RELEVANCE

Invitrogen’s Fetal Hemoglobin monoclonal antibody specifically recognizes fetal hemoglobin found in fetal erythrocytes. The antibody permits the enumeration of fetal erythrocytes, in the maternal circulation as a result of a fetal-maternal hemorrhage, or leakage, of fetal cells into the maternal circulation. Hemorrhage of cells from an Rh+ fetus into the circulation of an Rh- mother may result in the formation of Rh-reactive antibodies in the mother. Rh hemolytic anemia in a subsequent Rh+ fetus may result from placental transfer of antibodies formed in the mother to the fetus. Although the volume of fetal erythrocytes found in the maternal circulation during pregnancy and immediately post-partum is reported to be small and of uncertain clinical significance in many cases (3), substantial hemorrhage may result from a number of causes including fetal or maternal trauma and placental defects (2,3). Erythrocytes containing fetal hemoglobin may be found in individuals of any age, but with lower amounts of fetal hemoglobin compared to fetal red cells. These cells have been termed F cells. High levels of F cells may also exist in adults with a heterogeneous group of genetic disorders of uncertain etiology, referred to as Hereditary Persistence of Fetal Hemoglobin (14). Other clinical conditions causing significant levels of anemia may also result in elevated levels of F cells. Several clinical conditions have been described with increased levels of F cells. These conditions include hereditary anemic diseases such as sickle cell anemia and thalassemia major (11, 15, 16).

IV. PRINCIPLES OF THE TEST

An EDTA anticoagulated peripheral blood sample is drawn from an appropriate donor. The erythrocyte count is determined. Approximately 2.5 x 107 red cells are subjected to brief fixation in 0.05% gluteraldehyde solution as described in Section XI. Fixed and washed cells are permeabilized with a detergent in a manner that is frequently used to enable macromolecules such as monoclonal antibodies to penetrate cellular membranes. Invitrogen FITC, R-PE and TRI-COLOR -conjugated monoclonal antibodies bind specifically to fetal hemoglobin in fetal red cells. To identify cells containing fetal hemoglobin, fixed and permeabilized cells are incubated with the monoclonal antibody and washed to remove unbound antibody. Antibody stained cells are subsequently analyzed by flow cytometric methods.





Invitrogen’s FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies bind to fetal hemoglobin in F cells as well as fetal hemoglobin in fetal cells. F cells are displayed at the leading edge of the unstained autofluorescent leukocyte population and exhibit lower fluorescence intensity than fetal cells, enabling their exclusion from the fetal cells in flow cytometric analysis. The method of excluding F cells from the analysis of fetal cells is demonstrated in Section XI, Interpretation of Results. Positive and negative control samples should be used with sample analysis to establish that all reagents are performing in a consistent manner and that the positive fluorescence attributed to antibody-stained fetal red cells is differentiated from unstained normal red blood cells, leukocytes and any cellular debris. Invitrogen recommends the use of FDA-cleared FETALTROL TM fetal red cell controls for this purpose. These cells are most appropriate for establishing this assay and setting markers as their values are close to expected clinical ranges. FETATROL cells are produced by Trillium Diagnostics, LLC, Portland, ME, and distributed by Invitrogen Laboratories (Catalog # FH101). If FETALTROL cells are not available, mixtures of defined amounts of red cells from cord blood in adult blood may be used. In this case, the recommended positive control samples consist of both 1% and 5% fetal erythrocyte-containing placental cord blood in normal adult blood. The recommended negative control sample is a normal male or non-pregnant adult female.

V. REAGENTS

A. INVITROGEN MONOCLONAL ANTIBODY REAGENTS

FITC-conjugated monoclonal antibody directed to fetal hemoglobin (Code MHFH01 or MHFH01-4): Clone: HbF-1 (12)* Isotype: IgG1 Species: Mouse Composition: IgG1 heavy chain, Kappa light chain Source: Mouse ascites fluid Method of Purification: Column chromatography Fluorochrome: Fluorescein isothyocyanate (FITC) Excitation Wavelength 488 nm Emission Wavelength 525 nm

R-PE- conjugated monoclonal antibody to directed fetal hemoglobin (Code MHFH04 or MHFH04-4): Clone: HbF-1 (12) Isotype: IgG1 Species: Mouse Composition: IgG1 heavy chain, Kappa light chain Source: Mouse ascites fluid Method of Purification: Column chromatography Fluorochrome: R-Phycoerythrin (R-PE)** Excitation Wavelength 488 nm Emission Wavelength 575 nm





TRI-COLOR -conjugated monoclonal antibody directed to fetal hemoglobin (Code MHFH06):

Clone: HbF-1 (12) Isotype: IgG1 Species: Mouse Composition: IgG1 heavy chain, Kappa light chain Source: Mouse ascites fluid Method of Purification: Column chromatography Fluorochrome: R-PE-Cy5 (TRI-COLOR, TC) Excitation Wavelength 488 nm Emission Wavelength 667 nm

* The monoclonal antibody to HbF, HbF-1, is licensed from Trillium Diagnostics, LLC.

** U.S. Patent No. 4,520,110; Canadian Patent No. 1,179,942; European Patent No. 76,695

B. REAGENT SUPPLIED:

Monoclonal antibody for 100 or 400 tests 0.50 or 2.0 ml vial containing conjugate in PBS buffer with BSA (and sucrose for RPE and Tri-Color only), 0.1% sodium azide.

C. MATERIALS REQUIRED BUT NOT SUPPLIED:

Test tubes, polypropylene, 12 x 75 mm Micropipettes capable of dispensing appropriate blood volumes; Eppendorf Repeat Pipettor is recommended. Blood collection tubes with anticoagulant Vortex mixer Clinical centrifuge or an appropriate blood bank cell washer The temperature requirement for centrifugation with a swinging bucket rotor or appropriate cell washer is room temperature (22±3°C). Flow cytometers: Becton Dickinson FACscan TM and Coulter® EPICS®-XL or equivalent

D. REAGENTS REQUIRED BUT NOT SUPPLIED:

1% Triton X-100, Invitrogen Catalog No. HFH-10 A 0.1% working solution is prepared in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.1% sodium azide. Do not use this reagent beyond the stated expiration date. Alsever’s Solution, Invitrogen Catalog No. HFH-11 Intended for the preparation of positive and negative controls from cord blood. Do not use this reagent beyond the stated expiration date.





Phosphate Buffered Saline (PBS) with 0.1% Bovine Serum Albumin (BSA) The recommended PBS can be obtained from Sigma, Catalog #1000-3. Solubilize packet in 1 liter of deionized water. The recommended BSA can be obtained from Sigma, Catalog #A3294. Add 1 gram of BSA to PBS. Adjust Ph to 7.4. Store at 4°C until used. Discard after 7 days. Glutaraldehyde, 0.05% The recommended glutaraldehyde stock solution can be obtained from Sigma, Catalog #G5882. Gluteraldehyde stock solution should be stored at -20°C. Prepare 0.05% working solution by dilution of 100 ul of stock solution in 50 ml of PBS/BSA. Store working solution in refrigerator and retain at 4°C during use. Prepare fresh for use each day.

Formaldehyde, 1.0% The recommended 10% formaldehyde stock solution can be obtained from Polysciences, Inc., Catalog #04018, methanol-free. Store at room temperature. Prepare working solution of 1% in PBS/BSA and adjust to pH 7.4. Store working solution at 2-8°C and discard any unused portion after 7 days. Positive Reagent Controls Negative Reagent Control FETALTROL cells, catalog # FH101, are the preferred control standards

VI. STATEMENT OF WARNINGS

If FETALTROL cells or cord blood are not available for positive controls, the assay cannot be performed reliably. For cord blood-derived controls, the recommended positive samples consist of both 1% and 5% fetal erythrocyte-containing placental cord blood from normal adults. Monoclonal antibody reagents contain sodium azide. Sodium azide under acid conditions yields hydrazoic acid, an extremely toxic compound. Azide compounds should be discarded in an approved toxic disposal container. Do not expose reagents to a direct light source during incubation with cells and prior to analysis. Do not pipet by mouth. Samples should be handled as if capable of transmitting infection. Appropriate disposal methods should be used. The sample preparation procedure employs fixatives (gluteraldehyde and formaldehyde). Contact is to be avoided with skin or mucous membranes. Do not use antibodies beyond the stated expiration date of the product.





VII. APPROPRIATE STORAGE CONDITIONS

All reagents supplied by the manufacturer should be stored at 2-8°C when not in use. Freezing is not recommended. Reagents should be brought to room temperature (22±3°C) before use. Protect fluorochrome-conjugated monoclonal antibodies from light sources during storage, during incubation with cells and prior to analysis.

VIII. EVIDENCE OF DETERIORATION

Reagents should not be used if any evidence of deterioration or substantial loss of reactivity is observed. The normal appearance of the FITC-conjugated monoclonal antibody directed to HbF is a clear, yellow-orange liquid. The normal appearance of the R-PE-conjugated monoclonal antibody directed to HbF is a clear, pink-red liquid. The normal appearance of the TRI-COLOR-conjugated monoclonal antibody directed to HbF is a clear, deep pink liquid.

IX. SPECIMEN COLLECTION

Collect venous blood samples into blood collection tubes containing anticoagulant. EDTA is the preferred anticoagulant for use with the Invitrogen Fetal Hemoglobin Test. Other anticoagulants have not been thoroughly evaluated (17). For optimal results the sample should be processed within 6 hours of venipuncture. However, samples may still be used if the blood sample is processed for analysis within 30 hours of venipuncture. Unstained anticoagulated blood should be stored at 20-25°C prior to sample processing. Blood samples that are hemolyzed, clotted or appear to be lipemic, discolored or to contain interfering substances should be discarded.

Refer to "Standard Procedures for the Collection of Diagnostic Blood Specimens," published by the National Committee for Clinical Laboratory Standards (NCCLS) for additional information on the collection of blood specimens.

X. POSITIVE AND NEGATIVE REAGENT CONTROLS

Positive and negative reagent controls must be included with each test sample or group of test samples analyzed if the assay is to be performed reliably. Invitrogen recommends the use of FDA-cleared FETALTROL fetal red cell controls for this purpose. These cells are most appropriate for establishing this assay and setting markers as their values are close to expected clinical ranges. FETALTROL cells are produced by Trillium Diagnostics, LLC, Portland, ME, and distributed by Invitrogen Laboratories (Invitrogen catalog # FH101). Detailed instructions for the use of these cells are included with the kit or may be downloaded from our website (www.Invitrogen.com, keyword: fetal). These reagents assist in identifying the fetal red cell analysis region, and in distinguishing antibody stained fetal cells from leukocytes, monocytes, granulocytes, adult red cells and cellular debris. If these control cells are not available, cord blood may be substituted and prepared as described below. The 1 and 5% positive cord blood controls consist of 1 and 5% HbF positive cells in the cord blood of





normal adults. Positive controls should be prepared in Alsever’s Solution, and may be retained for repeat use for up to two weeks. Positive controls should be acquired as list mode files prior to test samples to verify that all reagents perform as expected under test conditions. The 1% positive cord blood reagent control is used to define the fluorescence intensity region for detection of fetal cells. An analysis region should be set to exclude nonspecific fluorescence and to include the brighter fluorescence attributed to fetal blood cells. The 1 and 5% positive controls should indicate approximately 1 and 5% of fetal cells, respectively, when properly prepared and analyzed. Failure of the positive controls to perform as expected is a basis for discarding test results and repeating the test. A negative reagent control sample, consisting of a normal male or non-pregnant adult female blood, must be used to define a negative result. Compensation settings between the FITC, R-PE and TRI-COLOR fluorescence signals should be optimized to separate the autofluorescence of leukocytes and the antibody-stained HbF-containing cells, which can be readily accomplished using the recommended 1% and 5% cord blood positive controls. POSITIVE CONTROL PREPARATION

1. The fetal red cells for the 1% and 5% positive controls should be taken from EDTA

anticoagulated cord or newborn whole blood that is ABO compatible with the adult blood to be used in the controls. The adult blood should be from a non-hospitalized, non-pregnant individual with normal Hematology CBC parameters. It is particularly important that the individual be negative for sickle cell anemia.

2. After collection and gentle mixing, spin the fetal and adult blood at 600 x g for 5 minutes in a

blood bank centrifuge (cell washer) and remove the plasma (supernatant). The cells should then be washed twice with Alsever’s solution. The wash volumes should be similar in volume of blood. Ultimately the cells should be resuspended in Alsever’s solution at approximately the same volume as the original blood sample.

3. Dilute 1 ml of both the cord or newborn and adult blood with 1 ml Alsever’s solution and

determine the TBC counts of each. Dilution of the cells increases the accuracy of counting. Once the cell numbers have been determined, correct the counts by multiplying by 2 to adjust for the dilution. Prepare the 1% and 5% positive control samples from the washed, undiluted blood based on the following formulas:

1% Mixture [Adult RBC count determined above] x .01 = A [A/Adult RBC count] x 1000 = µl of whole blood to remove from a 1 ml aliquot of undiluted adult blood. This volume should be discarded. This volume is always 10 µl. [A/Cord RBC count determined above] x 1000 = µl of cord blood to add back to the 990 µl of adult blood from the previous step. Add 1 ml of Alsever’s solution to the mixture, mix well, measure the new RBC count and record. Refrigerate until ready to test. 5% Mixture [Adult RBC count determined above] x .05 = B





[B/Adult RBC count] x 1000 = µl of whole blood to remove from a 1 ml aliquot of undiluted adult blood. This volume should be discarded. This volume is always 10 µl. [B/Cord RBC count determined above] x 1000 = µl of cord blood to add back to the 990 µl of adult blood from the previous step. Add 1 ml of Alsever’s solution to the mixture, mix well, measure the new RBC count and record. Refrigerate until ready to test.

4. Use 2 ml polypropylene tubes for mixture storage. 5. Store mixtures in the refrigerator when not in use.

6. When ready to test, resuspend mixtures by rolling the vial in the hands 10 times and then place

on a tube rocker to continue mixing while the vial reaches ambient temperature.

7. Under these conditions these mixtures should be stable for approximately 14 days. XI. SAMPLE PREPARATION

1. Determine the red blood cell count of the specimen and fix approximately 2.5 x 107 red cells (or alternatively 10 µl of whole blood) in 1 ml of prepared 0.05% glutaraldehyde solution. Glutaraldehyde must be fresh and stored properly at –20°C. Invitrogen suggests the use at 1.0 ml ampules from Sigma (Cat. No. G5882), shipped and stored frozen (-20°C). Thaw individual ampules rapidly before use (in hands, not water bath). Unused portion should be discarded. Prepare a 0.05% working solution using 1x PBS, no azide or BSA, as a diluent, keep cold. Vortex after the addition of cells.

2. Retain glutaraldehyde fixed cells for 10 minutes at room temperature. It is important to follow the recommended incubation time (minimum 10 minutes, no more that 12 minutes).

3. Wash fixed cells by centrifugation at 600 x g for 5 minutes with 2 ml of prepared PBS with 0.1% BSA. Alternatively, a blood bank cell washer may be used for all cell wash procedures.

4. Repeat step 3 twice. It is important to include all 3 washes. Some RBC lysis may be noted. 5. Remove supernate and resuspend the cell pellet by vortex in 0.5 ml of Triton X-100 working

solution. It is much easier to use Invitrogen’s Triton-X-100 (Invitrogen catalog # HFH-10) especially when establishing the procedure. Stock Triton-100 is highly viscous and difficult to measure in small volumes. Incubate cells for 3-5 minutes at room temperature.

6. Wash Triton treated cells by centrifugation for 5 minutes at 600 x g with 2 ml of PBS/BSA. 7. Remove supernate and resuspend the cell pellet in 0.5 ml of PBS/BSA. 8. Add 10 µl of the cell suspension (about 1-2 x 105 cells) to 5 µl of Invitrogen HbF monoclonal

antibody and 70 µl of PBS/BSA. 9. Incubate antibody stained cells for 15 minutes at room temperature in the dark. 10. Wash antibody stained cells by centrifugation for 5 minutes at 600 x g with 2 ml of PBS/BSA. 11. Remove supernate and resuspend the cell pellet in 0.5 ml of 1% formaldehyde in PBS/BSA.

Store cells in the dark in formaldehyde solution prior to analysis by flow cytometry.





XII. INTERPRETATION OF RESULTS

FLOW CYTOMETRY

1. List mode files of at least 50,000 events should be collected for log forward scatter (FSC), log side scatter (SSC) and log fluorescence signals for both the fluorochrome-conjugated HbF antibody (e.g. FL1 for FITC- conjugated antibody, FL2 for R-PE- conjugated antibody and FL3 for TRI-COLOR-conjugated antibody) as well as another signal used to detect the autofluorescence of leukocytes in the specimen (e.g., FL2 for FITC-conjugated antibody). Threshold on the FSC signal. Compensation is not an issue if only using one color.

2. Sample handling and flow rates should be selected to minimize doublets and coincidence signals.

It is suggested that a rinse tube be run for a short period between samples to prevent carryover. 3. It is best run the negative control first. If using cord blood-derived positive control samples, it

may be advantageous to run experimental samples next, followed by positive control samples. This will reduce the possibility of carryover of cord blood HbF-positive cells. Detection of fetal erythrocytes using this assay is based on a fluorescence intensity region established with the positive control samples. Visualization of the fluorescence intensity of all red cells is important to insure accurate determination of the percentage of fetal cells in the background of adult erythrocytes. Voltage settings for the detectors are used consistently to standardize results. A change in the laser, the FSC detector, the SSC PMT or the FL1, FL2 or FL3 PMT will require adjustments of these settings depending upon the Invitrogen HbF monoclonal antibody-fluorochrome is used.

4. When selecting a new voltage setting, the negative population should be placed fully within the

first decade.

DATA ANALYSIS

Data may be analyzed using conventional flow cytometry software or any third party offline program. The red cell population may be gated using light scatter parameters, but this method may be insufficient to ensure elimination of all leukocytes, white blood cells (WBC), from analysis. However, using the above staining procedure leukocytes show significant autofluorescence. This may be utilized to exclude leukocytes from analysis of HbF containing red cells. For example, when using a FITC- conjugated antibody, set an analysis gate on FSC vs. FL2, excluding FL2 bright positives. These are WBC’s with increased antofluorescence.





The following dot plot is representative of data generated from a sample stained with a monoclonal antibody directed to HbF and properly gated using fluorescence and light scatter parameters:

The following histogram is representative of a 1% HbF positive control of placental cord blood in normal adult blood stained with the Invitrogen FITC-conjugated monoclonal antibody directed to HbF. The distributions of F cells (normal adult red blood cells expressing varying amounts of fetal hemoglobin) and fetal cells are as indicated:

The following histogram is representative of a 1% HbF positive control of placental cord blood in





normal adult blood stained with the Invitrogen R-PE-conjugated monoclonal antibody directed to HbF. The distributions of F cells (normal adult red blood cells expressing varying amounts of hemoglobin F) and fetal cells are as indicated:

The following histogram is representative of a 1% HbF positive control of placental cord blood in normal adult blood stained with the Invitrogen TRI-COLOR-conjugated monoclonal antibody directed to HbF. The distributions of F cells and fetal cells are as indicated:





The following histogram is representative of a 5% HbF positive control of placental cord blood containing fetal erythrocytes in adult blood stained with the Invitrogen FITC conjugated monoclonal antibody directed to HbF:

The following histogram is representative of a 5% HbF positive control of placental cord blood in adult blood stained with the Invitrogen R-PE-conjugated monoclonal antibody directed to HbF:





The following histogram is representative of a 5% HbF positive mixture of placental cord blood in adult blood stained with the Invitrogen TRI-COLOR-conjugated monoclonal antibody directed to HbF:

The following histogram is representative of a negative control sample. F CELLS





Erythrocytes containing fetal hemoglobin at substantially increased levels may be found in several clinical conditions (11, 14). Fetal hemoglobin in these cells, termed F Cells, is characterized by lower fluorescence intensity and may be readily distinguished from the higher fluorescence intensity of fetal hemoglobin in fetal cells. The 1% cord blood positive reagent control should be used to properly define the fluorescence intensity region for the detection of fetal cells and to exclude from the analysis any F cells that may also be present in the patient’s sample. The following histograms are representative of maternal blood samples with substantially increased F Cells as typically seen in Hereditary Persistence of Fetal Hemoglobin and Sickle Cell Disease. The proper location of the any higher fluorescence intensity fetal cells is also identified in both histograms. F Cells in Hereditary Persistence of Fetal Hemoglobin

F Cells in Sickle Cell Disease

Anti-HbF-FL1 —>





CALCULATION OF RESULTS The percent and total volume of fetal red blood cells may be determined in the following manner: 1. Enumerate the total number of adult and fetal erythrocytes as described in Section XI under

“Flow Cytometry.” At least 50,000 total events (adult erythrocytes) should be counted. 2. Calculate the percent fetal cells in the total counted. 3. Determine the total volume of fetal erythrocytes in the maternal circulation as the % of fetal cells

x 50, assuming a total maternal circulatory volume of 5000 ml.

Example: Total adult red cells counted = 50,000 Total fetal red cells counted = 200 Percent of fetal cells in the total counted; 200 ÷ 50000 = 0.4% fetal cells Total volume of fetal erythrocytes in the maternal circulation; 0.4% x 50 = 20 ml

XIII. QUALITY CONTROL PROCEDURES

1. Positive and negative reagent controls should be included with each test sample or group of test samples analyzed to ensure proper laboratory working conditions and to establish that all reagents are performing consistently. In this manner, the positive fluorescence attributed to antibody-stained fetal red cells is differentiated from unstained normal red blood cells, leukocytes and any cellular debris. The recommended positive control samples are those found in the FETALTROL kit (Catalog # FH101). Alternately 1% and 5% fetal erythrocyte-containing placental cord blood in normal adult blood may be used.

2. Stability requirements for positive control samples are: (a) the initial (day 1) analysis should be

within 10% of target values, i.e. 1%±0.1% for the 1% positive control and 5%±0.5% for the 5% positive control, and (b) the subsequent day results with stored controls over approximately a 2 week period should be within 0.2% of the initial analysis, i.e. 0.75% to 1.15% and 4.7% to 5.1%.

3. A negative control sample must be used with sample analysis on a daily basis or as frequently as

needed to ensure proper laboratory working conditions. The recommended negative control is contained in the FETALTROL kit (Catalog # FH101). Alternatively, an anticoagulated normal adult blood sample from either a male or non-pregnant female may be used.

4. Each laboratory should establish their own normal ranges, because values obtained from normals

may vary from laboratory to laboratory. 5. Refer to the appropriate flow cytometer instrument manuals and other available references for

recommended instrument calibration procedures (18, 19).





XIV. LIMITATIONS OF THE PROCEDURE

1. Positive and negative reagent controls must be included with each test sample or group of test samples analyzed. If FETALTROL cells or cord blood is not available for the performance of positive controls, the assay cannot be performed reliably.

2. The following clinical conditions may result in an increased level of fetal hemoglobin-containing red cells or F cells detected, and should not be confused with fetal red cells seen with fetal-maternal hemorrhage:

Severe Anemia Hereditary Persistence of Fetal Hemoglobin Sickle Cell Anemia Thalassemia

3. Autofluorescence of leukocytes or nucleated cells may be of the same intensity as fetal cells. Therefore, the analysis of data by flow cytometric methods should be conducted in a manner to assure that such populations are excluded from fetal cell analysis.

4. Flow Cytometer sample lines should be thoroughly rinsed between sample analysis with PBS/BSA, to eliminate carryover between samples.

5. Positive controls should be acquired as list mode files prior to test samples to verify that all reagents perform as expected under test conditions, and should be analyzed after the analysis of test samples to minimize the potential for sample carryover.

6. Blood samples should not be refrigerated or stored at ambient temperature for an extensive period (longer than 24-30 hours) prior to incubation with monoclonal antibodies.

7. Accurate results with flow cytometric procedures depend on correct alignment and calibration of the laser as well as proper gate settings.

XV. EXPECTED VALUES

Blood samples were collected from a total of 161 adult female normal donors in an age range of 19-50 yrs., with a mean and median age of 33 yrs. These consisted of approximately 50 female normal donors in each of three independent laboratories. In each laboratory, normal donors were distributed approximately equally among the three age ranges of 19-29 yrs., 30-39 yrs. and 40-50 yrs. A single sample from each donor was analyzed for the determination of expected values using the Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF.

The normal donor population consisted of members of differing ethnic origins, including adult Caucasians, African Americans, Asian Americans and Hispanics.

The three independent laboratories were located in geographically diverse areas within the United States, including the Northern, South-central and Western regions.

Normal ranges are expressed as the 95% reference intervals (20) consisting of the mid-95% of values for all donors when rank ordered from the lowest to highest percent of fetal cells detected, as indicated on the following table:





EXPECTED VALUES IN ADULT FEMALE NORMAL DONORS

Format Mean% S.D. 95% Reference n Positive Intervals (Normal Range) ------------------------------------------------------------------------------------------------ FITC 0.03 0.04 0.00--0.15 153 R-PE 0.04 0.05 0.00--0.14 153 TRI-COLOR 0.03 0.04 0.00--0.14 153 The values obtained from normal individuals may vary from laboratory to laboratory; therefore, it is recommended that each laboratory establish its own normal range.

XVI. PERFORMANCE CHARACTERISTICS

SPECIFICITY

This study was conducted to evaluate any staining of blood elements other than fetal erythrocytes by the Invitrogen HbF monoclonal antibodies. Blood samples were obtained from healthy normal donors of Caucasian, African American, Hispanic and Asian American ethnic origins. Samples of each donor were stained with Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF. Cells contained in the lymphocyte, monocytes and granulocyte regions were selected for analysis. Separate sample were prepared for analysis of normal red blood cells (1% v/v) and platelets (platelet rich plasma) and were stained with each of the Invitrogen monoclonal antibodies.

The specificity study indicated no staining of any of the indicated blood elements by the Invitrogen HbF monoclonal antibodies in samples from normal donors. The possible staining of normal red blood cells at a concentration higher than 1% was not excluded by this study.

Studies have also been conducted to measure the binding specificity of the monoclonal antibody for hemoglobin types. These studies indicated that the antibody binds to hemoglobin F (fetal hemoglobin) in a dose-dependent manner but not to hemoglobins AO, A2 or S. The HbF monoclonal antibody did not bind to the isolated GG and AG globin chains of hemoglobin F or hemoglobin A, but only to the reconstituted globin chains of hemoglobin F. These studies indicate that antibody binding is specific to hemoglobin F, and suggest that the native conformational structure of hemoglobin F must be maintained for antibody binding. Additional studies to address the potential binding of the antibody to abnormal hemoglobin variants of HbA indicated no binding to hemoglobin HbA1c, a glycolyated form of hemoglobin A that is elevated in diabetes, or to the variant HbD.

REPRODUCIBILITY, INTRA-LAB

Intra-lab reproducibility for the Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF was determined by performing 6 replicate determinations for each antibody in each of three ranges: high, medium and low. Thus, a total of 18 determinations were performed for each HbF antibody. In this manner, reproducibility was demonstrated throughout the entire measurement range for the Invitrogen Fetal Hemoglobin Test. The 6 determinations for each range were performed by the staining, processing and analysis of 6 separate samples consisting of varying mixtures of placental cord blood in normal adult blood. Fetal cells were selected for the analysis of percent cells stained in each of the three ranges.





The study was performed in each of three independent laboratories, in the manner that each laboratory obtained, stained and analyzed separate blood samples. The following data are representative:

Format Level Mean% S.D. % CV n Positive

------------------------------------------------------------------------------------- HbF FITC high 9.51 0.22 2.42 6

mid 5.10 0.22 4.30 6 low 2.19 0.05 2.26 6


-------------------------------------------------------------------------------------

HbF R-PE high 9.61 0.40 4.25 6 mid 5.10 0.14 2.66 6 low 2.14 0.10 4.75 6


------------------------------------------------------------------------------------ HbF TC high 9.64 0.37 3.84 6 mid 5.13 0.21 4.16 6 low 2.23 0.10 4.58 6 REPRODUCIBILITY, INTER-LAB Inter-lab reproducibility for the Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF was determined by performing 6 replicate determinations for each antibody in each of three ranges; high, medium and low. In this manner, reproducibility was demonstrated throughout the entire measurement range for the Invitrogen Fetal Hemoglobin Test. The 6 determinations for each range were performed by the staining, processing and analysis of 6 separate samples consisting of varying mixtures of placental cord blood in normal adult blood. Fetal cells were selected for the analysis of percent cells stained in each of the three ranges. The study was performed in each of three laboratories. All laboratories stained and analyzed the same samples. Unstained and unfixed samples containing mixtures of cord blood in normal adult blood representing the appropriate ranges were prepared by one of the participating laboratories for staining and analysis by each of the participating laboratories. The following data were obtained: SITE 1






------------------------------------------------------------------------------------- HbF FITC high 6.34 0.09 1.48 6 mid 4.05 0.08 2.09 6 low 1.48 0.04 2.91 6


------------------------------------------------------------------------------------- HbF R-PE high 7.90 0.29 3.64 6 mid 5.35 0.30 5.60 6 low 2.84 0.21 7.34 6


------------------------------------------------------------------------------------- HbF TC high 7.43 0.10 1.37 6 mid 4.37 0.11 2.57 6 low 1.60 0.07 4.55 6 SITE 2


------------------------------------------------------------------------------------ HbF FITC high 8.84 0.21 2.32 6 mid 5.69 0.13 2.36 6 low 2.44 0.08 3.28 6


------------------------------------------------------------------------------------ HbF R-PE high 8.81 0.22 2.51 6 mid 5.70 0.14 2.45 6 low 2.36 0.04 1.89 6






------------------------------------------------------------------------------------ HbF TC high 8.82 0.12 1.39 6 mid 5.68 0.15 2.69 6 low 2.41 0.05 1.90 6

SITE 3


------------------------------------------------------------------------------------ HbF FITC high 8.95 0.36 4.04 6 mid 5.85 0.23 3.86 6 low 2.57 0.14 5.32 6


------------------------------------------------------------------------------------ HbF R-PE high 8.43 0.12 1.44 6 mid 5.60 0.41 7.40 6 low 2.38 0.07 3.16 6


------------------------------------------------------------------------------------- HbF TC high 8.70 0.46 5.24 6 mid 5.65 0.18 3.12 6 low 2.41 0.17 7.13 6

CORRELATION

The Correlation study was conducted in 3 independent laboratories. Samples were analyzed with the Invitrogen Fetal Hemoglobin Test and a commercially available test based on the Kleihauer-Betke (KB) microscopic staining method for the detection of fetal hemoglobin in fetal cells (4) in each site. In all studies, confidence intervals are expressed at the 95% confidence limit.

In the study conducted at site #1, the percent of fetal cells detected by each of the HbF monoclonal antibodies to fetal hemoglobin was correlated with the percent of fetal cells detected by the KB test on patient samples (n=30) and prepared samples (n=50). Patient samples were obtained from women having clinical indications that were consistent with fetal-maternal hemorrhage and prepared artificially mixed control samples consisted of fetal cord blood in normal adult blood prepared in differing cell ratios in the range of 0-10% fetal cells. This range included up to the equivalent of 500 ml of fetal blood in maternal blood and greatly exceeded the 150-300 ml of fetal blood encountered in the most severe cases of fetal-maternal hemorrhage (3).





In the study conducted at site #2, the percent of fetal cells detected by the FITC-conjugated monoclonal antibody directed to fetal hemoglobin was correlated with the percent of fetal cells detected by the KB test in patient samples (n=38) and prepared samples (n=15) in the range of 0-3.0%. The Invitrogen test was analyzed on the EPICS-XL flow cytometer. In the study conducted at site #3, the percent of fetal cells detected by the R-PE-conjugated monoclonal antibody directed to fetal hemoglobin was correlated with the percent of fetal cells detected by the KB test in prepared samples (n=15) in the range of 0-3.0% and analyzed on the EPICS-XL flow cytometer. In all study sites results are expressed as the mean % positive cells, 95% confidence interval of the mean and numbers of samples studied in each group. Also included is the linear regression (r2) correlation between the indicated comparison groups. In the study conducted at site #1, the same prepared samples were analyzed on both the Becton Dickinson FACScan and Coulter EPICS-XL flow cytometers, as follows:

Summary of Correlations on FACscan Flow Cytometer Prepared Samples

Comparisons Mean % Confidence r2 n Positive Interval (95%) value --------------------------------------------------------------------------------------------------------------------- HbF FITC 4.54 3.63 97.95 50 KB 4.41 3.52 HbF R-PE 4.50 3.59 98.16 50 KB 4.41 3.52 HbF TC 4.49 3.58 97.98 50 KB 4.41 3.52 HbF FITC 4.54 3.63 99.59 50 HbF R-PE 4.50 3.59 HbF FITC 4.54 3.63 99.63 50 HbF TC 4.49 3.58 HbF R-PE 4.50 3.59 99.70 50 HbF TC 4.49 3.58





Summary of Correlations on EPICS-XL Flow Cytometer Prepared Samples

Comparisons Mean % Confidence r2 n Positive Interval (95%) value

--------------------------------------------------------------------------------------------------------------------- HbF FITC 4.22 3.38 98.48 50 KB 4.40 3.52 HbF R-PE 4.24 3.40 98.41 50 KB 4.40 3.52 HbF TC 4.20 3.36 97.96 50 KB 4.40 3.52 HbF FITC 4.22 3.38 99.61 50 HbF R-PE 4.24 3.40 HbF FITC 4.22 3.38 99.54 50 HbF TC 4.20 3.36 HbF R-PE 4.24 3.40 99.67 50 HbF TC 4.20 3.36

In the following linear regression analyses, the correlations of the Invitrogen Fetal Hemoglobin test and KB test are presented separately for each study site with prepared and patient samples identified within each site. However, prepared samples analyzed in the study conducted at site #1 are as indicated in the tables above. Site 1 HbF FITC Patient Samples FACscan Flow Cytometer Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF FITC 0.51 0.04 96.80 30 KB Method 0.51 0.03 HbF R-PE Patient Samples Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF R-PE 0.50 0.05 96.84 30 KB Method 0.51 0.03





HbF TC Patient Samples Procedure Mean% Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF TC 0.47 0.05 97.50 30 KB Method 0.51 0.03 Site 2 HbF FITC Patient Samples EPICS-XL Flow Cytometer Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF FITC 0.22 0.07 98.34 38 KB Method 0.20 0.05 HbF FITC Prepared Samples EPICS-XL Flow Cytometer Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF FITC 1.52 1.04 85.50 15 KB Method 1.61 1.11 Site 3 HbF R-PE Patient Samples EPICS-XL Flow Cytometer Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF R-PE 0.08 9.43 64.00 13 KB Method 0.11 0.01 HbF R-PE Prepared Samples EPICS-XL Flow Cytometer Procedure Mean % Confidence r2 n Positive Interval (95%) value --------------------------------------------------------------------------------------------------





HbF R-PE 1.38 0.93 84.01 15 KB Method 1.40 0.81 LINEARITY Linearity of measurement was determined for samples consisting of mixtures of cord blood cells in normal adult blood in the range of 0.0-5.0% positive cord blood cells. This range was selected to equal and exceed the range of values for the percent of fetal cells that are likely to be experienced in fetal-maternal hemorrhage. The linear regression method was used to plot the known expected values versus the observed values for the percent of fetal cells determined by the flow cytometric method for the Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF, with the following result:

LINEARITY OF MEASUREMENT

Procedure Mean % Confidence r2 n Positive Interval (95%) value -------------------------------------------------------------------------------------------------- HbF FITC 1.08 0.04 99.97 10 HbF R-PE 1.06 0.05 99.96 10 HbF Tri-Color 1.05 0.07 99.98 10 FITC-conjugated monoclonal antibody directed to HbF; linearity of the expected versus observed percent of fetal cells detected in samples consisting of mixtures of cord blood cells in normal adult blood:

LINEARITY OF MEASUREMENT FITC-Conjugated Antibody Directed to Fetal Hemoglobin

R-PE-conjugated monoclonal antibody directed to HbF; linearity of the expected versus observed percent of fetal cells detected in samples consisting of mixtures of cord blood cells in normal adult





blood:

LINEARITY OF MEASUREMENT R-PE-Conjugated Antibody Directed to Fetal Hemoglobin

TRI-COLOR-conjugated monoclonal antibody directed to HbF; linearity of the expected versus observed percent of fetal cells detected in samples consisting of mixtures of cord blood cells in normal adult blood:

LINEARITY OF MEASUREMENT TC-Conjugated Antibody Directed to Fetal Hemoglobin





SATURATION An additional study was conducted to identify saturating conditions for each of the Invitrogen monoclonal antibodies, consisting of preparations of cord blood alone (100% cord blood). Five different cord blood samples were stained and analyzed in a single laboratory, to determine the ability of the Invitrogen Fetal Hemoglobin Test to detect all fetal cells present, and to assure that the concentration and potency of the HbF monoclonal antibodies is sufficient to detect all fetal cells in a test sample. Therefore, samples employed in this study contained a greater than tenfold higher content of fetal cells than would be expected to occur in clinical specimens. Summary of values obtained for the percent of fetal cells detected by the Invitrogen FITC-, R-PE- and TRI-COLOR-conjugated monoclonal antibodies directed to HbF in 100% cord blood samples: Format Mean% S.D. % CV n Positive ------------------------------------------------------------------------------------ HbF FITC 95.66 3.88 4.05 5 HbF R-PE 96.70 2.70 2.80 5 HbF TRI-COLOR 95.60 2.58 2.70 5 PERFORMANCE OF POSITIVE AND NEGATIVE REAGENT CONTROLS Positive and negative reagent controls were prepared by the individual study sites and were used with sample analysis in all studies. Control data were acquired in list mode prior to the analysis of test samples to establish that all reagents were performing properly. Control data were analyzed following the analysis of test samples to minimize the potential for sample carryover. The following table is a summary of the percent of fetal cells detected from the analysis of positive and negative controls, following staining with each of the Invitrogen monoclonal antibodies in the Expected Value Study. Comparable values were obtained in studies of abnormal samples. Format Mean% S.D. Range n Positive ±2 S.D. ---------------------------------------------------------------------------------- 1% + Control: HbF FITC 0.96 0.09 0.78--1.14 35 HbF R-PE 0.99 0.21 0.57--1.41 35 HbF TC 0.99 0.17 0.65--1.33 35 5% + Control: HbF FITC 4.70 0.47 3.76--5.64 35 HbF R-PE 4.81 0.60 3.61--6.01 35 HbF TC 4.85 0.43 3.99--5.71 35 Negative Control HbF FITC 0.02 0.02 0.00--0.06 35 HbF R-PE 0.05 0.09 0.00--0.23 35 HbF TC 0.04 0.12 0.00--0.28 35

XVII. BIBLIOGRAPHY





1. Freedman J., and A. H. Lazarus. 1995. Applications of flow cytometry in transfusion

medicine, Transfusion Medicine Reviews 9: 87-109. 2. Davis B. H. 1993. Flow Cytometric Analysis of Red Blood Cells in Clinical Flow

Cytometry: Principles and Application, pp 373-387, Williams & Wilkins. 3. Sebring E. S., and H. F. Polesky. 1990. Fetomaternal hemorrhage: incidence, risk factors,

time of occurrence and clinical effects, Transfusion 30: 344-357. 4. Kleihauer E., H. Braun, and K. Betke. 1957. Demonstration of fetal hemoglobin in

erythrocytes of a blood smear, Klin. Wochenschr. 35: 637-638. 5. Emery C. L., L. F. Morway, M. Chung-Park, J. Wyatt-Ashmead, J. Sawady, and T. D.

Beddow. 1995. The Kleihauer-Betke Test, Clinical utility, indication, and correlation in patients with placental abruption and cocaine use, Arch. Pathol. Lab. Med. 119: 1032-1037.

6. Towery R., T. P. English, and D. Wisner. 1993. Evaluation of pregnant women after blunt injury, J. Trauma 35: 731-736.

7. Corsetti J. P., C. Cox, J. F. Leary, M. T. Cox, N. Blumberf, and R. A. Doherty. 1987. Comparison of quantitative acid-elution technique and flow cytometry for detecting fetal maternal hemorrhage, Ann. Clin. Lab Sci., 17: 197-206.

8. Nance S. J., J. M. Nelson, and P. A. Arndt. 1989. Quantitation of fetal-maternal hemorrhage by flow cytometry, Am. J. Clin. Path. 91: 288-292.

9. Nance S. J., and G. Garraty. 1987. Application of flow cytometry to immunohematology, J. Immunol. Meth. 101: 127-131.

10. Medearis A. L., P. A. Hensleigh, D. R. Parks, L. A. Herzenberg. 1984. Detection of fetal erythrocytes in maternal blood post partum with the fluorescence-activated cell sorter, Am. J. Obstet. Gynecol. 148(3): 290-295.

11. Thorpe S. J., S. L. Thein, M. Sampietro, J. E. Craig, B. Mahon, and E. R. Huehns. 1994. Immunochemical estimation of hemoglobin types in red blood cells by FACS analysis, Brit. J. Hemat. 87: 125-132.

12. Davis B. H., S. Olsen, N. C. Bigelow, and J. C. Chen. 1998. Detection of fetal red cells in fetomaternal hemorrhage using an anti-hemoglobin F monoclonal antibody by flow cytometry, Transfusion 38: 749-756.

13. Davis, B. H., J. Ben-Ezra, R. M. Bohmer, M. Y. Clark-Stuart, J. N. Lowder, W. C. Mahoney, R. A. Sacher, and A. Van Agthoven. Fetal Red Cell Detection, Approved Guideline , NCCLS Document H52-A. 2001.

14. Stamatoyannopoulos G., and A. W. Nienhuis. 1987. Hemoglobin switching, in The Molecular Basis of Blood Diseases, pp. 67-105, Saunders & Co., Philadelphia.

15. Chen, J. C., N. C. Bigelow, and B. H. Davis. 2000. Proposed flow cytometric reference method for erythroid F cells counting. Cytometry 42: 239-246.

16. Mundee, Y., N. C. Bigelow, B. H. Davis, and J. B. Porter. 2000. Simplified flow cytometric method for fetal hemoglobin containing red blood cells. Cytometry 42: 389-393.

17. Nicholson J. K. A., and T. A. Green. 1993. Collaborative Laboratories, Selection of anticoagulants for lymphocyte immunophenotyping. J. Immunol. Methods 165: 31-35.

18. Brown M. C., R. A. Hoffman, and S. Kirchanski. 1986. Controls for flow cytometers in hematology and cellular immunology, Ann. N.Y. Acad. Sci. 468: 93-103.

19. Durrand R. E. 1981. Calibration of flow cytometer detector systems, Cytometry 2: 192-193. 20. How to define and determine reference intervals in the clinical laboratory; Approved Guideline,

NCCLS Document C28-A, Vol. 15, No. 4, June 1995.





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MONOCLONAL ANTIBODIES DIRECTED TO HUMAN FETAL …tools.thermofisher.com/content/sfs/manuals/L13012_ms_x_hu_HbF_mab.pdfof fetal red blood cells. Fetal cells are identified by the presence